Carátula 2da parte - FBMC

INTRODUCCION A LA
FISIOLOGIA MOLECULAR
Hombre contemplando. Runo Tamayo
GUIA DE SEMINARIOS 2012
Segunda parte
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IFM 2012
Día Fecha Prof Tema Seminarios TP/Seminarios
Lu 08-oct feriado
Ma 09-oct
Mie
10-oct FM
Sistema respiratorio. Sangre. Intercambio de
gases.
TP. Fisio del reposo A
Jue 11-oct TP. Fisio del reposo A
Lu
15-oct
FM
Sistema respiratorio. Sangre. Intercambio de
gases.
S9. Respiratorio
Ma 16-oct 14hs Recuperatorio 1er parcial S9. Respiratorio
Mie 17-oct LS Sistema Circulatorio: sangre y hemodinámica TP. Fisio del Reposo B
Jue 18-oct TP. Fisio del Reposo B
Lu 22-oct FM Sistema Circulatorio: corazón S10. Sangre-Corazón
Ma 23-oct S10. Sangre-Corazón
Mie
24-oct LS
Sistema Circulatorio: regulacion local del flujo
sanguíneo
TP.Fisio del ejercicio
A
Jue 25-oct TP.Fisio del ejercicioA
Lu 29-oct LS Sistema excretor S11. Hemodinamia
Ma 30-oct S11. Hemodinamia
Mie
31-oct LS
Sistema Circulatorio: regulacion neural del
flujo sanguíneo
TP. Fisio del ejercicio
B
Jue 01-nov TP.Fisio del ejercicioB
Lu
05-nov LS
Sistema endocrino. Control de glucemia.
Regulación de la Temperatura
S12. Excretor
Ma 06-nov S12. Excretor
Mie 07-nov LS Regulación autónoma. TP.Ejercicio-Análisis
Jue 08-nov TP.Ejercicio-Análisis
Lu 12-nov S13. Endocrino
Ma 13-nov S13. Endocrino
Mie 14-nov TP. Excretor
Jue 15-nov TP. Excretor
Lu 19-nov S14. Autónomo
Ma 20-nov S14. Autónomo
Mie 21-nov
Jue 22-nov
Lu 26-nov
Ma 27-nov
Mie 28-nov 2do parcial
Jue 29-nov
Lu 03-dic
Ma 04-dic
Mie 05-dic
Jue 06-dic
Lu 10-dic 14hs Recuperatorio 2do parcial
Ma 11-dic
Mie 12-dic
Jue 13-dic

IFM 2012
Seminario 9: Sistema respiratorio. Intercambio de gases.
-Bibliografía Sugerida. Del libro “Principles of Human Physiology” de Stanfield &
German: Capítulo 15, The respiratory system: pulmonary ventilation; y Capítulo 16:
The respiratory system: Gas exchange and regulation of breathing.
1. El sistema respiratorio se compone de una sucesión de compartimentos por los
cuales circula el aire. Indique en un esquema los compartimentos de conducción y los
canales en los que se produce el intercambio. ¿Cuál de estos compartimentos presenta
la mayor superficie?
2. ¿Qué función tienen y dónde se ubican las células goblet y las células ciliadas?
3. Durante la respiración se produce un cambio rítmico en la presión alveolar.
a. Dibuje la evolución de este ritmo e indique cuál fase corresponde a la inspiración y
cuál a la espiración.
b. ¿Qué fuerzas operan durante la inspiración y cuáles durante la espiración?
c. ¿Qué elementos anatómicos periféricos y del sistema nervioso central están involucrados
en la generación de estas fuerzas?
4. El compliance (distensibilidad) pulmonar deriva de las propiedades mecánica de
los alvéolos
a. ¿Cómo se define este parámetro pulmonar?
Utilizando un pulmón aislado se procede a inflarlo (Inf) y desinflarlo (Def) de aire
(air) para medir el compliance. El mismo pulmón es sometido a cambios de volumen
llenándolo y vaciándolo de solución salina (Saline). Las siguientes curvas muestran
los resultados.
b. Explique la diferencia del trayecto de las curvas de inflado y desinflado del
pulmón.
c. Explique la diferencia entre estas curvas y las mostradas por el pulmón cuando éste
es llenado de solución salina.
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5. Los surfactantes afectan el ‘compliance’ pulmonar
a. Explique de qué tipo de sustancia se trata.
b. ¿Cuál es el efecto de los surfactantes sobre el compliance?
c. ¿Cómo se alcanza este efecto?
d. Dibuje, sobre las curvas de inflado y desinflado que se muestra en la pregunta 4,
cómo se modificarían si se agregara un surfactante exógeno?
6. En la siguiente figura se muestra la relación entre los cambios en el volumen
pulmonar en función de la presión pulmonar para individuos sanos y para pacientes
con fibrosis o con enfisema. La fibrosis se produce por el depósito de mucosa en el
pulmón, mientras que en el enfisema se observa una destrucción de la matriz
extracelular.
¿Qué asociación hipotética puede realizar entre la breve descripción de la enfermedad
que se dio más arriba y los efectos sobre el compliance?
7. ¿Cuál es el problema respiratorio principal en bebés prematuros que carecen de
surfactantes?
8. Explicar cuáles son los pasos que permiten el transporte de oxígeno desde la
atmósfera hacia los tejidos, en un mamífero, siguiendo la siguiente guía:
a. ¿Cómo entra este gas a la sangre y cuál es la fuerza impulsora del transporte?
b. ¿Cómo se transporta en la sangre?
c. ¿Qué fuerza impulsora regula el flujo del oxígeno hacia los tejidos?
9. Confeccionar una tabla indicando los valores de las presiones parciales del O 2 y el
CO 2 en: el aire, la cavidad alveolar, la vena pulmonar y la arteria pulmonar. Explicar
cuáles son las razones por las que se producen las variaciones observadas en cada
compartimiento respecto del aire. ¿Por qué razón no tenemos en cuenta al gas
mayoritario en el aire, el N 2?
10. La hemoglobina es una proteína alostérica. Explique este concepto. ¿Cómo se
expresa esta propiedad en su relación con el O 2?
11. La mioglobina y la hemoglobina fetal tienen un comportamiento diferente como
transportadores de O 2. Dibuje esquemáticamente un gráfico que muestre la relación
entre el porcentaje de saturación de O 2 de la hemoglobina adulta, la fetal y la
mioglobina en función de la presión de O 2 y explique de qué manera se expresa en
este gráfico el carácter alostérico de la Hb.
12. Cuando se habla del contenido de O 2 en la sangre, ¿se puede dar esta medida en
mm de Hg? Explique su respuesta.
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13. El O 2 es transportado en la sangre por dos vías, mientras que el CO 2 es
transportado por tres vías. ¿Cuáles son cada una de estas vías y qué porcentaje de cada
gas es transportado por cada una de ellas?
14. El eritrocito juega un rol fundamental en la remoción de CO 2 de los tejidos. ¿Qué
fenómenos tienen lugar en estas células de la sangre que hacen más eficiente la
remoción de CO 2? ¿Por qué no suceden en el plasma?
15. En un vaso artificial que permite el flujo de O 2 a través de la pared del mismo
fluye sangre oxigenada. El vaso fue colocado en un medio desoxigenado y a medida
que la sangre fluía por el mismo se midió el nivel de saturación de oxígeno de la
misma. Los resultados se muestran en la figura:
a. ¿Cómo se explica la caída en la saturación de O 2 a lo largo del tubo?
b. ¿Dónde espera que se observen estos efectos en el sistema circulatorio? ¿Por qué?
Utilizando el mismo esquema experimental se midió cómo influye la velocidad del
flujo en la desoxigenación de la sangre y se observaron los siguientes resultados.
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c. ¿Cómo se explica la relación oxigenación – velocidad del flujo?
d. ¿Influye el hematocrito en esta relación?
16. El monóxido de carbono es potentemente venenoso. ¿Cuál es la razón?
17. El aumento en la concentración de dióxido de carbono trae aparejado un cambio
en el pH de la sangre. ¿Es este cambio un aumento o una disminución? ¿A qué se
debe que el cambio sea relativamente pequeño? El sistema de control de pH que
involucra al CO 2 ¿es abierto o cerrado?, explique.
18. El efecto Bohr es consistente con la necesidad de una mayor provisión de O 2 a los
tejidos. Explicar esta afirmación.
19. En un estudio se analiza el efecto del pH sanguíneo sobre el sistema respiratorio.
En un animal anestesiado el pH sanguíneo es manipulado farmacológicamente, al
tiempo en que se registra la actividad del nervio frénico (inerva al diafragma)
mediante electrodos extracelulares que permiten evaluar la tasa de disparo en el
mismo.
a. ¿Qué cambios espera registrar en la actividad eléctrica del nervio frénico ante
una disminución en el pH?
b. ¿Qué efectos produce el cambio de actividad del nervio frénico en las fibras
musculares del diafragma?
c. ¿Cuál es el mecanismo por el cual se transmite la información desde el nervio
frénico a las fibras del diafragma?
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IFM 2012
Seminario 10: Sangre. Corazón.
-Bibliografía Sugerida: ver Seminario 9. Capítulo 20: Cardiac electrophysiology and the
electrocardiogram; Capítulo 21: The heart as a pump. Del libro: Medical Physiology de
Boron & Boulpaep.
Sangre
1. Estudios experimentales mostraron que la altura respecto del nivel del mar produce un
aumento en el hematocrito. Si tomamos como dato que la altura implica una disminución
en la presión parcial de oxígeno, ¿cómo se explica el aumento en el hematocrito? ¿Qué
rol juega el 2,3-difosfoglicerato (DPG) en la adaptación del organismo a la altura? Utilice
las curvas de disociación en su explicación.
2. En las siguientes curvas se estudió los niveles de oxigenación de la sangre en
presencia de concentraciones crecientes de 2,3-difosfoglicerato.
a. Explique el efecto del DPG.
b. Indique cuál de las curvas
corresponde a la mayor
concentración de DPG, y cuál
a la menor.
3. La anemia nutricional se define como una deficiencia dietaria de hierro. En estos
pacientes se observa un hematocrito normal pero un menor contenido de O2 en sangre.
¿Cómo se pueden explicar estos resultados?
4. ¿Cuáles son las funciones principales de las proteínas plasmáticas? ¿Cuál es la más
abundante?
5. El técnico de laboratorio encargado de realizar los análisis de sangre confundió un
tubo de anticoagulante con otro que tenía el mismo anticoagulante con un agregado de
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NaCl. Al analizar el hematocrito del paciente obtuvo valores por debajo de los normales.
Si la sangre analizada fuera la suya ¿empezaría la dieta del guiso de hígado y lentejas?
6. Usted se inscribe para competir en el Tour de France y en un vestuario le recomiendan
extraerse sangre y reinyectarse los glóbulos rojos el día anterior a la competencia. ¿Cuál
es el objetivo de este “tip” deportivo (no se detecta en el antidoping)? ¿Sería realmente
ventajoso?
Corazón
1. El corazón puede ser considerado como dos bombas actuando en serie, ¿cómo se
explica esta aseveración?
2. Los siguientes son componentes funcionales del ciclo cardíaco. Indique con números
del 1 al 10 el orden en que se producen, siendo 1 el Llenado auricular.
__ Contracción isovolumétrica
__ Llenado ventricular
__ Apertura de las válvula auriculoventricular
__ Cierre de las válvulas auriculoventriculares
__ Contracción ventricular
__ Contracción auricular
1_ Llenado auricular
__ Apertura de las válvulas semilunares (de ventrículo a arterias)
__ Cierre de las válvulas semilunares (de ventrículo a arterias)
__ Relajación ventricular
3. La presión atmosférica es de 760 mm Hg, mientras que la presión ventricular en su
pico máximo es de 120 mm Hg. ¿Cómo se explica que el corazón no colapse bajo el gran
gradiente de presión?
4. Las fibras musculares cardíacas forman un sincicio funcional. Explique esta idea, y
cuál es la función del mismo. Ahora, si es así, ¿por qué es necesario que las fibras
musculares sean excitables, en lugar de seguir pasivamente al marcapasos?
5. ¿En qué se diferencian los potenciales de acción de una fibra marcapasos, de los
potenciales de acción de una fibra muscular cardiaca de la aurícula o el ventrículo?
Responda en términos de: i) las conductancias involucradas; ii) la duración del potencial
de acción
6. La contracción de la musculatura estriada, lisa y cardiaca se inicia por la
despolarización supraumbral de estas fibras musculares. Sin embargo, el fenómeno que
da origen a dicha despolarización es diferente en cada caso. Explique el origen de la señal
y su naturaleza.
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7. El acople excitación-contracción en el músculo cardíaco se da a través del aumento de
la concentración intracelular de Ca 2+ ¿cuáles son las fuentes de Ca 2+ ? ¿cómo se vehiculiza
el catión divalente desde estas fuentes al citosol? ¿cuál es su blanco de acción?
8. Relacione las siguientes estructuras con los siguientes fenómenos: Estructuras: canal
de calcio sensible a rianodina, canal de calcio de tipo L sensible al voltaje, troponina C.
Fenómenos: contracción muscular, entrada de calcio del medio externo al citoplasma,
“Ca ++ -spark”. En su respuesta ubique cada estructura en un compartimiento celular.
9. ¿Qué es un “Ca ++ -spark”? ¿Cuáles son sus propiedades funcionales? ¿Cómo sirven
estas propiedades a la función fisiológica que cumple el Ca ++ -spark?
10. ¿Cuáles son los principales marcapasos del corazón de los mamíferos? ¿Cómo se
comunican entre si? ¿Cuáles son sus respectivas funciones
11. Está investigando las características de las fibras marcapasos con técnicas
electrofisiológicas en el corazón de mamíferos. Ha impalado una fibra en un corazón
aislado en una cámara especial de registro y esta fibra dispara en forma rítmica de manera
espontánea. Tan excitado estás con este primer registro que asumís que, efectivamente,
estás registrando una fibra marcapasos. ¿Alcanzan estos datos para aseverarlo? Justificar
la respuesta.
12. ¿Qué es el gasto cardíaco (cardiac output)? Este puede definirse desde un punto de
vista hemodinámico o cardíaco ¿cuáles son las fórmulas que lo describen?
13. La contracción auricular y la ventricular cardíaca están desfasadas en el tiempo.
a. ¿Qué señal inicia la contracción auricular y cuál la ventricular?
b. ¿Por qué es necesario este desfase?
c. ¿Cómo se regula este desfase?
d. ¿Cómo afecta este desfase la frecuencia cardiaca?
e. ¿Qué efectos tiene sobre la frecuencia cardiaca el sistema autónomo?
14. El flujo de sangre en el corazón está regulado por válvulas ¿Qué regula el cierre y
apertura de las válvulas? Indique cuál de los siguientes parámetros cardíacos está más
afectado por la eficiencia de la actividad de las válvulas: ciclo cardíaco y gasto cardíaco.
Explique por qué.
15. Durante el ciclo cardíaco, los períodos de sístole y diástole se asocian
funcionalmente con los períodos de contracción y relajación ventricular, respectivamente.
Si bien el estado de las aurículas es ignorado en dicha clasificación, ¿qué sucede con la
presión auricular durante dichos períodos ventriculares?
16. Aborde los aspectos de ciclo cardíaco que se observan en el siguiente diagrama:
a. ¿Cómo se explica el retardo entre la fase de subida en la presión de la aorta y la fase de
subida de la presión en el ventrículo?
b. ¿Cómo se explica que la presión auricular y ventricular estén en antifase?
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c. Indique los períodos de contracción y relajación ventricular isovolumétricas.
17. El siguiente gráfico muestra la variación de presión vs. volumen ventricular para un
corazón normal (plano transparente) y para un corazón que padece estenosis mitral (plano
gris).
Nota: la válvula mitral es la que regula el flujo entre la aurícula y el ventrículo izquierdo.
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a. Indique cuál de las siguientes variables cambian con esta enfermedad:
frecuencia cardíaca
volumen sistólico
presión sistólica
b. De una explicación funcional a los cambios observados.
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IFM 2012
Seminario 11: Hemodinámica
-Bibliografía Sugerida: Medical Physiology de Boron & Boulpaep, Capítulo 17:
Organization of the cardiovascular system, Capítulo 18: Arteries and veins; Capítulo 19:
The microcirculation.
-Bibliografía obligatoria: “Role of Shear Stress and Endothelial Prostaglandins in Flow-
and Viscosity-Induced Dilation of Arteriole In Vitro”. Koller et al., Circulation Research
72: 1276-1284 (1993)
1. Los vasos del sistema circulatorio están conectados en configuraciones ‘en serie’ y ‘en
paralelo’. Explique estos términos y dé ejemplos de tramos del sistema que operan en una
y otra configuración.
2. La ley de Poiseuille indica que el flujo depende de la presión y de la resistencia.
a. ¿Cuál es la función que relaciona a estas dos variables? ¿qué factores de la sangre y del
sistema vascular influyen en la resistencia?
b. Se dice que el flujo de la sangre es mayoritariamente laminar ¿qué define a este tipo de
flujo?
c. ¿Cómo se define viscosidad?
3. El efecto Fahraeus-Lindquist describe que la viscosidad aparente de la sangre que
circula por tubos con diámetro muy pequeño (

