Departments of Molecular Physiology and Biological Physics and of Internal Medicine (Cardiology) and the Center for Structural Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906
THE RISE IN CYTOPLASMIC CALCIUM, identified first as
the trigger of muscle contraction (8), is now recognized as a universal signal that controls numerous processes in all eukaryotic cells. The
similar recognition that Ca2+ is
stored in and released from both the sarcoplasmic reticulum (SR) of
muscle (4) and the endoplasmic reticulum (ER) of nonmuscle cells (20,
23) led to major questions still being asked about the identity of the
molecular mechanisms of Ca2+
release from the SR/ER (2, 24). The first clear answer came from the
heart, when it was shown that, in cardiac myocytes,
Ca2+ is released from the SR by
Ca2+ that enters from the
extracellular space; this is the now widely recognized process known as
Ca2+-induced
Ca2+ release (CICR; Ref. 6). The
influx of Ca2+ opens the ryanodine
receptor (RyR) Ca2+-release
channels, localized to specialized regions of the cardiac SR and
characterized by their ability to bind ryanodine. The gating of RyRs in
skeletal muscle, where they also serve as the
Ca2+-release channels of the SR,
is correlated with electric charge movement (21) at the contact between
invaginations of the skeletal muscle cell membrane, the T tubules, and
the adjacent terminal cisternae of the SR, but its precise, molecular
mechanism is still to be elucidated. In nonmuscle cells in which the
mechanism was first recognized (26) and in smooth muscle (5, 9, 24, 25), a chemical messenger, inositol 1,4,5-trisphosphate
(IP3), releases
Ca2+ through
IP3 receptors/channels.
IP3 is produced on binding of excitatory agonists to their heptameric serpentine receptors that are
coupled to G In the face of apparently overwhelming evidence identifying
IP3 as the G protein-coupled
Ca2+-releasing messenger, Mathias
and co-workers, in the current article in focus (Ref. 18; see p. C1456
in this issue), now propose that an alternate pathway for releasing
intracellular Ca2+, independent of
IP3, can also be utilized by some
heptameric serpentine receptors. Their proposal is based on
measurements of cytosolic Ca2+
concentration and IP3 production
in Chinese hamster ovary (CHO) cells and L6 myoblasts transfected with
platelet-derived growth factor (PDGF) and fibroblast growth factor
(FGF) receptors and CHO-K1 cells containing a cloned endothelin-1
(ET-1) receptor. Control experiments confirm that, in these, as in many
other cells, intracellular heparin, a competitive inhibitor of
IP3, blocks Ca2+ release induced by
IP3, PDGF, and FGF. The surprise
is that neither heparin nor an antibody to the type 1 IP3 receptor blocks
Ca2+ release by either ET-1 or
Intracellular heparin, introduced by reversible permeabilization, can
block the rise in intracellular
Ca2+ concentration induced by a
muscarinic agonist in intact smooth muscle in the absence, but not in
the presence, of extracellular Ca2+, indicating that heparin
blocks Ca2+ release but not
Ca2+ influx (12). To exclude the
possibility that in their experiments the heparin-resistant effects of
ET-1 and The experiments of Mathias et al. (18), although subject to the above
reservations, raise important questions about a possible non-IP3-dependent, alternate
pathway of Ca2+ release activated
by some heptameric serpentine receptors. The first question, not
addressed by the study, is the identity of the organelle and the
Ca2+ channel involved in this
Ca2+-release mechanism. Many
nonmuscle cells, as well as muscle, contain both ryanodine and
IP3 receptors (7, 17, 27).
Therefore, one of the explanations that could account for
IP3-independent Ca2+ release, without doing gross
violence to the accepted paradigm, is that it takes place through RyRs
that are known to be insensitive to heparin. This is assuming that the
source of Ca2+ is the SR/ER or the
contiguous perinuclear space rather than a more esoteric source, such
as the Golgi apparatus (3, 13). A second question concerns the nature
of the messenger involved. Mathias et al. (18) suggest
that it may be cyclic ADP-ribose, a potent
Ca2+-releasing agent isolated from
sea urchin eggs, acting on sea urchin ER and not antagonized by heparin
(15). However, evidence for major physiological effects of cyclic
ADP-ribose in vertebrates, thought to be related to CICR, (15) is, at
best, limited (11, 14, 19, 22). Nevertheless, the presence of RyRs in
the interior of cells such as smooth muscle (16), where the local
concentration of Ca2+ due to
influx is unlikely to reach levels sufficient to trigger CICR (10),
raises intriguing questions about the function of these RyRs/channels
and their yet-to-be-identified physiological messenger.
A final question about the proposed
IP3-independent pathway is whether
it is limited to the types of cultured cells investigated by Mathias et
al. (18) or whether it also plays a significant role in other, not
cultured, cells. It would be unfair to ascribe these new findings to an
abnormal coupling mechanism restricted solely to use by transfected
receptors, because the thrombin receptor is endogenous to
CHO cells in which the
Ca2+-releasing effect of
ARTICLE
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Article
References
q/11 proteins or to
receptor tyrosine kinases, either of which can activate one of the
isoforms of phospholipase C that hydrolyzes phosphatidylinositol
4,5-bisphosphate to yield IP3 and
diacylglycerol.
-thrombin. These latter results lead to the novel proposal that
certain agonists (ET-1,
-thrombin) acting on G protein-coupled
receptors can release Ca2+ by an
alternate, IP3-independent pathway
that is unavailable to receptor tyrosine kinases.
-thrombin were mediated by
Ca2+ influx rather than release,
Mathias and colleagues (18) show that heparin fails to block
Ca2+ release by
-thrombin, even
when extracellular Ca2+
concentration is reduced (with 1 mM EGTA) to ~110 nM. They further suggest that the greater resistance of the effects
(Ca2+ release) of ET-1 and
-thrombin to heparin is unlikely to be due to the stimulation of
greater IP3 production by these
agonists, although they show that
-thrombin is a more potent
stimulator of IP3 production than
PDGF or EFG. However, as they recognize, measurements of
IP3 cannot exclude the possibility
that apparent heparin resistance may be due to
IP3 receptors that are
inaccessible to heparin and/or exposed to localized very high
concentrations of "IP3
sparks." Although compartmentalization may be the refuge of
editorial writers and other scoundrels, the fact remains that total
cellular IP3 need not correlate
well with physiological effects that can be exerted at concentrations
much lower than total cellular IP3
content (1, 28).
-thrombin was also not blocked by heparin. In this instance, the
question remains whether the greater, bulk or local, concentration of
IP3 released by activation of
-thrombin and other receptors could be the cause of apparent
insensitivity to heparin. It would be interesting to learn whether,
when transfected in CHO cells, activation of other G protein-coupled
receptors (e.g.,
1-adrenergic,
muscarinic) that are known to have their Ca2+-releasing activity inhibited
by heparin in noncultured cells (12) can also release
Ca2+ by an
IP3-independent mechanism. These
and other questions remain to be answered, but the present findings of
Mathias et al. (18) challenge existing dogma and, as such, the
hypothesis deserves to be further tested.
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FOOTNOTES |
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Address for correspondence: PO Box 10011, Charlottesville, VA 22906.
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REFERENCES |
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