Sarcoplasmic Reticulum Function in Smooth Muscle - Physiological ...

134 SUSAN WRAY AND THEODOR BURDYGA
A) CAFFEINE. Caffeine, an alkaloid methylxanthine, increases
the RyR Ca sensitivity by increasing its open
probability (P o) without changing its conductance, as
shown in single-channel experiments on RyR purified
from cardiac (601), skeletal (600), and smooth muscles
(258) reconstituted into planar lipid bilayers. The threshold
caffeine concentration for Ca release is 250 M (404).
The caffeine-induced Ca release is a two-step process: a
small Ca release and then a second regenerative phenomenon,
in which released Ca acts on a cluster of RyRs,
triggering further Ca release. Caffeine has long been used
to deplete SR Ca stores in smooth muscle; this is frequently
done in a Ca-free medium containing Ca chelators
such as EGTA. The transient contractile response obtained
under such conditions is a qualitative estimate of
the average size of the Ca stores in the SR. Caffeine,
however, does not deplete the SR Ca store in those tissues
expressing the RyR2 dominant negative splice variant,
e.g., the uterus.
In the guinea pig ureter, which expresses effectively
only RyR (91, 92), concentrations of caffeine for Ca release
ranged from 0.5 to 20 mM (89). The use of caffeine
in smooth muscle comes with several concerns: 1) it
inhibits phosphodiesterase and therefore raises cAMP
concentration (341), which at concentrations exceeding 1
mM inhibits voltage-gated Ca channels; 2) it has the potential
to augment capacitative Ca entry by depletion of
the SR; and 3) it inhibits the SR Ca release induced by IP 3
(480) and inositol phosphate formation (576).
B) RYANODINE. Ryanodine is a poisonous alkaloid found
in the plant Ryania speciosa (670). The compound has an
extremely high affinity for the RyR. The pharmacology of
RyRs has been elegantly reviewed (231). Ryanodine has
complex concentration-dependent effects on the conductance
and gating of single RyR channels (670). It binds
with such high affinity to the receptor that it was used as
a label for the first purification of RyRs and gave its name
to them. At nanomolar to low micromolar concentrations,
ryanodine locks the receptors in a half-open state,
whereas it closes them at micromolar concentration, irreversibly
inhibiting channel opening. There is an agreement
that high-affinity binding results in channel activation
or subconductivity, whereas low-affinity binding
leads to channel inhibition (670). Ryanodine at low concentration
is frequently used to deplete the SR by causing
the Ca release channels to remain in a semiconducting
state. This leak from the SR has several consequences,
such as preventing the SR from storing any Ca it may
accumulate, loss of Ca sparks and their effects on Casensitive
ion channels, and loss of ability to generate
IP 3R-mediated Ca waves and Ca oscillations in many
types of smooth muscle. This last effect is due to ryanodine
depleting common Ca stores, i.e., shared by RyR and
IP 3Rs. The binding of ryanodine to RyRs is use-dependent,
Physiol Rev VOL 90 JANUARY 2010 www.prv.org
that is, the channels have to be in the activated state for
it to be effective.
C) TETRACAINE. Tetracaine is a potent local anesthetic
and an allosteric blocker of RyR channel function. At low
concentrations, tetracaine causes an initial inhibition of
spontaneous Ca release events (137), while at high concentrations
it blocks release completely (236).
D) RUTHENIUM RED. Ruthenium red is an organic polycationic
dye that tightly binds to tubulin dimers and RyRs.
It is a potent (nM) inhibitor of RyR (783), but also inhibits
mitochondrial Ca uptake (242).
2. IP3Rs Pharmacological inhibitors of IP3Rs are less abundant
compared with RyRs, and unfortunately, agents used
to block these receptors are nonselective. The inhibitors
most commonly used include 2-aminoethoxy-diphenylborate
(2-APB), heparin, and xestospongins (for a more
detailed review, see Ref. 384).
A) 2-APB. 2-APB is a synthetic monomer that can form
a five-membered boroxazolidine heterocyclic ring (boroxazolidone),
when an internal coordinate bond is formed
between the nitrogen in the ethanolamine side chain and
the tricoordinated boron (664). 2-APB inhibits the IP3R channel opening without affecting IP3 synthesis or binding.
Despite earlier claims to the contrary, 2-APB is not
selective for IP3Rs as it reduces capacitative Ca entry and
Ca efflux from mitochondria by inhibition of the NCX, and
it may also block gap junctions (reviewed in detail in Ref.
384).
B) HEPARIN. Heparin is a cell-impermeant inhibitor of
IP3R and acts as a competitive inhibitor of IP3 in permeabilized
smooth muscles (91). At low concentrations (up
to 2 mM), heparin may be specific, but at higher concentrations
(20 mM), it chelates Ca and inhibits contraction
(358).
C) XESTOSPONGINS. The xestospongins A, C, and D, araguspongine
B, and demethylxestospongin B are alkaloids
from the Australian marine sponge Xestospongia sp.
(199). They are cell-permeant, potent inhibitors of IP3Rs. Xestospongin C is the most potent and widely used in
smooth muscle studies (34, 45, 169, 483). Although more
studies are required, accumulated data suggest that the
various isoforms of IP3Rs differ in their sensitivities to
xestospongins. Xestospongins are not selective and can
inhibit voltage-gated Ca channels, which complicates
their use.
3. cADPR
Cyclic ADP ribose (cADPR) is a cellular messenger
for calcium signaling (235). It is derived from nicotinamide
adenine dinucleotide (NAD) and ADP-ribosyl cyclase
(392). It is a physiological allosteric modulator of
RyRs and helps stimulate CICR at low cytosolic [Ca]. RyR