9. Con el fin de investigar el flujo sanguíneo en los vasos del riñón se analizó cómo
respondían segmentos de arterias aisladas ante flujos crecientes. Los parámetros que se
midieron fueron presión y diámetro. Los resultados se muestran en la figura que se
presenta más abajo. En condiciones control (Endothelium intact = endotelio intacto) se
observa un aumento en el diámetro a medida que aumenta la presión, debido al aumento
en el flujo.
En otra series de experimentos se repitió el experimento en arterias en las cuales se dañó
el endotelio (Endothelium disrupted = endotelio dañado) o en la que se expusieron las
arterias intactas a un inhibidor de la síntesis de NO (L-NMMA).
a. ¿Qué ilustran estos resultados?
Utilizando este esquema experimental, y a partir de estos resultados, usted quiere analizar
si el sistema autónomo tiene capacidad de regular este efecto local. Usted realiza esta
investigación en el marco de su Tesina de Licenciatura y dado que se quiere recibir
pronto sólo puede hacer dos series experimentales imprescindibles para dar una respuesta
básica a su pregunta.
b. ¿Qué experimentos realizaría? Explique el razonamiento que hizo al elegir cada
tratamiento.
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IFM 2012
Seminario 12: Sistema renal
-Bibliografía Sugerida: Capítulo 17: The Urinary System: Renal Function y Capítulo
18: The Urinary System: Fluid and Electrolyte Balance. Del libro: Human Physiology
de Germann & Stanfield.
1. Las presiones que se ponen en juego en el proceso de filtración glomerular son
la hidrostática y la oncótica.
a. Explicar el origen de cada una de estas presiones.
b. ¿En qué dirección actúa la presión hidrostática prevalente, y en cuál la presión
oncótica prevalente? Explicar esta direccionalidad.
c. La presión osmótica que ejercen los iones no juega un rol importante en la
filtración glomerular. Explicar la razón por la cual esta aseveración es correcta.
2. Si disminuye la resistencia en la arteriola aferente, ¿qué espera observar, un
aumento o una disminución en el volumen filtrado en el glomérulo? ¿Cómo varía su
respuesta si lo que se produce es una disminución en la resistencia en la arteriola
eferente? Explique sus respuestas.
3. Teniendo en cuenta las consideraciones realizadas en la pregunta 2, explique de
qué manera el reflejo miogénico puede ayudar a evitar que ante un aumento en la
presión arterial se produzca una mayor filtración glomerular. ¿En qué arteriola espera
que se produzca el reflejo miogénico, en la aferente o en la eferente, para lograr el
efecto mencionado?
4. ¿En qué porción del nefrón se produce la mayor reabsorción de H 2O, y en cuál se
produce una regulación específica de la reabsorción de H 2O?
5. La formación de un gradiente osmótico en la médula renal es función del:
a. glomérulo
b. vasa recta
c. asa de Henle
d. mácula densa
Explicar la elección de la(s) respuesta(s) correcta(s).
6. La hormona antidiurética (o vasopresina) participa en la regulación de la presión
arterial. ¿Cómo se produce esta regulación? Incluya en su respuesta los conceptos de
baroreceptores, neurohormona, acuaporina y osmolaridad de la orina.
7. La renina es una hormona cuya acción se ejerce (directa o indirectamente) a nivel
de la vasculatura, del riñón y del sistema nervioso central. Explique los tres niveles de
acción de la renina.
8. ¿Cuál es el rol del canal de Na + en las células epiteliales del riñón? ¿En qué región
del tubo renal se encuentra, y qué cara de la célula epitelial ocupa?
9. En base al siguiente gráfico conteste las preguntas que se formulan.
a. ¿Qué parámetro puede utilizar para deducir la velocidad máxima de transporte en
el sistema ejemplificado en este gráfico?
b. Identifique el umbral de excreción renal.
c. Explique cómo se relacionan las curvas de reabsorción/excreción.
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d. ¿Qué variación esperaría observar en estos gráficos en un animal en el cual se
inhibe la acción de la insulina?
10. El pH sanguíneo puede ser compensado a nivel respiratorio y renal. ¿Qué
proteínas membranales permiten regular el pH en el epitelio renal? Indique de qué
manera la función de estas proteínas regula el pH.
11. Animales mutantes nulos (knock-out, KO) para el gen que codifica al canal de Na +
epitelial SGK1 mostraron diferencias con el wild-type (wt) solamente cuando fueron
sometidos a una dieta con bajo contenido de Na + . ¿Cuál(es) de estas observaciones
es(son) correcta(s)?
a. La orina de los animales KO presentó mayores niveles de Na + que la orina de los
animales wt.
b. La sangre de los animales KO presentó menores niveles de Na + que la sangre de
animales wt.
c. Los animales KO respondieron mejor al tratamiento con aldosterona que los wt.
12. La aldosterona potencia la reabsorción de Na + aumentando la expresión de los
canales de Na + epiteliales y modificando la actividad de la bomba de Na + /K + . En
experimentos en que se midió la corriente eléctrica sensible a ouabaína (bloqueante
específico de esta bomba) se observó que la aldosterona aumenta dicha corriente. ¿Por
qué razón no alcanzó con modificar la densidad de canales de Na + sino que, además,
fue necesario aumentar la actividad de la bomba?
13. ¿Cuánto tarda un volumen de sangre igual a la volemia (5000 ml) en pasar por los
riñones si el flujo sanguíneo renal es del 25% del volumen minuto (5000 ml)? ¿Qué
cantidad de veces por día pasa un volumen como éste por los riñones?
14. El flujo plasmático renal (FPR) es la cantidad de plasma que pasa por minuto por
los riñones. Utilizando los datos de la pregunta anterior y considerando un
hematocrito del 45 %, ¿cuál es su valor?
15. En un estudio experimental se administra una droga que inhibe completamente la
reabsorción de glucosa por el riñón. Complete la información faltante en la siguiente
tabla considerando que la droga no afecta la VFG (velocidad de filtrado glomerular).
Nota: La velocidad de excreción de una sustancia es la velocidad de aparición de
dicha sustancia en la orina que se acumula en la vejiga.
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antes de la droga después de la droga
[inulina] plasma 1 mg / ml 1 mg / ml
[glucosa] plasma 1 mg / ml 1 mg / ml
vel. excreción inulina 100 mg / min 100 mg / min
vel. excreción glucosa 0 mg / min mg / min
aclaramiento inulina ml / min ml / min
aclaramiento glucosa ml / min ml / min
16. Considere dos tipos de sustancias:
Sustancia A: sometida solamente a filtración glomerular, no se reabsorbe ni secreta.
Sustancia B: sometida a filtración glomerular y reabsorción.
Para cada sustancia dibuje las tres siguientes curvas:
a. Relación entre la tasa de filtrado glomerular y concentración en plasma
b. Relación entre la tasa de excreción y concentración en plasma
c. Relación entre la tasa reabsorción y concentración en el plasma.
Superponga en un mismo par de ejes las tres curvas, donde el eje x es la
concentración de la sustancia en plasma y el eje y es el valor relativo de c/u de las tres
tasas de movimiento de la sustancia.
Concentración de A en plasma (mg/ml) Concentración de B en plasma (mg/ml)
Se provoca una vasoconstricción en la arteria aferente del glomérulo. Dibuje
nuevamente las curvas a y b anteriormente requeridas en condiciones control y luego
del tratamiento.
17. Las acuaporinas (AQPS) son una familia de canales de membrana que permiten
el flujo de agua a favor de su gradiente. AQP2 es el tipo predominante de acuaporina
sensible a AVP (vasopresina) en el ducto colector, localizados en la membrana apical.
Su regulación está involucrada en el balance osmótico corporal a corto y largo plazo.
AVP se une a sus receptores basolaterales (tipo V2) activando adenilato ciclasa vía
proteína G, esto a su vez induce un incremento en cAMP y PKA. PKA fosforila a
AQP2 incrementando la cantidad de AQP2 en la membrana apical, presumiblemente
por estimulación de la fusión de vesículas de AQP2 con la membrana. El incremento
de AQP2 en membrana aumenta instantáneamente la reabsorción de agua y la
concentración de la orina.
16

Usted se propone evaluar el efecto de la dopamina (Dop) en el tráfico
vesículas/membranas de AQP2. Para ello usted mantiene fetas de médula interna renal
canina (zona rica en ductos colectores) en cultivo y las estimula con AVP, Dop o
combinaciones de ambas. Luego de incubar, homogeiniza las células y por
centrifugación diferencial separa fracciones ricas en membrana apical o vesículas. Las
muestras se analizan por SDS-PAGE seguido por inmunoblot y densitometría para
cuantificar la cantidad de proteína.
a. En base a sus resultados ¿cuál es el efecto de la dopamina en el tráfico vesicular
de AQP2?
b. ¿La dopamina actuará directamente sobre AQP2 o será dependiente de la
activación previa por AVP?
c. Proponga un mecanismo de acción de dopamina y el experimento que haría para
ponerlo a prueba.
Además del mecanismo propuesto por usted, un colega le regala un anticuerpo que
reconoce específicamente AQP2 fosforilada (p-AQP2). Usted corre a preparar otro
experimento donde nuevamente estimula a sus cultivos con AVP, AVP y dopamina o
dopamina sola. Luego corre un gel de extratos totales de células del colector.
d. ¿Está sorprendido por el incremento de p-AQP2 por AVP?
17

e. ¿Cómo explica el efecto de la dopamina más AVP?
f. Teniendo en cuenta que la dopamina también baja p-AQP2, ¿se sustenta la
hipótesis de que actúe sobre la vía de AVP?
g. ¿Qué hipótesis alternativa se le ocurre? Plantee el experimento para ponerla a
prueba.
18

IFM 2012
Seminario 13: Endocrinología
-Bibliografía obligatoria: “Measurements of cytoplasmic Ca2+ in islet cells clusters
show that glucose rapidly recruits ß-cells and gradually increases the individual cell
response” Jonkers y Henquin. Diabetes 50: 540-550. (2001)
-Bibliografía Sugerida: “Organization of Endocrine Control”, capítulo 46 Del libro:
Medical Physiology de Boron & Boulpaep.
1. a. ¿En qué se distinguen las secreciones paracrinas de las endocrinas?
b. explique el concepto de secreción autocrina
c. distinga las secreciones endocrinas de las exócrinas. Para cada caso dé un par de
ejemplos. ¿Cómo diferenciaría una glándula endocrina de una exócrina?
2. ¿En qué se diferencia la secreción endocrina de la que tiene lugar en una sinapsis?
¿Qué rol juega el Ca 2+ en los procesos de secreción y cuáles son los reservorios de
calcio que se ponen en juego en dichos procesos?
3. ¿Qué diferencias existen entre las hormonas y las neurohormonas? ¿Cuál es el
vehículo de las señales en el sistema nervioso, y cuál el de las señales en el sistema
endocrino?
4. ¿Puede establecer alguna relación entre la estructura química de una hormona y la
localización celular de su receptor? En caso afirmativo indique cuál es esa relación.
¿En qué tipo de hormonas la estimulación de la síntesis y de la secreción no pueden
disociarse?
5. Haga un cuadro comparativo diferenciando las hormonas liberadas por la
neurohipófisis y la adenohipófisis. Incorpore en su cuadro los reguladores de la
liberación de cada hormona, su blanco y el efecto. ¿Cuáles son las hormonas liberadas
por la neurohipófisis?¿Qué regula su liberación?¿Cuáles son sus blancos de acción?
6. ¿Qué es un reflejo neuroendocrino? ¿Cuántos tipos existen y en qué se diferencian?
Dar ejemplos de retroalimentacion negativa de lazo corto y lazo largo en el control de
la secreción hormonal.
7. La glándula suprarrenal se divide en dos regiones, la corteza y la médula.
Identifique las hormonas liberadas por cada una de estas regiones y sus funciones e
indique qué regula la liberación de las mismas.
8. Dibujar esquemáticamente la anatomía general del páncreas y describir sus partes
principales. ¿Cuáles son los tipos celulares del páncreas endócrino y qué hormonas
secretan? ¿Cuáles son las acciones primarias de la insulina y el glucagon?
9. En la unidad funcional células α-células β del páncreas, ¿cuál de las células integra
la información? ¿Cómo se transmite dicha información al otro tipo celular?
19

10. ¿Cuál es la diferencia principal entre un paciente cuyo páncreas tiene células ß
disfuncionales y aquél cuyo páncreas tiene células ß con baja sensibilidad a la
glucosa? ¿Qué tipo de tratamiento puede paliar los síntomas en cada uno de estos
casos?
11. La diabetes tipo 2 está caracterizada por altos niveles de glucosa en sangre debido
a una secreción reducida de insulina por parte de las células beta del páncreas o bien
por una reducida acción de la hormona. Misaki y colaboradores (J. Pharmacol. Exp.
Ther. 332: 871-878) decidieron probar la acción de una nueva droga recientemente
sintetizada, la iptakalim en cultivos de células beta para evaluar su posible empleo
como droga hipoglucemiante. El experimento de la figura 1 muestra los resultados de
exponer celulas beta previamente cargadas con Fura-2 (sensor de calcio) a distintas
sustancias. Los cultivos se crecieron en un medio que contenía 5 mM de glucosa
(normoglucemia).
Datos: el diazoxide produce la apertura del canal de K + sensible a ATP, la nifedipina bloquea
los canales de calcio sensibles a voltaje tipo L.
En la figura 2 se mide la liberación de insulina por parte de las células beta, por medio
de un RIA del perfusato en el control y luego de los tratamientos.
Figura 1
Figura 2
a. Explique en detalle los resultados de la figura 1 y proponga el mecanismo de
acción de la droga.
b. Teniendo en cuenta la figura 2, ¿piensa que la iptakalim tiene un futuro en la
terapéutica de esta enfermedad?
c. Diseñe un experimento para probar la droga in vivo.
20

