EDITORIAL FOCUS
Diversity of Ca2+-mobilizing mechanisms Focus on "cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle"

Piero Biancani

Department of Medicine, Brown University, Rhode Island Hospital, Providence, Rhode Island 02902-0001

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THE CURRENT ARTICLE IN FOCUS by Murthy and Makhlouf (Ref. 19; see p. C1199 in this issue) reports and discusses in some detail cGMP-mediated Ca2+ release in smooth muscle cells isolated from the circular layer of the rabbit stomach. The authors note that 8-bromo-cGMP, nitric oxide (NO), and vasoactive intestinal peptide (VIP), agents that normally cause relaxation of smooth muscle, can be converted to contractile agents when cAMP- and cGMP-dependent protein kinases (PKA and PKG, respectively) are inhibited. It has been reported elsewhere that sodium nitroprusside (SNP) causes contraction of longitudinal esophageal muscle (10, 21) through cGMP-dependent mechanisms. These agents (SNP, NO, VIP) have in common the ability to increase the intracellular levels of cGMP. The authors show that, when PKA and PKG are inhibited, cGMP causes mobilization of Ca2+ from intracellular stores. It is therefore reasonable to examine how cGMP, and possibly other cyclic and noncyclic nucleotide phosphates, may function as Ca2+-mobilizing agents.

Mobilization of intracellular Ca2+ is a common signal transduction mechanism that can be triggered by diverse signals such as hormones binding to specific surface receptors, cell-cell fusion during fertilization, or membrane depolarization during muscle contraction. This diversity of signals culminating in Ca2+ mobilization is reflected by diversity of Ca2+-mobilizing messengers. To date several Ca2+-release activators have been discovered, including inositol trisphosphate (IP3) (3), sphingosine 1-phosphate (7), and several cyclic and noncyclic nucleotide phosphates (14).

cGMP is a member of an expanding family of nucleotide phosphates that act as Ca2+-mobilizing agents. These include GTP, cGMP, cyclic ADP-ribose (cADPR), a metabolite of beta NAD+, and nicotinic acid adenine dinucleotide phosphate (NAADP). These agents differ in their properties and in the Ca2+ stores they are capable of discharging.

GTP. GTP stimulates Ca2+ release in neuronal, smooth muscle, and fibroblast cell lines (see Refs. 5, 10, 14, and 40 of Ref. 19). GTP-induced Ca2+ release is additive to that of IP3 and appears to be mediated by a product of the hydrolysis of GTP, since Ca2+ release could not be induced by nonhydrolyzable analogs of GTP. The GTP-dependent mechanism, so far observed in transformed cell lines, is different from the cGMP-mediated Ca2+ release observed in the present study on freshly dispersed smooth muscle cells. In smooth muscle cells GTP did not cause Ca2+ release.

cADPR. cADPR is a product of the activity of ADP-ribosyl cyclase, which is homologous to the mammalian CD38 ectoenzyme, first recognized as a lymphocyte antigen but subsequently found to be present in numerous cell types (14). These enzymes are bifunctional in that they are capable of catalyzing the conversion of beta NAD+ to cADPR via an ADP-ribosyl cyclase reaction and to catalyze the exchange of the nicotinamide group of NADP with nicotinic acid to generate NAADP (2). The ability of cADPR to stimulate Ca2+ release independently of IP3 was first determined in sea urchin eggs (5), a well-established and convenient model for studying Ca2+ release by these agents (14). Subsequently, it was shown that cADPR-induced Ca2+ release was pharmacologically similar to Ca2+-induced Ca2+ release (6), which is thought to be mediated by ryanodine receptors (18). In smooth muscle cells of the longitudinal muscle layer, clear evidence for the ability of endogenous cADPR to induce Ca2+ mobilization was obtained by Kuemmerle and Makhlouf (12) as follows: a contractile agonist (CCK-8) was shown to activate ADP-ribosyl cyclase and to stimulate the formation of cADPR in longitudinal smooth muscle in a concentration-dependent manner; cADPR bound to and selectively activated ryanodine receptor/Ca2+ channels (13) and had no effect on IP3 receptor/Ca2+ channels; and cADPR mobilized Ca2+ and acted further to enhance Ca2+-induced Ca2+ release via ryanodine receptor/Ca2+ channels (12).

It is worth noting that production of cADPR, leading to Ca2+ release, occurs through different mechanisms in sea urchin eggs and in smooth muscle. In sea urchin eggs ADP-ribosyl cyclase is found in a soluble form that is cGMP and ATP dependent and in a membrane-bound form that is a homologue of CD38 (8), fertilization is accompanied by production of cGMP and activation of PKG, and the latter is thought to activate ADP-ribosyl cyclase to generate cADPR and thus stimulate Ca2+ release. In smooth muscle, however, activation of ADP-ribosyl cyclase appears to depend on an initial influx of Ca2+ into the cell, which causes Ca2+-induced Ca2+ release and activates ADP-ribosyl cyclase. The resultant formation of cADPR enhances Ca2+-induced Ca2+ release (6, 11, 12, 17).

