Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

Mechanisms of Pulmonary Vasoregulation:In Vivo and In Vitro StudiesThe primary focus of our research is toelucidate fundamental mechanisms ofpulmonary vascular regulation. Thesestudies provide a foundation for investigating theacute effects of inhalational and intravenousanesthetics on pulmonary vasoregulation, as wellas chronic changes in the pulmonary circulationthat occur following lung transplantation.The pulmonary circulation is unique inthat it receives 100% of the right ventricularoutput. Thus, it represents the total “afterload”against which the right ventricle must ejectblood. Any increase in pulmonary vascularresistance will increase the work of the rightventricle, which under normal circumstances hasrelatively little inotropic reserve. An acute orchronic increase in pulmonary vascular resistancecan be particularly deleterious in the setting ofright ventricular hypertrophy and heart failure.Compared with cellular mechanisms thatregulate the systemic circulation, those thatregulate pulmonary vasomotor tone are much lesswell understood. An increase in pulmonaryvasomotor tone can be achieved by increasingintracellular calcium concentration and/orincreasing myofilament calcium sensitivity. Wehave compelling evidence that endogenousvasoconstrictor stimuli (alpha adrenoreceptoractivation, angiotensin II, endothelin) that workthrough the same signal transduction pathwaycan have markedly different effects on these twocellular mechanisms that regulate pulmonaryvasomotor tone.We use a variety of in vitro techniques todelineate the cellular mechanisms that mediatethe effects of endogenous vasoconstrictorstimuli, as well as to elucidate the cellularmechanisms by which anesthetic agents and lungtransplantation modify these responses. Thesetechniques include:• Isolated sarcoplasmic reticulum (SR)vesicles to directly measure pulmonary arterysmooth muscle (PASM) SR calcium uptake,release, and content;• Purified myofibrils to directly measurePASM actomyosin ATPase activity;• Individual PASM cells to measureintracellular calcium concentration, membranepotential, ion currents, inositol phosphateproduction, and phosphorylation of contractileproteins;• Western blot analysis to measureprotein tyrosine phosphorylation;• Immunofluorescence techniques tomeasure translocation of protein kinase Cisoforms; and• PASM strips to simultaneously measurechanges in the calcium-tension relationship andintracellular pH.Our results indicate that anesthetic agentsand lung transplantation can alter multiplecellular mechanisms of pulmonary vasoconstriction.We have also developed a technique tomeasure the effects of vasoconstrictor stimuli,alone and in combination with anesthetic agents,on the intact pulmonary microcirculation. Themajor focus of these studies is to assess the roleof endogenous endothelial dilator mechanisms inmodulating microvascular responses to vasoconstrictorstimuli and to determine whetheranesthetics exert their effects on these endothelialmechanisms or directly on pulmonary vascularsmooth muscle. These studies represent the firstsystematic investigation of the effects ofanesthetics on the pulmonary microcirculation.Finally, we perform in vivo studies inchronically instrumented dogs to assess the effectsof anesthetics on the pulmonary vasoconstrictorresponses to alveolar hypoxia, circulatoryhypotension, and normovolemic hemodilution.We believe it is imperative to characterize theseresponses in the whole animal, with all mechanismsthat either mediate or modulate thepulmonary vascular responses to these stimuliintact. It is important to note that hypoxia,hypotension, and hemodilution are frequentoccurrences during anesthesia for cardiac surgery.We believe that our experimental approachis unique in that it integrates the in vitro, microcirculatory,and in vivo methodologies. The resultsfrom each type of study are complementary andare used to delineate the effects and mechanismsof action of anesthetics on the pulmonarycirculation. These studies yield fundamentalinformation about cellular mechanisms ofpulmonary vascular regulation, which providesinsight about mechanisms of pulmonary vasculardisease. Our results also elucidate cellularmechanisms of anesthetic action, which providesinsight about the optimal choice of anestheticagent to minimize increases in right ventricularafterload in patients with right ventriculardysfunction or failure.THE MURRAYLABORATORYASSISTANT STAFFBalakrishnan Gopakumaran, Ph.D.RESEARCH FELLOWSXueqin Ding, M.D., Ph.D.Hiroshi Ito, M.D.Woon-Seok Roh, M.D.Sachiko Shimizu, M.D.TECHNOLOGISTDawn Farrar, B.S.CLINICAL RESEARCH NURSESCarrie Beven, R.N.Michael Beven, B.AChristine Cribbs, R.N.Deborah Manke R.N.Stephanie Ziegman, R.N.Paul A. Murray, Ph.D.Ogawa, K., Tanaka, S., and P.A. Murray PA (2001) Propofol potentiates phenylephrine-inducedcontraction via cyclooxygenase inhibition in pulmonary artery smoothmuscle. Anesthesiology 94:833-839.Tanaka, S., Kanaya, N., Homma, Y., Damron, D.S., and P.A. Murray (2002) Propofolincreases pulmonary artery smooth muscle myofilament calcium sensitivityvia the protein kinase C signaling pathway. Anesthesiology 97:1557-1566.Damron, D.S,, Kanaya, N., Homma, Y., Kim, S.O., and P.A. Murray (2002) Roleof PKC, tyrosine kinase and rho-kinase in a-adrenoreceptor-mediated pulmonary arterysmooth muscle contraction. Am. J. Physiol. 283:L1051-L1064.Sato, K., Seki, S., and P.A. Murray (2002) Effect of halothane and enflurane onsympathetic b adrenoreceptor-mediated pulmonary vasodilation in chronically-instrumenteddogs. Anesthesiology 97:478-487.Sohn, J.-T., and P.A. Murray (2003) Inhibitory effects of etomidate and ketamineon ATP-sensitive K + channel relaxation in canine pulmonary artery. Anesthesiology98:104-113.153