Glosario del trabajo de Jonkers y Henquin.
Fura. Sustancia derivada del quelante de calcio BAPTA, que al unirse a calcio sufre
un corrimiento en su espectro de absorción a pesar de que mantiene su espectro de
emisión. Este indicador de calcio es utilizado para medir cambios en la concentración
del cation en tiempo real. Esto se hace iluminando a la preparación con la longitud de
excitación del indicador no unido al calcio (340 nm) y con la onda de excitación del
indicador unido al calcio (380 nm), y midiendo la intensidad de luz con filtros que
capten las frecuencias de onda típicas de la emisión del indicador (510 nm). La razón
entre la emisión lograda con 340 dividido la emisión lograda a 380 permite estimar
los cambios en la concentración intracelular del calcio.
Tinción con yoduro de propidio (propidium iodide, PO) + naranja de acridina
(acrydine orange AO). Tinción que distingue células vivas (fluorescen en verde,
AO) de células que tienen sus membranas dañadas (fluorescen en rojo, PO).
Tolbutamide: Esta droga estimula la secreción de insulina. Actúa bloqueando los
canales de K + sensibles al ATP.
1) ¿Cuál es la pregunta que intenta dilucidar este trabajo?
2) Haga un diagrama del protocolo experimental que incluya: a. preparación, b. ¿qué
se mide,? c. ¿cómo se estimula?, d. ¿qué controles se realizan?
3) Discutir la Figura 1, haciendo énfasis en las diferencias observadas entre células
aisladas y grupos de células (clusters); y entre las diferentes concentraciones.
Consejo: mire los experimentos graficados en la Figura 5 para entender las
mediciones que se realizan.
4) Discutir la Figura 2. ¿Cuál es el parámetro que se analiza? ¿En qué se diferencia
de lo que se grafica en la Figura 3 (A y B)?
5) ¿Qué argumento agregan los datos graficados en las Figuras 3C y 3D?
6) ¿Cuál es el sentido de medir el efecto de la tolbutamida en cada caso?
7) ¿A qué pregunta responden los experimentos cuyos resultados se grafican en las
Figuras 4 y 5? ¿Cuáles son los resultados y qué indican?
8) ¿Qué indican los resultados graficados en la Figura 6?
9) Describa los resultados que se presentan en la Figura 7. ¿Qué se concluye?
21

Measurements of Cytoplasmic Ca 2 in Islet Cell Clusters
Show That Glucose Rapidly Recruits -Cells and
Gradually Increases the Individual Cell Response
Françoise C. Jonkers and Jean-Claude Henquin
The proportion of isolated single -cells developing a metabolic,
biosynthetic, or secretory response increases
with glucose concentration (recruitment). It is unclear
whether recruitment persists in situ when -cells are
coupled. We therefore measured the cytoplasmic free
Ca 2 correction ([Ca 2 ] i) (the triggering signal of glucose-induced
insulin secretion) in mouse islet single
cells or clusters cultured for 1–2 days. In single cells,
the threshold glucose concentration ranged between 6
and 10 mmol/l, at which concentration a maximum of
65% responsive cells was reached. Only 13% of the
cells did not respond to glucose plus tolbutamide. The
proportion of clusters showing a [Ca 2 ] i rise increased
from 20 to 95% between 6 and 10 mmol/l glucose,
indicating that the threshold sensitivity to glucose differs
between clusters. Within responsive clusters, 75%
of the cells were active at 6 mmol/l glucose and 95–100%
at 8–10 mmol/l glucose, indicating that individual cell
recruitment is not prominent within clusters; in clusters
responding to glucose, all or almost all cells participated
in the response. Independently of cell recruitment,
glucose gradually augmented the magnitude of
the average [Ca 2 ] i rise in individual cells, whether
isolated or associated in clusters. When insulin secretion
was measured simultaneously with [Ca 2 ] i, a good
temporal and quantitative correlation was found between
both events. However, -cell recruitment was
maximal at 10 mmol/l glucose, whereas insulin secretion
increased up to 15–20 mmol/l glucose. In conclusion,
-cell recruitment by glucose can occur at the stage of
the [Ca 2 ] i response. However, this type of recruitment
is restricted to a narrow range of glucose concentrations,
particularly when -cell association decreases the
heterogeneity of the responses. Glucose-induced insulin
secretion by islets, therefore, cannot entirely be ascribed
to recruitment of -cells to generate a [Ca 2 ] i
response. Modulation of the amplitude of the [Ca 2 ] i
response and of the action of Ca 2 on exocytosis (amplifying
actions of glucose) may be more important.
Diabetes 50:540–550, 2001
From the Unité d’Endocrinologie et Métabolisme, University of Louvain
Faculty of Medicine, Brussels, Belgium.
Address correspondence and reprint requests to Dr. J.-C. Henquin, Unité
d’Endocrinologie et Metabolisme, UCL 55.30, avenue Hippocrate 55, B-1200
Brussels, Belgium. E-mail: henquin@endo.ucl.ac.be.
Received for publication 14 April 2000 and accepted in revised form
1 December 2000.
[Ca 2 ] i, cytoplasmic free Ca 2 concentration.
The control of insulin secretion by glucose involves
two major pathways that both require
metabolism of the sugar by -cells (1). The
triggering pathway serves to raise the cytoplasmic
free Ca 2 concentration ([Ca 2 ] i), which stimulates
exocytosis of insulin granules. This rise essentially
depends on the influx of Ca 2 through voltage-dependent
Ca 2 channels activated by a membrane depolarization
that is underlain by closure of ATP-sensitive K channels
(2–4). The amplifying pathway, which does not imply
changes in the activity of ATP-sensitive K channels and in
[Ca 2 ] i, serves to produce as yet incompletely identified
signals that augment the action of Ca 2 on exocytosis
(5–9).
The glucose dependency of insulin secretion and many
other events occurring in -cells is characterized by a
sigmoidal relationship. This type of response may result
from an increase in the individual response of functionally
homogeneous -cells, from the progressive recruitment
into an active state of -cells with distinct glucose sensitivities,
or both (10).
Isolated -cells differ in their individual sensitivity to
glucose. Measurements of insulin secretion (11–14), insulin
biosynthesis (15,16), and glucose metabolism (17,18)
have shown a large heterogeneity in the glucose responsiveness
of single -cells. The threshold glucose concentration
is variable, hence the percentage of cells developing a functional
response increases with the glucose concentration.
Physiologically, -cells are not isolated but associated
within the islets of Langerhans, where intercellular coupling
or paracrine influences may erase their individual
differences to constitute a functionally homogeneous population.
Thus, in contrast to the heterogeneous responses
produced in isolated -cells, glucose induced a uniform
increase in NAD(P)H autofluorescence in -cells residing
within intact islets (19). However, studies of the nucleus
size (20), of the insulin gene promoter activity (21), of protein
synthesis (16), and of the rough endoplasmic reticulum
size (22) suggest that some degree of -cell heterogeneity
persists in situ. There also exist differences in the threshold
for glucose-induced electrical activity in -cells within islets,
but the range is limited to 5.5–11 mmol/l glucose (23–25),
and interislet variability partly accounts for these differences.
Whether glucose-induced recruitment of -cells into an
active secretory state persists or is abolished when the
cells are coupled is still unresolved. Sustained stimulation
of insulin secretion in vivo by hours of hyperglycemia 22
or
540 DIABETES, VOL. 50, MARCH 2001

y glibenclamide revealed differences in the degree of
-cell degranulation within individual islets—a picture
that is compatible with heterogeneity of secretion (26).
Unfortunately, the question is difficult to address directly
in vitro because no current technique permits measurements
of insulin secretion from individual cells within
islets or clusters. The [Ca 2 ] i rise is the most important
event that can be monitored to identify -cells stimulated
to secrete amidst inert but associated cells.
In this study, therefore, we characterized the effects of
increasing concentrations of glucose on [Ca 2 ] i in small
clusters of mouse islet cells to determine whether -cells
are progressively recruited to produce the signal triggering
insulin secretion. We compared these effects with those in
islet single cells and correlated them with the changes in
insulin secretion.
RESEARCH DESIGN AND METHODS
Solutions. The control medium used for islet isolation and for the experiments
was a bicarbonate-buffered solution that contained (in mmol/l) 120
NaCl, 4.8 KCl, 2.5 CaCl 2, 1.2 MgCl 2, and 24 NaHCO 3. It was gassed with O 2-CO 2
(94:6) to maintain pH 7.4 and was supplemented with 0.5 mg/ml bovine serum
albumin (fraction V). The Ca 2 -free solution used to disperse islets in isolated
cells and clusters contained (in mmol/l) 138 NaCl, 5.6 KCl, 1.2 MgCl 2, 5
HEPES, and 1 EGTA, with 100 IU/ml penicillin and 100 g/ml streptomycin,
and its pH was adjusted to 7.35 with NaOH. The medium used for cultures was
RPMI 1640 medium containing 10 mmol/l glucose (except in experimental
series 2, in which 7 mmol/l glucose was used), 2 mmol/l glutamine, 10%
heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 g/ml streptomycin.
Preparation. Islets were aseptically isolated by collagenase digestion of the
pancreas of fed female NMRI mice, followed by hand selection (27). To obtain
isolated cells and clusters, the islets were incubated for 5 min in a Ca 2 -free
solution. After centrifugation, this solution was replaced by culture medium,
and the islets were disrupted by gentle pipetting through a siliconized glass
pipette. Clusters and isolated cells were then cultured for 1 or 2 days on
22-mm circular glass coverslips (28).
Experimental series. Four independent series of experiments were performed.
In the first series, we compared the effects of a wide range of glucose
concentrations (6–20 mmol/l) on [Ca 2 ] i changes in islet single cells and
clusters of 2–20 cells from the same preparations (same coverslips). The
second series was similar except for the glucose concentration in the culture
medium (7 instead of 10 mmol/l). In the third series, we investigated the
characteristics of [Ca 2 ] i changes in the different cells of selected clusters.
Because one cell showing a [Ca 2 ] i change in a cluster may mask the presence
of a nonresponding cell located above or below, clusters of 2–15 cells forming
monolayers (no superimposed nuclei) were selected. In the fourth series, we
directly compared the changes in [Ca 2 ] i and insulin secretion in the same
preparations (same coverslip).
Measurements of [Ca 2 ] i. Clusters and cells attached to the coverslips were
loaded for 60 min with fura-2 (series 3) or for 90 min with fura-PE3 (series 1,
2, and 3) in control medium containing 10 mmol/l glucose and 1 mol/l fura-2
or fura-PE3 acetoxymethylester. The coverslip was then transferred into a
temperature-controlled perifusion chamber (Intracell; Royston, Herts, U.K.) of
which it formed the bottom. The chamber was placed on the stage of an
inverted microscope (40 objective) and perifused (1.5 ml/min) at 37°C with
control medium containing increasing glucose concentrations. Cells and
clusters were successively excited at 340 and 380 nm, and the fluorescence
emitted at 510 nm was captured by a CCD camera (Photonic Science,
Turnbridge Wells, U.K.). The images were analyzed by the MagiCal system
(Applied Imaging, Sunderland, U.K.). The intervals between successive [Ca 2 ] i
measurements (ratios of the 340- and 380-nm images) were 2.4 s for the
experiments lasting 40 min (series 3) and 4.8 s for the longer experiments
(series 1, 2, and 3). Other details of the technique, including the method for in
vitro calibration of the signals, can be found elsewhere (28,29).
At the end of each experiment, the perifusion was stopped and the
chamber was filled with 1 ml phosphate-buffered saline containing 75 mol/l
propidium iodide and 0.67 mol/l acridine orange (Sigma, St. Louis, MO)
during 5 min (30). Excitation at 490 nm and reading of the emitted fluorescence
at 510 nm visualizes living cells in green and dead cells in red. After the
test of cell viability, the chamber was filled with 1 ml of control solution
containing 1 mol/l bisbenzimide (Sigma) for 30 min. The preparation was
F.C. JONKERS AND J.-C. HENQUIN
excited at 365 nm to reveal fluorescent nuclei, permitting unambiguous
identification of single cells and counting of the number of cells within
clusters.
Combined measurements of insulin secretion and [Ca 2 ] i. Clusters and
isolated cells cultured for 2 days on a coverslip were loaded with fura PE3
before being transferred into the recording system, as described above, except
that a 20 objective was used. The chamber was then perfused at a flow rate
of 1.5 ml/min, and the effluent was collected in 2-min fractions that were
immediately centrifuged to eliminate cells detached from the preparation.
Insulin was measured in duplicate 400-l aliquots of each fraction. The
characteristics of the assay have previously been reported (27). It should be
borne in mind that [Ca 2 ] i was measured over a window covering 0.1% of the
coverslip area. The [Ca 2 ] i signal was thus representative of the changes
occurring in the many more cells and clusters from which insulin secretion
was measured. The possible presence of dead cells and the number of cells in
the observed field were not evaluated in this series.
Immunodetection of somatostatin and glucagon cells. To determine the
proportion of non–-cells in the preparations, coverslips with cells and
clusters cultured for 2 days were fixed in Bouin Allen’s fluid (European
Laboratory Supplies, Bienvere, Belgium) during 6hatroom temperature.
They were then processed to immunostain - and -cells with a mixture of
antiglucagon and antisomatostatin serum, each at a dilution of 1:25,000 (Novo
Biolabs, Bagsvaerd, Denmark). Positive cells were identified by a peroxidase
method using 3,3-diaminobenzidine as the substrate for staining. The preparations
were then counterstained with hemalun. The method has been
described in full elsewhere (31). Labeled non–-cells were counted for five
different cultures, and their proportion was determined by counting the
number of nuclei.
Presentation of results. The experiments are illustrated by representative
recordings, and quantified data are presented as means SE.
RESULTS
Cellular composition of the preparations. On average,
the cell populations attached to the coverslips comprised
15% single cells and 85% of cells within clusters of different
sizes. Preparations from five different cultures were immunostained
with a mixture of antiglucagon and antisomatostatin
serum. The proportion of non–-cells was 13
1.4% in the whole preparations but was higher among
isolated cells (33 3.8%) than within clusters (9 1.1%),
of which 58% did not contain non–-cells. However, the
probability that single non–-cells were studied is less
because of our selection of fields containing relatively
large cells. Mouse -cells are larger than - and -cells
(32). The clusters used for the experiments of series 1–3
were selected on their size, which comprised between 2
and a maximum of 15–20 cells (mean 7.2 0.3 cells, n
220).
Influence of increasing glucose concentrations on
[Ca 2 ] i in islet single cells and clusters. Islet single
cells and clusters cultured for 1–2 days in the presence of
10 mmol/l glucose were stimulated by stepwise increases
in the glucose concentration while their [Ca 2 ] i was measured.
No recordings were obtained during perifusion with
solutions containing 6 mmol/l glucose. However, in other
experiments, [Ca 2 ] i was consistently low and stable in
the presence of 4 mmol/l glucose (F.C.J., unpublished
data). The typical response to higher glucose concentrations
was characterized by repetitive transient elevations
of [Ca 2 ] i (Fig. 1). Sustained elevations of [Ca 2 ] i were not
observed, even at 20 mmol/l glucose. In some single cells,
[Ca 2 ] i oscillations were induced by 6 mmol/l glucose (cell
1), whereas other cells only responded to a higher glucose
concentration (cell 2) or did not respond at all (cell 3). In
glucose-sensitive cells, tolbutamide consistently increased
the [Ca 2 ] i rise produced by 20 mmol/l glucose. Most
glucose-insensitive cells responded to tolbutamide (e.g.,
cell 3 in Fig. 1). In clusters, [Ca 2 ] i responses 23
were also
DIABETES, VOL. 50, MARCH 2001 541