The enzyme responsible for synthesizing cADPR, ADP-ribosyl cyclase, is found in numerous cell types (14, 16, 20). The wide distribution of the enzyme and the complexity of the associated metabolic pathways suggest that there may be multiple regulatory mechanisms controlling the concentration of cADPR (8).

NAADP. NAADP+ is a recently identified metabolite of NADP+ that is as potent as IP3 and cADPR in mobilizing intracellular Ca2+ in sea urchin eggs and microsomes (the usual model for studying Ca2+ release by these agents). Like cADPR, NAADP is also a catalytic product of ADP-ribosyl cyclase (or CD38), this time catalyzing the exchange of the nicotinamide group of NADP with nicotinic acid to generate NAADP (2). The switch of the catalysis to the exchange reaction requires acidic pH and nicotinic acid (2). The mechanism of Ca2+ release activated by NAADP+ and the Ca2+ stores it acts on are different from those of IP3 and cADPR. The NAADP+-sensitive Ca2+ release is likely to be a new pathway for mobilizing internal Ca2+, since it is unaffected by pharmacological agents of known Ca2+-release mechanisms (1). It is insensitive to 8-amino-cADPR, a specific antagonist of the cADPR receptor (22). Heparin, an antagonist of the IP3 receptor, antagonists of the ryanodine receptor, high concentrations of Mg2+, procaine, and ruthenium red (4, 5, 9, 15) also have no effect on the release. Fractionation studies show that the Ca2+ stores discharged by NAADP are different from those discharged by cADPR or IP3 as well as from mitochondria (5, 15). Furthermore, these stores are not thapsigargin sensitive and may thus possess a distinct Ca2+-ATPase uptake mechanism. Binding studies show that the NAADP+ receptor is distinct from that of cADPR and that, at subthreshold concentrations, NAADP+ can fully inactivate subsequent binding to the receptor in a time-dependent manner; this desensitization does not affect cADPR (1). Thus the NAADP+-sensitive Ca2+-release process has novel regulatory characteristics, which are distinguishable from Ca2+ release mediated by either IP3 or cADPR.

cGMP. The ability of cGMP to stimulate Ca2+ release was detected in smooth muscle cells from the circular layer of the stomach after blockade of PKA and especially PKG activity. In smooth muscle these protein kinases act at different locations to attenuate the intracellular levels of Ca2+. They block Ca2+ release from sarcoplasmic stores (phosphorylation of IP3 and ryanodine receptors), stimulate Ca2+ uptake by the stores (phosphorylation of Ca2+-ATPase), inhibit plasma membrane Ca2+ channel activity (block Ca2+ influx), and stimulate plasma membrane K+ channel activity (hyperpolarization and thus inhibition of Ca2+ influx). When the effect of these kinases is blocked, cGMP could be shown to induce Ca2+ release. The release occurred from thapsigargin-sensitive Ca2+ stores. These stores, however, were distinct from IP3- or ryanodine-sensitive Ca2+ stores, since cGMP-induced Ca2+ release could not be blocked by either heparin or ruthenium red (the latter blocks ryanodine receptor/Ca2+ channels, which are found in intestinal longitudinal muscle rather than circular muscle). Furthermore, Ca2+ release induced by cGMP was additive to that induced by IP3, consistent with an effect of cGMP on a distinct store. The fact that kinase inhibitors were required to demonstrate the effect of cGMP implies that kinases are able to inhibit (presumably by phosphorylation) the cGMP-dependent Ca2+-release pathway, just as they inhibit the IP3-dependent pathway in circular muscle and the ryanodine-dependent pathways in longitudinal muscle.

It is important to note that, whereas PKA and PKG inhibit Ca2+ release in vascular and visceral smooth muscle and cerebellar neurons, they seem to stimulate Ca2+ release in other cell types (e.g., hepatocytes). Whether cGMP independently of its kinase is able to stimulate Ca2+ release in hepatocytes, for example, is not known.

The functional significance of some of these Ca2+-mobilizing agents, in cells other than sea urchin eggs, remains to be established. cADPR-induced Ca2+ release appears to play a physiological role in agonist-induced contraction of intestinal longitudinal muscle (12) by causing Ca2+ release from ryanodine-sensitive stores and amplifying Ca2+-induced Ca2+ release. cGMP-induced Ca2+ release, while clearly demonstrated in this article (19), occurs in smooth muscle only after blockade of PKA and PKG, which are otherwise present and functioning in cells. As the authors speculate, it is possible that these kinases may not be present at some stage of development, when cGMP may mediate release of cGMP-dependent Ca2+ stores. It is also possible that SNP-induced contraction of esophageal longitudinal muscle, which is cGMP dependent and mediated through an indomethacin-dependent pathway (10), may in part depend on cGMP-induced Ca2+ mobilization. This possibility, however, has not yet been examined and remains speculative.

It is becoming increasingly clear, however, that multiple and distinct Ca2+ stores may be present in a variety of cells and that these stores may be regulated by multiple and distinct mechanisms. This battery of release mechanisms may provide versatility of response to diverse signals requiring Ca2+ mobilization to obtain a specific result.

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AJP Cell Physiol 274(5):C1196-C1198
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