EDITORIAL FOCUS
How many messengers to send calcium? Focus on "IP3 receptor blockade fails to prevent intracellular Ca2+ release by ET-1 and alpha -thrombin"

Andrew P. Somlyo

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

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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 Galpha 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.

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 alpha -thrombin. These latter results lead to the novel proposal that certain agonists (ET-1, alpha -thrombin) acting on G protein-coupled receptors can release Ca2+ by an alternate, IP3-independent pathway that is unavailable to receptor tyrosine kinases.

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 alpha -thrombin were mediated by Ca2+ influx rather than release, Mathias and colleagues (18) show that heparin fails to block Ca2+ release by alpha -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 alpha -thrombin to heparin is unlikely to be due to the stimulation of greater IP3 production by these agonists, although they show that alpha -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).

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 alpha -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 alpha -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., alpha 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.

    FOOTNOTES

Address for correspondence: PO Box 10011, Charlottesville, VA 22906.

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Am J Physiol Cell Physiol 274(6):C1453-C1455
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