-CELL RECRUITMENT BY GLUCOSE
FIG. 1. Examples of [Ca 2 ] i responses to increasing glucose (G) concentrations in islet single cells or clusters. The recording of [Ca 2 ] i was
started (time 0) after 10 min of perifusion with a medium containing 4 mmol/l glucose, simultaneously with the rise of the glucose concentration
to 6 mmol/l. Each concentration of glucose was applied for 12 min. The experiment was terminated by addition of 100 mol/l tolbutamide (Tolb)
to the medium containing 20 mmol/l glucose. Cells 1 and 2 and clusters 1 and 2 showed different sensitivities to glucose. Cell 3 responded to
tolbutamide but not to glucose. All preparations were cultured for 1 day. The quantification of these different responses is presented in Figs. 2
and 3.
characterized by large oscillations (no sustained elevation)
but showed a lesser variability than in single cells.
However, differences in the threshold concentration of
glucose were also observed between clusters (Fig. 1).
These differences were noted within the same preparation
and did not simply reflect interpreparation variability.
The percentage of islet single cells showing a [Ca 2 ] i
rise in the presence of different glucose concentrations is
shown in Fig. 2A. It was slightly less on day 2 than day 1,
but the overall glucose dependency was not influenced by
culture time. Whereas [Ca 2 ] i was consistently low and
stable in the presence of 4 mmol/l glucose (F.C.J., 24
unpub-
542 DIABETES, VOL. 50, MARCH 2001

FIG. 2. Influence of the glucose concentration on the percentage of islet single cells (A) and clusters (B) showing a [Ca 2 ] i response
(recruitment). The preparations were cultured for 1 day (D1) or 2 days (D2) in the presence of 10 mmol/l glucose before being stimulated by
increasing glucose concentrations and eventually by 100 mol/l tolbutamide (Tolb.), as shown in Fig. 1. The total number of studied single cells
were 190 (D1) and 190 (D2), and those of studied clusters were 47 (D1) and 53 (D2), from 10 different cultures. Values are means SE. The inset
shows the results from an independent series of experiments including 40 clusters from seven cultures for 1 day (D1) in the presence of only 7 mmol/l
glucose (Cult. G7).
lished data), 20% islet single cells responded to 6
mmol/l glucose. The proportion increased with the glucose
concentration and reached a plateau of 60% between
10 and 15 mmol/l glucose. In the presence of
tolbutamide, only 13% of the cells remained inert. None of
these cells were dead according to the propidium iodide/
acridine orange technique (see RESEARCH DESIGN AND METH-
ODS); they were probably -cells because these cells do not
respond to tolbutamide in the mouse (33). The proportion
of islet cell clusters showing a [Ca 2 ] i response also
increased between 6 and 10 mmol/l glucose and reached a
maximum of 90–95% (Fig. 2B). The few clusters that were
still inert in 20 mmol/l glucose alone all responded to
tolbutamide. The results of this first series of experiments
indicate that glucose recruits islet single cells and clusters
to generate a [Ca 2 ] i response.
We also measured the influence of the glucose concentration
on the amplitude of the [Ca 2 ] i response. In both
single cells and clusters, glucose induced a concentrationdependent
rise in mean [Ca 2 ] i that reached a maximum at
15 mmol/l glucose (Fig. 3A and B). This rise was partly
accounted for by the recruitment of active cells and
clusters. However, when only those cells or clusters
active at a given glucose concentration were taken into
consideration, a rise in mean [Ca 2 ] i was still observed
as the glucose concentration was raised (Fig. 3C and D).
The phenomenon can be seen in Fig. 1. In none of the
242 glucose-responsive cells and 95 glucose-responsive
clusters did [Ca 2 ] i suddenly switch from an oscillatory
pattern to a sustained elevation. This indicates that glucose
augments the amplitude of the individual response,
and, as shown in Fig. 3C and D, this effect is larger in
clusters than in single cells.
A second independent series of experiments was performed
with clusters of islet cells cultured for 1 day in the
presence of a lower concentration of glucose (7 instead of
10 mmol/l glucose). As shown by the insets in Figs. 2B and
3D, the recruitment of clusters and the rise in mean [Ca 2 ] i
F.C. JONKERS AND J.-C. HENQUIN
in active clusters were similar to those observed in the first
series. This indicates that our findings are not dependent
on a specific duration of the culture or glucose concentration
during the culture.
Influence of the glucose concentration on the [Ca 2 ] i
response in individual cells within clusters. The
above data have shown that the glucose sensitivity of
different clusters is variable but did not provide any information
about the homogeneity of the response within each
cluster. The third series of experiments, therefore, characterized
the individual cell response in monolayer
clusters. The upper trace in Fig. 4A illustrates the global
[Ca 2 ] i response in a cluster of 10 cells that started to
respond in 6 mmol/l glucose. The four lower traces show
that the signal was synchronous and of similar amplitude
in individual cells. Figure 4B illustrates the response of
another cluster (14 cells) that was also active at 6 mmol/l
glucose. Although synchronous in all cells, the [Ca 2 ] i
response was of smaller amplitude in some cells (C3–C4)
than in others (C1–C2). When this difference in amplitude
was observed, it usually persisted even at higher glucose
concentrations.
The characteristic synchronous [Ca 2 ] i response to glucose
is illustrated by the series of pseudocolor images
of Fig. 5A, recorded in a cluster of eight cells. All cells
showed simultaneous increases or decreases in [Ca 2 ] i.
Cell recruitment within an active cluster was only rarely
observed. In the cluster illustrated by Fig. 5B, one cell was
active at 6 mmol/l glucose, whereas the other three cells
remained silent. At 7 mmol/l glucose, a synchronized response
occurred in the four cells, with sometimes a slightly
greater amplitude in the first cell than in the others.
[Ca 2 ] i waves propagating across this or other clusters
were not observed. In summary, one can consider that
the progressive increase in the glucose concentration
recruited clusters 4A, 4B, and 5B first, and then cluster 5A,
but that no recruitment of individual cells occurred 25
within
DIABETES, VOL. 50, MARCH 2001 543

-CELL RECRUITMENT BY GLUCOSE
FIG. 3. Influence of the glucose concentration on average [Ca 2 ] i in islet single cells and clusters. A and B: Average [Ca 2 ] i was measured in all
cells and clusters regardless of the presence or absence of a [Ca 2 ] i response. The n values are thus identical at all glucose concentrations: 190
single cells 1 day (D1), 190 single cells 2 days (D2), 47 clusters D1, and 53 clusters D2. The observed increase in [Ca 2 ] i therefore reflects both
the increase in the proportion of responding cells and clusters and the increase in the individual responses. C and D: Only those cells and clusters
showing a [Ca 2 ] i response were included in the calculations. Hence, the n values are different at each glucose concentration. The curves
therefore illustrate the increase in the individual responses. Values are means SE. The inset shows the increase in [Ca 2 ] i measured in the
clusters cultured for 1 day in the presence of only 7 mmol/l glucose (Cult. G7). The preparations used for these calculations were the same as
those shown in Fig. 2B. Tolb., tolbutamide.
the active clusters, except in cluster 5B, in which one cell
became active before the others.
The incidence of these different types of responses is
presented in Fig. 6A. The whole columns show that the
percentage of clusters with a [Ca 2 ] i response increased
with the glucose concentration to reach 100% at 10 mmol/l
glucose. The black section of each column corresponds to
those clusters in which not all cells were active. This
proportion was small and decreased as the glucose concentration
was raised (Fig. 6A). In other words, when a
cluster responded to glucose by a [Ca 2 ] i rise, the vast
majority of cells or all cells contributed to the response.
These observations indicate that individual cell recruitment
within clusters is an infrequent phenomenon.
However, it remained possible that the synchronization
of [Ca 2 ] i oscillations was affected by glucose. The results
of this analysis are shown in Fig. 6B. In the clusters that
were active at 6-7 mmol/l glucose, [Ca 2 ] i oscillations
were synchronous in 85–90% of the cells. As the glucose
concentration was raised, the regularity of the responses
increased to characterize 95% of the clusters at 10 mmol/l
glucose (Fig. 6B). The few nonresponsive or asynchronous
cells within the active clusters may be non–-cells (33).
Correlations between glucose-induced [Ca 2 ] i responses
and insulin secretion. In a fourth series of
experiments, we directly compared the effects of glucose
on cytosolic [Ca 2 ] i and insulin secretion in the same
preparations of islet single cells and clusters. Figure 7A
shows the mean changes induced by six glucose concentrations
tested in sequence. The [Ca 2 ] i trace corresponds
to the average changes in all single cells and clusters
present in the observation field, and the insulin secretion
profile reflects the activity of all cells attached to the
coverslip. Raising the glucose concentration from 4 to 7
mmol/l caused a large peak followed by a smaller sustained
elevation of both [Ca 2 ] i and insulin secretion.
Subsequent increases in the glucose concentration induced
progressive parallel elevations of [Ca 2 ] i and insulin
secretion. The average integrated increases in [Ca 2 ] i and
insulin secretion above basal levels are shown 26
in Fig. 7B
544 DIABETES, VOL. 50, MARCH 2001

FIG. 4. Examples of the homogeneity of the [Ca 2 ] i responses to
increasing glucose (G) concentrations in islet cell clusters. Clusters
forming monolayers were selected to permit analysis of [Ca 2 ] i in
individual cells without the confounding problem of active cells masking
inactive ones in another layer. Successive [Ca 2 ] i measurements
were obtained at 2.4-s intervals. The upper trace shows the global
response of the cluster and the lower traces show the response of four
individual cells (C1–C4). The changes in [Ca 2 ] i were synchronous in
all cells of both clusters, but their amplitude was either similar (A) or
variable (B) between cells of the cluster.
and C. Both parameters displayed a similar glucose dependency.
Finally, the glucose dependency of insulin secretion was
compared with that of the recruitment of islet single cells
and clusters from the same preparations (Fig. 8). A [Ca 2 ] i
response was induced in the majority of clusters and
responsive cells already by 7 mmol/l glucose, whereas
insulin secretion kept increasing up to 15–20 mmol/l
glucose. Glucose-induced insulin secretion, therefore, cannot
entirely be ascribed to the recruitment of -cells to
generate a [Ca 2 ] i signal. The increase in the individual
cell response (Fig. 7) certainly plays a major role.
DISCUSSION
Isolated single -cells display heterogeneous metabolic,
biosynthetic, and secretory responses to glucose. Because
of their different threshold sensitivities to glucose, increasing
numbers of cells become active (are recruited) as the
sugar concentration is raised (10). It is also widely
F.C. JONKERS AND J.-C. HENQUIN
accepted that single -cells exhibit heterogeneous [Ca 2 ] i
responses to glucose (34–38). However, only one aspect of
this heterogeneity is well established: the pattern of the
response to a stimulatory concentration of glucose is
irregular and variable. In contrast, only limited evidence,
based on the use of few glucose concentrations, suggests
the existence of a variable sensitivity to glucose (35,37).
The present study clearly establishes that the threshold
glucose concentration inducing a [Ca 2 ] i rise is variable
between individual isolated mouse -cells. The proportion
of -cells showing an elevation of [Ca 2 ] i increases with
the rise in glucose concentration. The phenomenon of
recruitment thus also exists at the [Ca 2 ] i level. Interestingly,
the proportion of 60–65% active cells in 15 mmol/l
glucose is in agreement with the percentage of rat -cells
secreting insulin (as shown by reverse hemolytic plaque
assay) in response to a similar glucose concentration (14,
15,39). This similarity suggests that, when glucose recognition
(metabolism and subsequent steps) is sufficient to
lead to a [Ca 2 ] i rise, it also leads to insulin secretion in
individual cells.
The major aim of our study, however, was not to characterize
glucose-induced [Ca 2 ] i changes in isolated cells
but to assess whether recruitment also exists in a more
physiological situation, when -cells are associated in clusters
that may be more representative of their situation
within islets. The results show that the threshold glucose
concentration inducing a [Ca 2 ] i response is also variable
between clusters. Raising the glucose concentration recruited
more and more active clusters. The difference with
single cells did not reside in the glucose sensitivity (Km between 7 and 8 mmol/l for both types of preparations) but
in the maximum response. All or practically all clusters
responded to glucose compared with 60–65% of single
cells. The inclusion of unrecognized -cells in the studied
single cells probably contributes to but cannot entirely
explain the difference.
Whereas some heterogeneity was observed between
clusters, the individual cell response within clusters was
more homogeneous. No more than 25% of the clusters
responding to 6 mmol/l glucose included unresponsive
cells. This proportion decreased close to zero as the
glucose concentration was raised to 8–10 mmol/l. Moreover,
the synchrony of the response was the rule; asynchronous
[Ca 2 ] i changes in neighboring cells occurred in
no more than 15% of the active clusters at 6-7 mmol/l
glucose, and this proportion decreased with the rise in
glucose. Recruitment of individual cells within clusters
rarely occurs; when a cluster is recruited, all or nearly all
its cells respond. [Ca 2 ] i measurements by confocal microscopy
in intact mouse islets have shown that - and
-cells (subsequently identified by immunocytochemistry)
display distinct responses from those of -cells (33,40). It
is thus possible that the few nonresponsive or asynchronous
cells within clusters are non–-cells. On the other
hand, because -cells can be coupled with -cells in vitro
(41), we cannot exclude the possibility that some non–cells
are entrained by -cells within clusters.
Our results further show that average [Ca 2 ] i in single
cells and clusters increases with the glucose concentration.
This increase corresponds to both the recruitment of
active cells and a change in the magnitude of the 27
individual
DIABETES, VOL. 50, MARCH 2001 545

-CELL RECRUITMENT BY GLUCOSE
FIG. 5. Illustration of the homogeneity or heterogeneity of the [Ca 2 ] i responses to increasing glucose (G) concentrations in islet cell clusters.
The left-hand pictures show the analyzed clusters after staining of the nuclei with bisbenzimide. The series of color pictures show pseudocolor
images of [Ca 2 ] i in the clusters at the time indicated by arrows (blue corresponds to low [Ca 2 ] i and red to high [Ca 2 ] i). The traces show the
integrated [Ca 2 ] i changes with time. A: Cluster of eight cells, with one nucleus slightly out of focus; all cells responded synchronously. B: Cluster
of four cells; the upper right cell only responded to 6 mmol/l glucose; subsequently all four cells responded synchronously.
cell response. In previous studies using cultured -cells
from ob/ob mice (36,37), the glucose-dependent increase in
[Ca 2 ] i was ascribed to recruitment of individual cells
showing abrupt transitions, at variable glucose concentrations,
between three states: low basal [Ca 2 ] i, oscillatory
[Ca 2 ] i, and steadily elevated [Ca 2 ] i (reached by 17 and
40% of the cells in 11 and 20 mmol/l glucose, respectively)
(36). Such abrupt changes between oscillations and sustained
elevations of [Ca 2 ] i were never observed in our
preparations, in which the glucose-dependent28increase in
546 DIABETES, VOL. 50, MARCH 2001

FIG. 6. Homogeneity of the [Ca 2 ] i responses induced by increasing glucose concentrations in islet cell clusters. Preparations cultured for 1 or
2 days were stimulated by increasing glucose concentrations as shown in Fig. 4. Because the distribution of the different groups was similar after
1 and 2 days, the data were pooled. A: Percentage of clusters in which the [Ca 2 ] i response was present in all cells or in some cells only. The total
number of studied clusters was 120. B: Percentage of the active clusters in which the [Ca 2 ] i response was synchronous or asynchronous. The sum
of the two columns is thus 100%, but the number of clusters was different at each glucose concentration.
[Ca 2 ] i was more gradual. There is no doubt that the
[Ca 2 ] i response of individual -cells to glucose is not of
an all-or-none type. Finally, we did not observe [Ca 2 ] i
waves propagating across the clusters. This is in contrast
with a previous study (42) that described such propagations
in clusters of -cells from ob/ob mice tested 3 h after
dispersion of the islets; two examples were shown, but the
incidence of the phenomenon was not given. These waves
were attributed to electrical coupling of the cells. In
another study, glucose-induced [Ca 2 ] i waves propagating
within monolayers of cultured -cells and also between
physically separated clusters have been ascribed to
rhythmic release and diffusion of unknown stimulating
factors (43). We cannot exclude the possibility that we
have missed [Ca 2 ] i waves propagating too fast for the
resolution of our system or propagating only over distances
exceeding the size of the studied clusters.
An important observation of the present study is that the
recruitment of -cells occurs over a narrow range of
glucose concentrations: 6–10 mmol/l. Our findings are in
complete agreement with the glucose dependency of the
appearance of electrical activity in -cells within intact
mouse islets (23–25). This electrical activity indeed reflects
Ca 2 influx, the major mechanism underlying the
glucose-induced [Ca 2 ] i rise (2–4,29). By synchronizing
the changes in membrane potential, electrical coupling
(44,45) synchronizes Ca 2 influx and thereby minimizes
the heterogeneity of the triggering signal of -cells in situ.
However, the quantitative correlation is not perfect. As
already pointed out by others (36), Ca 2 -dependent electrical
activity in intact islets is more finely regulated by the
changes in glucose concentration than are the [Ca 2 ] i
oscillations in single -cells or clusters.
We do not believe that paracrine effects explain the high
incidence and good synchronization of the [Ca 2 ] i responses
in clusters. Thus, no more than 42% of the clusters
contained at least one non–-cell, whereas over 95% of the
F.C. JONKERS AND J.-C. HENQUIN
clusters stimulated by 10 mmol/l glucose displayed a
synchronous response. Moreover, isolated non–-cells
were scattered among single cells and clusters. There is
thus no reason why the small amounts of hormone that
they release (little glucagon is expected to be secreted
under our experimental conditions) should differentially
influence isolated -cells and clusters of -cells in a
constantly perifused system. It has also been suggested
that oscillations of the K concentration in the confined
extracellular space of the islet might contribute to the
synchronization of the membrane potential changes in
-cells (46). Such a mechanism is unlikely to remain
operative in our model of perifused monolayer clusters.
Recruitment of -cells into an active secretory state by
increases in the glucose concentration has been described
in preparations of isolated single -cells maintained in
culture (11–15) or tested several hours after islet isolation
(47). Whether the phenomenon exists under physiological
conditions, when -cells are associated within islets, has
not been established because secreting and nonsecreting
cells cannot be readily distinguished. The problem was
approached by recording the triggering signal of glucoseinduced
insulin secretion—the rise in [Ca 2 ] i. In fresh and
cultured mouse islets, half-maximum and maximum stimulation
of insulin secretion are produced by 15 and 30
mmol/l glucose, respectively (48). The concentration dependency
of insulin secretion by our preparations of
mouse islet cells and clusters was clearly shifted to the
left. We have no explanation for this difference, which
does not seem to have been reported (and studied) previously.
Nevertheless, the recruitment of -cells to induce a
[Ca 2 ] i response was even more sensitive to glucose,
which indicates that the phenomenon only partially accounts
for glucose-induced insulin secretion.
This conclusion is based on the evidence that, except
under artificial conditions of combined and strong activation
of protein kinase A and protein kinase 29
C (9,49),
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-CELL RECRUITMENT BY GLUCOSE
FIG. 7. Simultaneous measurements of [Ca 2 ] i and insulin secretion in preparations of islet single cells and clusters. A: Mean changes of [Ca 2 ] i
and insulin secretion in 24 experiments with preparations from 12 cultures. Peaks and nadirs of [Ca 2 ] i oscillations are erased by averaging. The
glucose (G) concentration was increased stepwise as indicated, from 4 to 30 mmol/l (G4–G30). B: Increase of [Ca 2 ] i above basal concentration
at 4 mmol/l: mean [Ca 2 ] i was calculated for the 14 min or only last 8 min of stimulation with each glucose concentration. C: Increase of insulin
secretion above the basal rate at 4 mmol/l glucose: mean secretion rate was calculated for each period of stimulation (14 min or only last 8 min).
Values are means SE for 24 experiments.
glucose does not increase insulin secretion if [Ca 2 ] i does
not rise in -cells or is not already elevated above basal
levels (1). However, the reverse is not necessarily true.
There is no proof that all -cells displaying a [Ca 2 ] i rise in
a glucose-stimulated islet or cluster secrete insulin. Three
theoretical possibilities can be envisaged. First, the [Ca 2 ] i
signal is present in all cells, but its magnitude is different.
It is not exceptional to observe -cells within a cluster
(e.g., Fig. 4B) or groups of -cells within whole islets
(29), in which [Ca 2 ] i is elevated to a lesser extent than
elsewhere in the preparation. This difference usually persists
throughout the whole range of glucose concentrations
and its significance is unclear. Second, glucose
metabolism may be similar in all -cells, as suggested by
cell-sized NAD(P)H measurements in intact islets (19), but
the efficacy of a similar [Ca 2 ] i signal on secretion may be
modulated by paracrine influences. However, it is not
immediately apparent which paracrine signal could positively
affect increasing numbers of -cells as the glucose
concentration is raised. Third, subtle metabolic differences
exist between -cells in situ, which either are
beyond the resolution of NAD(P)H measurements or involve
signals undetected by this method. In this case, the
action of Ca 2 on exocytosis could be different; in other
words, a similar triggering [Ca 2 ] i signal could lead to
different secretory responses because of differences in the
amplifying action of glucose (1,5–8).
In conclusion, -cell recruitment by glucose can occur
at the stage of the [Ca 2 ] i response. However, this type of
recruitment is restricted to a narrow range of glucose
concentrations, particularly when -cell association decreases
the heterogeneity of the responses. Recruitment 30
of
548 DIABETES, VOL. 50, MARCH 2001

FIG. 8. Influence of the glucose concentration on the appearance of a
[Ca 2 ] i response (recruitment) in islet single cells (E) and clusters
() and on insulin secretion for the 14 min (F) or only the last 8 min
of stimulation (f). All data were obtained from the same preparations
as those shown in Fig. 6. Note that the vast majority of -cells (at least
85%) are situated within clusters, which, therefore, are the major
contributors to insulin secretion.
-cells to generate a [Ca 2 ] i response contributes to, but
does not entirely explain, glucose-induced insulin secretion.
The increase in [Ca 2 ] i in individual -cells plays a
major role. If heterogeneity of insulin secretion exists in
situ, it probably occurs at a step of stimulus secretion
coupling downstream of the [Ca 2 ] i rise. This step could
be a modulation of the action of Ca 2 on exocytosis
through the amplifying effect of glucose (1).
ACKNOWLEDGMENTS
This work was supported by the Interuniversity Poles of
Attraction Program (P4/21), Belgian State Prime Minister’s
Office, Federal Office for Scientific, Technical, and Cultural
Affairs; by grant 3.4552.98 from the Fonds de la Recherche
Scientifique Médicale, Brussels; and by grant 95/00–188
from the General Direction of Scientific Research of the
French Community of Belgium. F.C.J. holds a research
fellowship from the Fonds pour la Formation à la Recherche
dans l’Industrie et dans l’Agriculture, Brussels.
We are grateful to Dr Y. Guiot and Prof. J. Rahier for
their help with the immunostaining, to Fabien Knockaert
for technical assistance, and to Stéphanie Roiseux for
editorial help.
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32

IFM 2012
Seminario 14: Regulación autónoma
-Bibliografía obligatoria: “Autonomic control of cardiovascular performance and
whole body O2 delivery and utilization in swine during treadmill exercise.” De
Stubenitzky et al. Cardiovascular Research 39: 459–474 (1998).
Bibliografía Sugerida: Capítulo 15: The autonomic nervous system. Capítulo 22:
regulation of Arterial Pressure and Cardiac Output; del libro: Medical Physiology de
Boron & Boulpaep.
1. Esquematice un circuito simpático y un circuito parasimpático desde la neurona
ubicada en el sistema nervioso central hasta su blanco (un blanco genérico), e indique
qué neurotransmisores y qué receptores se ponen en juego en este circuito.
2. ¿Cuáles son los blancos de acción del sistema simpático en el contexto del sistema
circulatorio? ¿Cuáles son los mecanismos por los que afectan a estos blancos (no se les
pregunta por la cascada de segundos mensajeros sino por los efectos que tienen sobre los blancos en
sí)?
3. Describa el reflejo barorreceptor e indique:
a. ¿Qué tipo de estímulos lo originan?
b. ¿Dónde se ubica el detector?
c. ¿Cuál es el efector?
d. ¿Qué función cumple?
4. Describa el reflejo quimioreceptor e indique
a. ¿Qué tipo de estímulos lo originan?
b. ¿Dónde se ubica el detector?
c. ¿Cuál es el efector?
d. ¿Qué función cumple?
5. Esquematice un reflejo espinal y defina qué elementos del reflejo barorreceptor se
corresponden con qué elementos de este esquema.
6. La deformación mecánica de las terminales nerviosas es considerada el mecanismo
primario de la activación de los barorreceptores. Sin embargo se cree que la
sensibilidad de estos receptores podría estar modulada de forma paracrina por la
liberación de factores desde las células endoteliales.
a. En la figura A se muestra el efecto de concentraciones crecientes de prostaciclina
(PGI2) en el seno carotideo aislado de conejos, sobre la actividad eléctrica de los
barorreceptores. ¿Qué puede concluir de la sensibilidad de estas terminales nerviosas
en función de la presión arterial? ¿Cómo se ve modificada esta propiedad en presencia
de PGI2?
b. En la figura B se observa el % de cambio en el diámetro del seno carotídeo en
función del aumento de la presión, tanto para las condiciones control como las del
tratamiento con una alta concentración de PGI2. ¿De acuerdo a los resultados
observados en la figura A, es coherente este resultado? ¿Por qué?
33

c. ¿Cuál sería el valor fisiológico del efecto descripto en estos experimentos? En
condiciones fisiológicas el PGI 2 podría provenir del endotelio vascular. ¿Qué
experimentos podría realizar para llegar a esta conclusión?
7. ¿Cómo se explica que el efecto del sistema simpático sea considerado
vasoconstrictor, mientras que sobre la vasculatura que irriga al músculo esquelético
tiene el efecto opuesto?
8. Se realizaron experimentos en gatos descerebrados (pero con reflejos autónomos
intactos), en los que, mediante un catéter ubicado en la arteria carótida, se aplicaron
escalones de presión en el seno carotídeo (CSP). La presión arterial fue registrada
mediante otro catéter ubicado en la arteria carótida (CAP). La actividad cardíaca fue
monitoreada mediante un electrocardiograma (ECG).
34

a. ¿Qué efecto produjo la elevación de la presión en el seno carotídeo sobre el ritmo
cardíaco? ¿Cómo explica este efecto?
b. ¿Cómo explica la caída en la presión arterial?
c. Los autores reportan que la aplicación de atropina, un inhibidor de receptores
colinérgicos muscarínicos, inhibió el efecto. Explique este resultado.
9. Estás investigando, en ratones, si el aumento de la actividad cardíaca producido
por el ejercicio físico está mediado i) por los terminales simpáticos que inervan al
corazón o ii) por la glándula adrenal.
a. ¿Qué experimento (y su debido control) realizarías para responder a la pregunta?
Indicar qué parámetro cardiovascular vas a utilizar para monitorear la actividad
cardiaca. Explicá el experimento y los resultados esperados en caso que la hipótesis i
o la hipótesis ii no sean rechazadas (en forma excluyente… aunque en la realidad
nunca lo es).
Supongamos que encontrás que la hipótesis i es la correcta (o sea, la activación de los
terminales simpáticos son suficientes para explicar la activación del corazón debida al
ejercicio y la activación de la glándula adrenal no tiene al corazón como blanco de
acción) y decidís publicar el hallazgo. Los reviewers de la revista reclaman que
investigues si el nivel de ejercicio al que fueron sometidos los animales fue suficiente
para producir una activación de la glándula adrenal.
b. ¿Qué experimento sencillo realizarías para comprobar (o descartar) que la glándula
adrenal sufrió una activación sustancial durante el ejercicio, y aún así esto no fue
importante para modificar el parámetro cardíaco elegido?
35

Análisis del artículo
Glosario:
Propranolol es un antagonista que tiene igual afinidad por los receptores β1 y β2. La
fentolamina bloquea receptores del tipo α.
Consejo: para poder seguir este artículo se les recomienda confeccionar un glosario
con el significado de las siglas.
1) Realice un esquema de los parámetros que se registran en este artículo y cuál es el
método que se utiliza. En el marco del sistema circulatorio qué se espera del bloqueo
de receptores β y α. Identifique los efectos sobre la vasculatura y sobre el corazón.
2) Explique las observaciones de la Figura 1 relacionando cada parámetro con el
resto:
a. ¿A qué se debe el aumento en el gasto cardíaco (CO) durante el ejercicio?
b. ¿Qué factor explica que la presión arterial no cambia a pesar del aumento en CO?
c. ¿Cómo se relaciona este factor con la actividad física?
d. La presión arterial no cambia pero sí lo hace la presión de la arteria pulmonar,
¿cómo se explica este resultado?
e. ¿Cómo se explica que no varíe el volumen sistólico (SV) cuando se observa un
aumento en la presión del ventrículo izquierdo?
3) En el texto se especifica que la presión parcial arterial de CO 2 disminuye durante el
ejercicio, ¿cómo se explica? Analizar la Figura 2.
4) Explique las observaciones de la Figura 3 relacionando cada parámetro con el
resto. ¿Cuáles son los efectos del propranolol sobre el sistema circulatorio? Analice
los resultados de la Figura 4.
5) Explique las observaciones de la Figura 5 relacionando todos los parámetros.
¿Cuál es el efecto de la fentolamina? ¿Cómo se diferencian de los efectos del bloqueo
de los receptores β? Explique las observaciones que indican la Figura 6 y compárelas
con la 4.
6) Analice la Figura 9 relacionando cada parámetro con el resto (ignore atropina +
propranolol). ¿Cuál es el efecto de la atropina y cómo se diferencia del efecto de
bloquear los receptores adrenérgicos α y β? Explique los resultados de la Figura 10.
36

Cardiovascular Research 39 (1998) 459–474
Autonomic control of cardiovascular performance and whole body O 2
delivery and utilization in swine during treadmill exercise
Rene´ Stubenitsky, Pieter D. Verdouw, Dirk J. Duncker*
Experimental Cardiology, Thoraxcenter, Cardiovascular Research Institute COEUR, Erasmus University Rotterdam, 3000 DR Rotterdam, Netherlands
Abstract
Received 22 October 1997; accepted 4 March 1998
Objective: The present study determined the role of the autonomic nervous system (ANS) in the regulation of systemic and pulmonary
circulation and of O delivery and utilization in swine at rest and during graded treadmill exercise. Methods: Instrumented swine (n512)
2
were subjected to treadmill exercise (1–5 km/h) under control conditions and in the presence of single and combined b-adrenergic,
a-adrenergic and muscarinic (M) receptor blockade. Results: Exercise produced a four-fold increase in body O consumption, due to a
2
doubling of both cardiac output and the arterio–mixed-venous O content difference. The latter resulted from an increase in O extraction,
2 2
from 4561% at rest to 7461% at 5 km/h, as the O carrying capacity [haemoglobin concentration (Hb)] increased by only |10%. The
2
increase in cardiac output resulted from a doubling of the heart rate and a small (,10%) increase in stroke volume. The mean aortic
pressure (MAP) was unchanged, implying a 50% decrease in systemic vascular resistance (P#0.05). In contrast, exercise had no
significant effect on pulmonary vascular resistance. The sympathetic division of the ANS controlled O delivery via b-adrenoceptors
2
(heart rate and contractility) and Hb concentration via a-adrenoceptor-mediated splenic contraction. In addition, the sympathetic division
modulated systemic vascular tone via a- and b-adrenoceptors, but also exerted a vasodilator influence on the pulmonary circulation via
b-adrenoceptors. The parasympathetic division controlled O delivery in part directly (heart rate) and in part indirectly via inhibition of
2
b-adrenoceptor activity (heart rate and contractility), even during heavy exercise. In addition, the parasympathetic division exerted a
direct vasodilator influence on the pulmonary, but not on the systemic, circulation. Conclusions: Thus, in swine, in a manner similar to
that in humans, both the sympathetic and parasympathetic division of the ANS contribute to cardiovascular homeostasis during exercise
up to levels of high intensity. © 1998 Elsevier Science B.V. All rights reserved.
Keywords: Autonomic nervous system; Exercise; O consumption; O extraction; Pulmonary circulation; Systemic circulation; Swine
2 2
1. Introduction closely. Thus, both swine and humans have a low initial
exercise capacity but can be very well trained and show
The large animal most frequently used in exercise similar Hb increments and pH decreases in response to
studies is the dog, despite differences in their response to exercise [1–4]. Despite the increase in the use of swine in
exercise compared to that of humans. For example, dogs exercise studies, there is a paucity of information on
(i) possess a much higher initial exercise capacity than cardiovascular control mechanisms in swine during exerhumans,
(ii) are capable of elevating the haemoglobin cise. Consequently, the role of the autonomic nervous
concentration (Hb) and, hence, the O -carrying capacity of system (ANS) in the regulation of the systemic and
2
their blood to a greater extent than humans, and (iii) tend pulmonary circulation and of O transport and utilization
2
to maintain arterial pH during heavy exercise [1–3]. in chronically instrumented swine during treadmill exercise
However, swine are now becoming increasingly more were examined in this study. The importance of sympapopular
in studies of exercise; their cardiovascular re- thetic circulatory control was assessed by studying animals
sponse to exercise resembles the response in humans more in the absence and presence of either non-selective badrenoceptor
blockade or a-adrenoceptor blockade. Since
* Corresponding author. Tel.: 131 10 408 8029; Fax: 131 10 436
5607; E-mail: duncker@tch.fgg.eur.nl Time for primary review 33 days.
0008-6363/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.
PII: S0008-6363(98)00102-3
37

460 R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474
presynaptic a -adrenoceptor blockade can result in en- cal wires and catheters were tunnelled subcutaneously to
2
hanced catecholamine release from sympathetic nerve the back, the chest was closed and the animals were
endings [5], we also studied the effect of a-adrenoceptor allowed to recover. All electrical wires and catheters were
blockade in the presence of b-adrenoceptor blockade. To protected with an elastic vest.
determine the contribution of parasympathetic circulatory
control, animals were also studied in the absence and 2.2. Post-surgical period
presence of non-selective muscarinic (M) receptor blockade.
Finally, the contribution of modulation of b-adren- On the first two days post-surgery, the animals received
ergic activity by the parasympathetic system was evaluated daily injections of buprenorphine (0.3 mg i.v.). During the
by determining the effects of combined b-adrenoceptor first week after surgery, the animals received intravenous
and M-receptor blockade. injections of 25 mg/kg amoxicillin and 5 mg/kg gentamycin
on a daily basis to prevent infection. Catheters
were flushed daily with physiologic saline containing 2000
2. Methods IU/ml heparin.
Twelve crossbred Landrace3Yorkshire swine (six male 2.3. Experimental protocols
and six female; three–four months of age and 2461 kgat
the time of surgery; 3062 kg at the time of study) were Studies were performed two–four weeks after surgery,
used in this study. All experiments were performed in with the animals exercising on a motor driven treadmill.
accordance with the ‘‘Guiding Principles in the Care and For each animal, experimental protocols were performed
Use of Laboratory Animals’’, as approved by the Council on different days and in random order.
of the American Physiological Society and with prior
approval of the Animal Care Committee of the Erasmus 2.3.1. Reproducibility of responses to exercise (n59)
University Rotterdam. Adaptation of animals to the labora- With swine lying quietly on the treadmill, resting
tory conditions started one week prior to surgery and was haemodynamic measurements, consisting of LV, left atrial,
continued until one week post-surgery. aortic and pulmonary artery blood pressures and cardiac
output were obtained and arterial and mixed venous blood
2.1. Surgical procedures samples were collected. Haemodynamic measurements
were repeated and the rectal temperature was measured
Full details of all experimental procedures have been while the animals were standing on the treadmill. Subpublished
previously [6–9]. Briefly, swine were sedated sequently, a five-stage exercise protocol was begun (1, 2,
with ketamine (30 mg/kg, i.m.), anaesthetized with 3, 4 and 5 km/h) with each stage lasting 2–3 min.
thiopental (10 mg/kg, i.v.), intubated and mechanically Haemodynamic variables were measured and blood sam-
ventilated with a mixture of O and nitrous oxide (1:2), to ples were collected during the last 30 s of each exercise
2
which 0.2–1% (v/v) isoflurane was added. Anaesthesia stage, when haemodynamics had reached a steady state. At
was maintained with midazolam (2 mg/kg11 mg/kg/h, the conclusion of the exercise protocol, the animals were
i.v.) and fentanyl (10 mg/kg/h, i.v.). Under sterile con- allowed to rest for 90 min. Then, the animals received an
ditions, the chest was opened via the fourth left intercostal intravenous infusion of saline (10 ml), which was infused
space and an 8 French (Fr) fluid-filled polyvinylchloride over 5 min. Five minutes after completion of the infusion,
(PVC) catheter was inserted into the aortic arch, for the resting measurements were obtained and the five-stage
measurement of blood pressure and collection of arterial exercise protocol was repeated.
blood samples, and was secured with a purse string suture.
After the pericardium was opened, a precalibrated electro- 2.3.2. Effects of b-adrenergic receptor blockade (n512)
magnetic flow probe (Skalar, Delft, Netherlands) was Ninety minutes after swine underwent the five stage
positioned around the ascending aorta for the measurement exercise protocol under control conditions, the animals
of cardiac output. A high fidelity pressure transducer received intravenous propranolol (0.5 mg/kg dissolved in
(Konigsberg Instruments, Pasadena, CA, USA) was in- 10 ml of saline and infused over 5 min). Five minutes after
serted into the left ventricle (LV) via the apical dimple, for completion of the infusion, resting measurements were
recording of LV pressure and its first derivative (LVdP/dt). obtained and the five-stage exercise protocol was repeated.
An 8 Fr PVC catheter was inserted into the LV for The dose of propranolol results in .95% inhibition of
calibration of the Konigsberg transducer signal, and into isoprenaline-induced increases in heart rate and LVdP/
the left atrium via the left atrial appendage for the dt [6], and produces stable haemodynamic alterations
max
measurement of local blood pressure. Similar catheters over a 30-min period [7] in awake swine.
were also inserted into the pulmonary artery for the
measurement of blood pressure, administration of drugs 2.3.3. Effects of a-adrenergic receptor blockade (n58)
and for collection of mixed venous blood samples. Electri- Ninety min after swine underwent the five stage exercise
38

R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474 461
protocol under control conditions, a-adrenergic receptor ter LV pressure signal. CO was measured with an electroblockade
was produced by intravenous administration of magnetic flowmeter system (Transflow 601 System, Skaphentolamine
(1 mg/kg in 10 ml of saline, infused over 5
min). Five minutes after completion of the infusion, resting
lar, Delft, Netherlands).
measurements were obtained and the exercise protocol was
repeated. The dose of phentolamine used results in .95%
2.5. Blood gas measurements
inhibition of the noradrenaline-induced (0.3 mg/kg, i.a.)
increase in carotid vascular resistance [10], and produces
stable haemodynamic alterations over a 30-min period [11]
Blood specimens were maintained anaerobically in iced
heparinized syringes until the conclusion of each exercise
in anaesthetized swine.
trial. Measurements of arterial and mixed venous
2.3.4. Effects of a-adrenergic receptor blockade in b-
adrenergic receptor blocked swine (n59)
Swine received intravenous propranolol (0.5 mg/kg in
10 ml of saline, infused over 5 min). Five minutes after
completion of the infusion, resting measurements were
obtained and the five-stage exercise protocol was performed.
After 90 min of rest, propranolol (0.2 mg/kg in 10
ml of saline) was re-administered. Five minutes later,
a-adrenergic receptor blockade was produced by infusing
phentolamine at a dose of 1 mg/kg (in 10 ml of saline),
and 5 min later, the exercise protocol was repeated. We
have previously shown that the propranolol dose regimen
produces identical responses during two consecutive exercise
protocols [9].
2.3.5. Effects of M-receptor blockade and the effects of
P O (mmHg), P CO (mmHg) and pH were then performed
2 2
with a blood gas analyzer (Acid–Base Laboratory Model
505, Radiometer, Copenhagen, Denmark), while arterial
and mixed venous O2 saturation (S O , %) and arterial
2
haemoglobin concentration (Hb, g%) were measured with
a haemoximeter (OSM2, Radiometer). From these mea-
surements, arterial and mixed venous blood O2 contents
(mmol/l) were computed as (Hbarterial30.6213O 2
saturation)1(0.001313P O ). Body O2 delivery (D O ) was
2 2
computed as the product of CO and arterial blood O2 content; body O2 consumption (V O ) was computed as the
2
product of CO and the difference in O2 content between
arterial and mixed venous (pulmonary artery) blood. Body
O2 extraction was computed as V O /D O ?100%.
2 2
2.6. Data acquisition and analysis
b-adrenergic receptor blockade in M-receptor-blocked Haemodynamic data were recorded and digitized on-line
swine (n512) using an eight channel data-acquisition program AT-
Ninety min after swine underwent the five stage exercise CODAS (Dataq Instruments, Akron, OH, USA) and stored
protocol under control conditions, M-receptor blockade on a computer for off-line analysis using a program written
was produced by the continuous intravenous infusion of in MatLab (The Mathworks Inc., MA, USA). A minimum
atropine (30 mg/kg/min, i.v.). Ten minutes after the start of 15 consecutive beats were used for analysis of the
of the infusion, when haemodynamics had reached a new digitized haemodynamic signals. From the individual
steady state, resting measurements were obtained and the beats, heart rate (HR), LV peak systolic blood pressure
five-stage exercise protocol was repeated. After completion (LVSP), LV pressure at end-diastole (defined as the onset
of the exercise protocol, the atropine infusion was stopped of positive LVdP/dt; LVEDP), mean left atrial- (MLAP),
and the animals allowed to rest for another 90 min. Then, mean aortic- (MAP) and mean pulmonary artery blood
swine received an intravenous infusion of propranolol (0.5
mg/kg dissolved in 10 ml of saline). After completion of
(MPAP) pressures, as well as LVdP/dtmax and cardiac
output (CO), were determined, while stroke volume (SV5
propranolol administration, the infusion of atropine (30 CO/HR), systemic vascular resistance (SVR5MAP/CO)
mg/kg/min, iv) was restarted, and 10 min later, the and pulmonary vascular resistance [PVR5(MPAP2
exercise protocol was repeated. The dose of atropine was MLAP)/CO] were calculated.
considered to produce complete vagal blockade, as a Statistical analysis was performed using three-way (sex,
doubling of the dose did not produce further increases in exercise and treatment) analysis of variance for repeated
heart rate. measures. Since no significant differences were observed
between males and females in any of the protocols, the
2.4. Haemodynamic measurements data from males and females have been pooled and
analyzed together. Subsequently, we performed two-way
Blood pressure in the aorta, left atrium and pulmonary (exercise and treatment) analysis of variance for repeated
artery were measured using Combitrans® pressure trans- measures. When a significant effect of exercise was
ducers (Braun, Melsungen, Germany), with the reference observed, post-hoc testing was done using Dunnett’s test.
point at mid-chest level. LV pressure was measured using a When a significant effect of treatment was observed, post-
Konigsberg micromanometer, which was calibrated using hoc testing was done using either a paired t-test or
the fluid filled LV catheter; LVdP/dt was obtained via Wilcoxon Signed Rank test, as appropriate. To compare
electrical differentiation of the Konigsberg micromanome- the effects of different treatments that were studied on
39

462 R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474
different days (e.g. the changes produced by phentolamine 3. Results
versus the changes produced by phentolamine in b-adrenoceptor-blocked
animals), we performed two-way analysis 3.1. Exercise responses in male and female swine
of variance of the changes produced by the interventions.
A P-value of less than or equal to 0.05 was considered to Analysis of variance indicated that there were no
be statistically significant (two-tailed). All data are pre- significant differences between the haemodynamic and
sented as mean6S.E.M. blood gas responses to exercise in the six male and six
female swine, either during control conditions, or in the
presence of adrenergic or muscarinic receptor blockade
2.7. Drugs (not shown). Therefore, data from male and female swine
were pooled and analyzed together.
Phentolamine (10 mg/ml, Regitine®, Ciba-Geigy, Arnhem,
Netherlands) was dissolved in water containing 3.2. Reproducibility of responses to exercise
glucose (35 mg/ml) and further diluted in saline to
produce a final concentration of 1 mg/kg/10 ml. Propran- 3.2.1. Haemodynamics
olol (Sigma-Aldrich, Bornem, Belgium) was dissolved in During exercise, CO increased from 3.660.3 l/min at
308C saline, to a concentration of 0.5 mg/kg/10 ml. rest (lying down) to 7.860.5 l/min at 5 km/h (P#0.01),
Atropine (Sigma-Aldrich) was dissolved in 308C saline, to which was principally due to an increase in HR, from
a concentration of 30 mg/kg/ml. Fresh drug solutions 11564 to 24163 bpm (P#0.01), as the SV only increased
were prepared on the day of each experiment. significantly (|10%) at lower levels of exercise (Fig. 1).
Fig. 1. Reproducibility of haemodynamic responses to graded treadmill exercise in chronically instrumented swine. Data points were obtained at rest [lying
(0 L) and standing (0 S)]
and during five levels of treadmill exercise (1–5 km/h). LV5left ventricle; PA5pulmonary artery; LA5left atrium. Data are
expressed as the mean6S.E.M.; n59, except for mean LA pressure and pulmonary vascular resistance (n54); *P#0.05 vs. rest (lying), †P#0.05 vs.
control at the corresponding time point.
40

R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474 463
Since preload did not change during exercise (reflected by hence, arterial O content increased by 1262% (P#0.05)
2
a maintained LVEDP, not shown) and afterload was at the highest level of exercise compared to resting
elevated [reflected by the increase in LVSP, from 12265 to conditions (Fig. 2). Mixed venous P decreased from
O 2
15265 mmHg (P#0.01)], the maintained SV was the 4361 mmHg at rest to 2761 mmHg at 5 km/h (P#0.01);
result of an increase in LV contractility, reflected by the similarly, the S and O content decreased during exer-
O 2
2
increase in LVdP/dt , from 31206210 to 65006350 cise, from 5361% at rest to 2561% at 5 km/h and from
max
mmHg/s (P#0.01). MAP decreased slightly when the 2.8660.11 to 1.5060.12 mmol/l, respectively (both P#
animals changed position from lying down to standing up, 0.01), while P increased from 5461 to5962 mmHg
CO 2
but, during exercise, MAP increased from 9163 to9863 and pH decreased from 7.3960.01 to 7.3660.01 (both
mmHg (P#0.05). The modest change in MAP in the P#0.05). Since both CO and the arterio–venous O 2
presence of a doubling of CO implies that the SVR content difference nearly doubled, whole body V in-
O 2
decreased. In contrast, MPAP increased, from 1461 to creased four-fold, from 8.260.6 to 33.162.1 mmol/min
3262 mmHg (P#0.01), while the difference between (P#0.01). All variables returned to baseline resting values
MPAP and MLAP increased virtually in parallel with CO, within 90 min; a second period of exercise resulted in
so that the PVR was not significantly altered. After 90 min highly reproducible responses.
of rest, when all haemodynamic variables had returned to
baseline resting values, a second period of exercise resulted
in nearly identical haemodynamic responses, with 3.3. Effects of b-adrenergic receptor blockade
the exception of a slightly lower HR and a larger SV
during the second exercise period (Fig. 1). Unpublished 3.3.1. Haemodynamics
data from our laboratory indicate that three consecutive Under resting conditions, propranolol decreased CO by
exercise trials in four swine did not reveal haemodynamic 1063%, which was bradycardia-mediated (Fig. 3). SV did
differences between the three exercise tests (not shown). not change despite a 3063% decrease in LVdP/dt and
max
unaltered LV systolic pressure and was, therefore, most
likely due to the increase in LVEDP, from 862 to1362
3.2.2. Blood gases and V mmHg (P#0.05). Propranolol had no effect on MAP,
O 2
Exercise resulted in a slight decrease in arterial P , implying that SVR had increased, while PVR was not
CO 2
from 4361 mmHg at rest to 4061 mmHg at 5 km/h and affected. Propranolol blunted the exercise-induced in-
an increase in pH from 7.4460.01 to 7.4760.01 (both creases in CO, HR, LVdP/dt and LV systolic pressure
max
P#0.05) (not shown). Arterial S did not change compared to control exercise, while SVR was higher at
O 2
(9661% at rest and 9661% at 5 km/h), but Hb and, each level of exercise compared to control conditions.
Fig. 2. Reproducibility of systemic O transport responses to treadmill exercise. Cardiac output (left panel), haemoglobin (Hb) concentration (upper right
2
panel), arterial O saturation (middle right panel) and mixed venous O saturation (lower right panel), plotted as a function of whole body O consumption
2 2 2
(V ), are shown. Data points were obtained at rest (lying) and during five levels of exercise before (control) and after administration of 10 ml of saline.
O 2
Data are expressed as the mean6S.E.M., n59; *P#0.05 vs. control.
41

464 R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474
Fig. 3. Effects of b-adrenergic receptor blockade on haemodynamic responses to graded treadmill exercise in chronically instrumented swine. Data points
were obtained at rest [lying (0 L) and standing (0 S)]
and during five levels of treadmill exercise (1–5 km/h) before (control) and after administration of
propranolol (0.5 mg/kg i.v.). LV5left ventricle; PA5pulmonary artery; LA5left atrium. Data are expressed as the mean6S.E.M.; n512, except for mean
LA pressure and pulmonary vascular resistance (n58); *P#0.05 vs. rest (lying), †P#0.05 vs. control at the corresponding time point.
Exercise in the presence of propranolol resulted in an and 7.3760.01 at 5 km/h, respectively) (not shown).
increase in SV that was maintained at higher levels of Arterial S and Hb were also not altered, but mixed
O 2
exercise, probably as a result of the propranolol-induced venous S values were lower at rest and particularly
O 2
elevation of LVEDP. Despite the lower CO during exercise during exercise compared to control conditions (Fig. 4),
after propranolol compared to control exercise, the pres- reflecting an increase in O extraction, from 4361 to
2
sure drop across the pulmonary bed was not different from 4862% at rest and from 6962 to7762% at 5 km/h (both
that of control conditions, implying that exercise in the P#0.05). The widening of the arterio–venous O content
2
presence of propranolol resulted in a higher PVR com- difference could only in part compensate for the lower CO,
pared to control exercise. so that body V was 13–17% lower during exercise at
O 2
3–5 km/h compared to that found during control exercise
(Fig. 4).
3.3.2. Blood gases and V O2
Propranolol had no effect on arterial P and pH at rest
CO 2
(4461 mmHg and 7.4660.01, respectively) or during 3.4. Effects of a-adrenergic receptor blockade
exercise (4061 mmHg and 7.4860.01 at 5 km/h, respectively).
Similarly, propranolol had no effect on mixed 3.4.1. Haemodynamics
venous P and pH either at rest (5461 mmHg and Under resting conditions phentolamine decreased MAP
CO 2
7.3860.01, respectively) or during exercise (5861 mmHg by 1163%, which was due to a 3066% decrease in SVR,
42

R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474 465
Fig. 4. Effects of b-adrenergic receptor blockade on the systemic O transport responses to treadmill exercise. Cardiac output (left panel), haemoglobin
2
(Hb) concentration (upper right panel), arterial O saturation (middle right panel) and mixed venous O saturation (lower right panel), plotted as a function
2 2
of whole body O consumption (V ), are shown. Data points were obtained at rest (lying) and during five levels of exercise before (control) and after
2 O 2
administration of propranolol (0.5 mg/kg i.v.). Data are expressed as the mean6S.E.M., n57; *P#0.05 vs. control.
as CO increased by 1766% (all P#0.05); the latter was
exclusively mediated by an increase in HR and was
the arterio–venous O2 content difference was balanced by
the increase in CO both at rest and during exercise,
accompanied by an increase in LVdP/dt max (Fig. 5).
During exercise, these effects of phentolamine persisted,
with the phentolamine-induced increase in CO being even
resulting in whole body VO levels that were not different
2
from those observed during control conditions (Fig. 6).
more pronounced at 5 km/h than at rest. Despite increasing
CO at rest and during exercise, phentolamine had no
effect on MPAP at rest and even caused a decrease at
3.5. Effects of a-adrenergic receptor blockade in b-
adrenergic receptor-blocked swine
higher levels of exercise compared to exercise under
control conditions, indicating that PVR was slightly lower
during exercise in the presence of phentolamine, which
3.5.1. Haemodynamics
In b-blocked animals, phentolamine caused decreases in
reached levels of statistical significance at 3 and 5 km/h.
MAP, LV systolic pressure, LVEDP and MLAP at rest and
during treadmill exercise, while HR and LVdP/dtmax did
not change and CO increased only at the highest levels of
3.4.2. Blood gases and V exercise, compared to exercise in the presence of proprano-
O2
Phentolamine had no effect on arterial P and pH at
lol alone (Fig. 7). Phentolamine resulted in a lower SVR at
CO2 rest and during exercise, but did not decrease PVR in the
rest (4462 mmHg and 7.4460.01, respectively) or during
exercise (3961 mmHg and 7.4660.01 at 5 km/h, respectively).
Similarly, phentolamine had no effect on mixed
b-blocked animals.
venous P and pH either at rest (5361 mmHg and 3.5.2. Blood gases and V
CO O
2 2
7.3860.01, respectively) or during exercise (5561 mmHg In b-blocked animals, phentolamine had no effect on
and 7.3760.01 at 5 km/h, respectively; not shown). arterial or mixed venous P and pH (not shown) or
CO 2
Arterial S and Hb were also not altered, but phen- arterial S , but increased mixed venous S during
O O O
2 2 2
tolamine increased mixed venous S at rest and during exercise, compared to exercise in the presence of proprano-
O 2
exercise, compared to control (Fig. 6), reflecting a de- lol alone (Fig. 8). In addition, phentolamine abolished the
crease in O2 extraction, from 4461 to3962% at rest and
from 6962 to 6462% at 5 km/h (both P#0.05). In
addition, phentolamine abolished the exercise-induced
exercise-induced increase in Hb, resulting in a lower
arterial O2 content during exercise compared to exercise
with propranolol alone. The resultant narrowing of the
increase in Hb, resulting in a lower arterial O2 content
during exercise in the presence of phentolamine
arterio–venous O2 content difference balanced the slight
increase in CO, so that, compared to propranolol alone,
(5.3060.22 mmol/l) compared to control exercise
(5.7360.23 mmol/l, P#0.05). The resultant narrowing of
whole body VO was not affected by phentolamine, either
2
at rest or during exercise.
43

466 R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474
Fig. 5. Effects of a-adrenergic receptor blockade on haemodynamic responses to graded treadmill exercise in chronically instrumented swine. Data points
were obtained at rest [lying (0 L) and standing (0 S)]
and during five levels of treadmill exercise (1–5 km/h) before (control) conditions and after
administration of phentolamine (1.0 mg/kg i.v.). LV5left ventricle; PA5pulmonary artery; LA5left atrium. Data are expressed as the mean6S.E.M.;
n58, except for mean LA pressure and pulmonary vascular resistance (n54); *P#0.05 vs. rest (lying), †P#0.05 vs. control at the corresponding time
point.
3.6. Effects of M-receptor blockade and effects of b- control. In M-blocked animals, administration of propranoadrenergic
receptor blockade in M-receptor-blocked
swine
lol resulted in decreases in CO, HR, LVdP/dtmax and LV
systolic pressure, and increases in LVEDP and SVR at rest
and during exercise, but had no effect on MAP. PVR was
3.6.1. Haemodynamics not affected at rest but was higher during exercise com-
Under resting conditions, atropine increased CO, HR, pared to exercise with atropine alone (Fig. 9).
LV systolic pressure and LVdP/dt max (Fig. 9). The increase
in CO (3166%) was considerably less than the increase in
HR (8867%), as SV decreased by 3063%; the latter was
probably due to the decrease in LVEDP. Atropine had no
effect on MAP, implying that SVR had decreased. In
3.6.2. Blood gases and VO2
Atropine had no effect on arterial P CO (4561 mmHg at
2
rest and 4162 mmHg at 5 km/h), or pH (7.4560.01 and
contrast, PVR increased, from 2.860.5 to 3.460.7 mmHg/
l/min (P#0.05). The effects of atropine on all haemo-
7.4860.01) (neither shown), or SO and Hb (Fig. 10),
2
either at rest or during exercise. Similarly, atropine had no
dynamic variables gradually waned during exercise, with
the exception of HR and LVdP/dt max,
which were significantly
elevated at all levels of exercise compared to
effect on mixed venous P CO (5161 mmHg at rest and
2
5661 mmHg at 5 km/h), pH (7.4060.01 and 7.3760.01),
or S O (Fig. 10). Since the arterio–venous O2 content
2
44

R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474 467
Fig. 6. Effects of a-adrenergic receptor blockade on the systemic O transport responses to treadmill exercise. Cardiac output (left panel), haemoglobin
2
(Hb) concentration (upper right panel), arterial O saturation (middle right panel) and mixed venous O saturation (lower right panel), plotted as a function
2 2
of whole body O consumption (V ), are shown. Data points were obtained at rest (lying) and during five levels of exercise before (control) and after
2 O 2
administration of phentolamine (1 mg/kg i.v.). Data are expressed as the mean6S.E.M., n57; *P#0.05 vs. control.
difference was also not affected, body VO increased in
2
parallel with CO at rest and during exercise at 1 km/h
O2 uptake [12], our findings suggest that the maximum O2
uptake for these animals would be approximately 36
compared to control conditions, but was not different from
control at higher exercise levels. Although atropine had no
mmol/min (|30 ml/min/kg). Maximum VO values in
2
swine (weighing approximately 30 kg) have been reported
significant effect on the relation between VO and SVR (not
2
shown), the relation between VO and mixed venous O
2
2
saturation was shifted slightly upwards (P#0.05), suggesting
that M-activation exerted a small vasoconstrictor
to be in the range of 25–50 ml/min/kg [2,12,14–16].
Ciccone et al. [15] reported that the maximally attainable
O2 uptake during exercise in miniswine was approximately
25 ml/min/kg. However, in the same study, stimulation
influence on the systemic bed (Fig. 10).
with an electric cattle prod (which was not employed in the
In M-blocked animals, administration of propranolol had
negligible effects on arterial and mixed venous PCO and
2
pH, or arterial SO and O content, but mixed venous S
2 2 O2
was lower at rest and during exercise compared to that
present study) during exercise resulted in maximum VO
2
levels of approximately 40 ml/min/kg, which suggests
that these high values of O2 uptake might have been at
least in part the result of exogenously imposed mental
found with atropine alone, reflecting an increase in O2 extraction (Fig. 10). The widening of the arterio–venous
stress. Nonetheless, values of 30–50 ml/min/kg are
similar to those obtained in normal man [17], but are still
O2 content difference could only in part compensate for
the lower CO, so that body VO was reduced at rest and
2
during exercise compared to atropine alone (Fig. 10).
much lower than those obtained in dogs [18–20], ponies
[21] and horses [22], in which maximum VO is in the
2
range of 60–130 ml/min/kg. Although other factors, such
as body composition, may contribute to these interspecies
differences in maximum V O , it would appear that the
2
exercise capacity of sedentary swine, like that of sedentary
4. Discussion man, is comparatively moderate [15,23]. Thus, similar
4.1. Responses to treadmill exercise
values for maximum VO have been reported for domestic
2
swine and the leaner miniswine [2,12,14–16]. In addition,
swine, like humans, can be easily trained and show a
Exercise resulted in increases in V O , from 8 mmol
2
O 2/min (|6 ml O 2/min/kg) at rest to 32 mmol O 2/min
(|25 ml O 2 /min/kg) at 5 km/h. HR was 238–251 bpm at
5 km/h, which represents 85–90% of the reported maximum
HR in swine (275–285 bpm) [12–14]. Since HR is
dramatic improvement in exercise capacity during training
[4,14,16,23], whereas dogs show a more modest increase
in exercise capacity during exercise training [23,24].
Arterial O2 tension and saturation are unaltered during
submaximal and maximal exercise in normal humans [17],
an excellent indication of VO as a percentage of maximum
2
and in chronically instrumented dogs [2,25] or horses [26].
45

468 R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474
Fig. 7. Effects of a-adrenergic receptor blockade on haemodynamic responses to graded treadmill exercise in chronically instrumented swine in the
presence of b-adrenergic receptor blockade. Data points were obtained at rest [lying (0 L) and standing (0 S)]
and during five levels of exercise after
administration of propranolol (0.5 mg/kg i.v.) and after propranolol (0.2 mg/kg i.v.) and phentolamine (1 mg/kg i.v.). LV5left ventricle; PA5pulmonary
artery; LA5left atrium. Data are expressed as the mean6S.E.M.; n59, except for mean LA pressure and pulmonary vascular resistance (n53); *P#0.05
vs. rest (lying), †P#0.05 vs. propranolol at the corresponding time point.
In contrast, 7–10 mmHg decreases in arterial O tension detectable changes in arterial O saturation. The arterial
2 2
have been reported in chronically instrumented swine P decreased slightly during exercise, which was associ-
CO 2
[2,27], although this is not an ubiquitous finding [16]. An ated with a small increase in arterial pH at higher levels of
explanation for a decrease in arterial O tension in swine is exercise. Similarly, previous studies in swine also reported
2
not readily found. For example, the thoracotomy pro- a decrease in arterial P , although arterial pH did not
CO 2
cedure, necessary for instrumentation, is an unlikely change at exercise levels up to 50–70% of maximum O 2
explanation, since, in the aforementioned studies, ponies uptake [2,16,27]; exercise at maximum O uptake was
2
and dogs were also instrumented. That swine have a much associated with a significant decrease in pH [2,16,27],
lower O uptake capacity than dogs or ponies is also an which was probably due to anaerobic metabolism, as
2
unlikely explanation, as, in humans, a decrease in arterial reflected by increased lactate production [16,27]. These
P is sometimes noted in trained individuals with a findings suggest that, in the present study, swine were
O 2
markedly elevated O uptake capacity, rather than in performing aerobic exercise.
2
sedentary individuals [17]. In the present study, we did not The exercise-induced four-fold increase in V was the
O 2
observe significant changes in arterial P in going from result of a 60–65% increase in O extraction, in combina-
O 2
2
rest to exercise, although in some of the protocols, a trend tion with a 130–150% increase in O delivery, the latter
2
towards a decrease was noted (P.0.10). Importantly, the being met by a 120–140% increase in CO and a 10–15%
changes in arterial P were never sufficient to produce increase in Hb. In exercising dogs [28–30], horses
O 2
46

R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474 469
Fig. 8. Effects of a-adrenergic receptor blockade on the systemic O transport responses to treadmill exercise in the presence of b-adrenergic receptor
2
blockade. Cardiac output (left panel), haemoglobin (Hb) concentration (upper right panel), arterial O saturation (middle right panel) and mixed venous O
2 2
saturation (lower right panel), plotted as a function of whole body O consumption (V ), are shown. Data points were obtained at rest (lying) and during
2 O 2
five levels of exercise after administration of propranolol (0.5 mg/kg i.v.) and after administration of propranolol (0.2 mg/kg i.v.) and phentolamine (1
mg/kg i.v.). Data are expressed as the mean6S.E.M., n57; *P#0.05 vs. propranolol; ‡P50.07 vs. propranolol.
[26,31,32] and sheep [33], O delivery is facilitated by an tained despite an increase in afterload (LVSP), which must
2
increase in the O -carrying capacity of arterial blood due have been due to the increase in contractility (LVdP/dt ),
2 max
to an increase in Hb (by 20–50%). The latter is a result of as preload (LVEDP) was not altered. The increase in CO
splenic contraction, which expresses erythrocyte-rich blood did not result in appreciable increases in MAP, indicating
into the general circulation [26,30,31]. In swine and that SVR decreased commensurately.
humans, an increase in Hb also occurs but is usually During exercise, MPAP increased in excess of the
smaller (,15%) [2,16,27,34,35] and may, in part, result increase in MLAP. The increase in pressure difference
from extravasation of plasma during exercise resulting across the pulmonary bed tended to be smaller than the
from increased capillary plasma filtration, thereby leading increase in CO, so that PVR tended to decrease (P.0.05).
to haemoconcentration [36]. Under resting conditions, In man, PVR decreases by more than 50% during exercise,
mixed venous S is considerably higher in ponies (74% but in quadrupeds such as sheep and horses, there is only a
O 2
[26]) and dogs (64% [29] and 72% [37]) than in swine 25% decrease in PVR [42,43]. The present study is the first
(55%, present study). Consequently, ponies and dogs can to report the responses of the pulmonary vascular bed in
nearly triple their fractional O2 extraction during heavy
exercise compared to resting conditions [26,29], whereas it
swine to treadmill exercise. Our findings indicate that, in
swine, PVR decreases even less during exercise than in
can at most double in swine.
The exercise-induced increase in CO was principally the
other quadrupeds.
result of an increase in HR, as SV increased only at the 4.2. Sympathetic control of cardiovascular function
lower three levels of exercise. Previous studies in swine
have documented either no change [1,38] or a small
during exercise
(,15%) increase in SV [2,12] during exercise. SV also 4.2.1. b-Adrenoceptor blockade
increases slightly in ponies [21,39] or dogs [28,40], but it b-adrenoceptors are present in the heart where they
can increase by up to 100% in man during exercise in the modulate HR and contractility, and are located on blood
upright position [41]. However, when humans exercise in vessels, where they mediate vasodilation. In awake resting
the supine position, which is more comparable to the body swine, b-adrenoceptor blockade resulted in lower HR and
position of quadrupeds, SV increases are much less
(|25%) [41]. This is the result of an increase in SV to
LVdP/dtmax and higher SVR under resting conditions,
confirming previous observations from our laboratory that
approximately 80% of the maximum SV when changing significant b-adrenergic activity is present in swine in the
from the upright to the supine position, so that only a resting state [9]. This contrasts with studies in awake
slightly further increase is possible during subsequent resting dogs [44,45] and resting (supine) humans [46,47],
supine exercise [41]. In the present study, SV was main- in which b-adrenergic activity can be minimal. The effect
47

470 R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474
Fig. 9. Effects of M-receptor blockade and the effects of combined b-adrenergic and M-receptor blockade on haemodynamic responses to graded treadmill
exercise in chronically instrumented swine. Data points were obtained at rest [lying (0 L) and standing (0 S)]
and during five levels of exercise under control
conditions (open circles), during atropine infusion (30 mg/kg/min i.v.; solid circles), and during atropine infusion (30 mg/kg/min i.v.) after administration
of propranolol (0.5 mg/kg i.v.; open and solid squares). LV5left ventricle; PA5pulmonary artery; LA5left atrium. Data are expressed as the
mean6S.E.M.; n512, except for mean LA pressure and pulmonary vascular resistance (n57); *P#0.05 vs. rest (lying), †P#0.05 vs. control at the
corresponding time point, closed squares indicate P#0.05 atropine1propranolol vs. atropine at the corresponding time point.
of b-adrenoceptor blockade on HR, contractility and CO Propranolol produced an increase in SVR that could
became progressively greater during exercise. Consequent- have been caused, at least in part, by unopposed aly,
the lower CO was no longer compensated for by an adrenergic vasoconstriction. However, comparison of the
increase in O2 extraction, so that whole body VO was
2
13–17% lower compared to that found when animals were
SVR in the phentolamine-treated swine (22.761.0 mmHg/
l/min under resting conditions, Fig. 5) and the phenundergoing
control exercise. Our observations correlate tolamine plus propranolol-treated animals (26.260.9
well with data obtained in humans, which indicate that mmHg/l/min, Fig. 7), representing a propranolol-induced
there is impaired exercise capacity during b-adrenoceptor increase in SVR of 3.5 mmHg/l/min, to the increase in
blockade [48]. In the present study, we failed to find SVR produced by propranolol alone (from 28.061.7 to
evidence for anaerobic metabolism during the higher levels 31.262.0 mmHg/l/min, Fig. 3), which was almost the
of exercise, however, it is possible that lactate production same [3.2 mmHg/l/min, P5nonsignificant (NS)], sug-
was only modest, due to the relatively short duration of gests that the increase in SVR was primarily due to
each exercise stage (2–3 min) and was, therefore, buffered blockade of b-adrenoceptors per se. Interestingly, propran-
by the blood. It is likely that longer periods of exercise at olol increased PVR during exercise, demonstrating for the
the highest level of exercise would have resulted in first time that, in swine, exercise is associated with an
decrements of arterial pH.
increased b-adrenergic vasodilator influence in the pul-
48

R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474 471
Fig. 10. Effects of M-receptor blockade and the effects of combined b-adrenergic and M-receptor blockade on the systemic O transport responses to
2
treadmill exercise. Cardiac output (left panel), haemoglobin (Hb) concentration (upper right panel), arterial O saturation (middle right panel) and mixed
2
venous O saturation (lower right panel), plotted as a function of whole body O consumption (V ), are shown. Data points were obtained at rest (lying)
2 2 O 2
and during five levels of exercise under control conditions, during atropine infusion (30 mg/kg/min i.v.), and during atropine infusion (30 mg/kg/min i.v.)
after administration of propranolol (0.5 mg/kg i.v.). Data are expressed as the mean6S.E.M., *P#0.05 vs. control, †P#0.05 vs. atropine.
monary circulation. The observation of b-adrenergic vaso- [14,16], or ponies [26]. The phentolamine-induced indilation
in the pulmonary bed is in agreement with findings creases in HR and contractility were b-adrenoceptor
in several other species, including the cat [49], dog [50] mediated, as they were abolished by propranolol. Phenand
sheep [51]. tolamine decreased SVR during exercise but also at rest,
indicating that, even under resting conditions, significant
4.2.2. a-Adrenoceptor blockade systemic a-adrenergic tone is present in resting swine, a
Blockade of a-adrenergic receptors influences car- finding that is consistent with observations in resting dogs
diovascular performance and O transport through several [56,57] and man [46]. The phentolamine-induced decrease
2
mechanisms. First, blockade of prejunctional a -adrenergic in SVR may have been, at least in part, mediated by
2
receptors interrupts the negative feedback control of increased b-adrenergic vasodilation (possibly secondary to
catecholamine release (norepinephrine) [5,52]. The resul- increased catecholamine release), as pretreatment with
tant increase in norepinephrine levels augments cardiac propranolol blunted the phentolamine-induced decrease at
b-adrenergic stimulation, thereby increasing HR and con- rest by approximately 50% (P#0.05). Phentolamine detractility.
Second, a-adrenergic blockade can decrease creased PVR during exercise at 3 and 5 km/h. The slight
vascular tone by interrupting post-junctional a - and a - vasodilation produced by phentolamine was blocked by
1 2
receptor-mediated vasoconstriction of resistance vessels of propranolol, indicating that it was probably the result of
inactive skeletal muscle and visceral organs [35,53]. increased b-adrenoceptor-mediated vasodilation. The lack
Conversely, a -adrenergic receptors on coronary vascular of effect of a-adrenoceptor blockade on PVR in the
2
endothelium stimulate the release of nitric oxide, which presence of propranolol may be due to simultaneous
opposes the direct vasoconstrictor effect [54]. Third, a- blockade by phentolamine of vascular smooth muscle a -
1
adrenergic blockade decreases Hb concentrations by inter- receptors and endothelial a -adrenoceptors that mediate
2
rupting post-junctional a-mediated splenic contraction pulmonary vasoconstriction and vasodilation, respectively
[33,55]. [58]. Future studies using selective a-receptors are needed
In the present study, phentolamine increased HR and to determine the importance of the a-receptor subtype in
contractility at rest (which were probably reflex-mediated the control of pulmonary vasomotion in swine during
in response to the hypotension) but also during exercise. exercise.
The latter cannot be explained by activation of the In this study, we observed that the exercise-induced
baroreceptor reflex, since, at higher levels of exercise and increase in Hb was abolished when swine were pretreated
hence sympathetic activity, systemic hypotension produced with the nonselective a-adrenergic receptor blocker, phenby
vasodilators like adenosine or dipyridamole do not tolamine, which is in agreement with earlier observations
produce further increases in HR during exercise in swine in exercising sheep [33]. If the increase in Hb had been
49

472 R. Stubenitsky et al. / Cardiovascular Research 39 (1998) 459 –474
due to extravasation of plasma, phentolamine would have down), which may have contributed to the increase in V O .
2
been expected to further increase Hb, as arteriolar vasodi- The atropine-induced decrease in SVR was probably
lation will lead to increased intracapillary filtration caused by the higher VO levels, although a small vasodilat-
2
pressures [36]. Sato et al. [55] demonstrated in dogs that or influence may have been exerted via an increase in
catecholamine-induced splenic contraction is elicited via b-adrenergic activity. In contrast, the increase in PVR that
a-adrenergic receptor stimulation. Thus, the increase in Hb followed blockade of M-receptors was not mediated via
during exercise in swine is most likely due to a-adreno- increased b-adrenoceptor vasodilation. The finding of a
ceptor-mediated splenic contraction. direct vasodilator effect of M-receptor activation is consistent
with the presence of vasodilator M-receptors on
pulmonary vascular endothelium [58].
4.3. Parasympathetic control of cardiovascular function
during exercise
M-receptors are located in the heart, particularly in the
5. Conclusions
atria, where they modulate HR directly and indirectly by
influencing the release of catecholamines from the sympa-
thetic nerve endings. Muscarine receptors are also present
in blood vessels, where they can produce vasoconstriction
via direct vascular smooth muscle contraction or vasodilat-
ion via endothelium-dependent mechanisms. Atropine
nearly doubled HR in awake resting swine, an effect that
was blunted in animals in which the b-adrenoceptors were
blocked, indicating that the parasympathetic nervous sys-
tem controls HR both indirectly, via b-adrenoceptor activity,
and directly. These findings also indicate that, in resting
swine, the parasympathetic part of the ANS dominates
over the sympathetic part, as it does in other large
mammalian species, such as the dog [53], horse [39] and
man [59], but not in rodent species [60,61]. M-receptor
blockade also markedly increased LVdP/dt max, but this
effect was abolished when b-adrenoceptors were blocked.
The observation that combined M-and b-adrenoceptor
blockade produced similar decreases in LVdP/dtmax com-
pared to b-adrenoceptor blockade alone suggests that the
parasympathetic system did not directly modulate contrac-
Autonomic control of global LV function, systemic and
pulmonary circulation and whole body O2 balance in
awake swine at rest and during treadmill exercise are
described in this study. The sympathetic division controlled
O2 delivery via b-adrenoceptors (HR and contractility)
and Hb concentration via a-adrenoceptor-mediated
splenic contraction. Also, besides modulating systemic
vascular tone via a- and b-adrenoceptors, the sympathetic
division exerted a vasodilator influence on the pulmonary
circulation via b-adrenoceptors. The parasympathetic divi-
sion controlled O2 delivery in part directly (HR) and in
part indirectly, via inhibition of b-adrenoceptor activity
(HR and contractility). In addition, the parasympathetic
division exerted a direct vasodilator influence on the
pulmonary, but not on the systemic, circulation. Thus, in a
manner similar to that in man, in swine, both the sympa-
thetic and parasympathetic division of the ANS contribute
to cardiovascular homeostasis at rest and during exercise,
even at high intensity.
tility. It is more likely that modulation occurred via
alterations in HR (‘‘Treppe’’ effect [62]) or via inhibition Acknowledgements
of b-adrenoceptor activity. Interestingly, atropine still
resulted in significant increases in HR, even at 5 km/h
when HR was 238 bpm (.85% of maximum HR),
indicating that vagal withdrawal was not complete, even
during heavy exercise. In humans and dogs, the parasympathetic
division also exerts an influence on the heart,
up to near maximum levels of exercise [63–65]. HR, after
The authors gratefully acknowledge Mr Rob van Bre-
men for technical assistance. The research of Dr. Duncker
has been made possible by a Research Fellowship of the
Royal Netherlands Academy of Arts and Sciences.
b-adrenoceptor blockade alone (17665 bpm), was not
different from that after combined b-adrenoceptor and
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