Stimulation of glycogenolysis in hepatocytes by angiotensin II may involve both calcium release and calcium influx

The stimulation of [³H]glucose release (a measure of glycogenolysis) from isolated guinea pig hepatocytes by Ca-mobilizing agonists can be resolved into two phases. The initial transient phase is independent of extracellular Ca, and is probably a result of Ca released from an intracellular pool. The second phase occurs only in the presence of extracellular Ca, which suggests that Ca-influx is also involved in the mechanism of Ca-mobilization by these agents in the guinea pig hepatocyte.     

The mechanism for synergism between phospholipase C- and adenylylcyclase-linked hormones in liver. Cyclic AMP-dependent kinase augments inositol trisphosphate-mediated Ca2+ mobilization without increasing the cellular levels of inositol polyphosphates.

The ability of cAMP-dependent hormones to modulate the actions of Ca2(+)-mobilizing hormones was studied in single fura-2-injected guinea pig hepatocytes. In 91% of cells the cAMP-linked hormone, isoproterenol, applied alone, did not alter cytosolic Ca2+ concentration. In 78% of cells which had been pre-exposed to a low concentration of angiotensin II, isoproterenol was able to increase cytosolic Ca2+. Isoproterenol did not, however, increase inositol 1,4,5-trisphosphate or inositol tetrakisphosphate on its own, or in the presence of angiotensin II. Isoproterenol was also able to raise cytosolic Ca2+ concentration in cells microinjected with inositol 2,4,5-trisphosphate or a photoactivatable derivative of inositol 1,4,5-trisphosphate. The elevation of cytosolic Ca2+ concentration induced by isoproterenol in angiotensin II-treated cells and cells injected with caged inositol 1,4,5-trisphosphate was blocked by heparin, implying that the effect was mediated by an inositol 1,4,5-trisphosphate receptor agonist. In permeabilized hepatocytes, inositol 1,4,5-trisphosphate-induced Ca2+ release was enhanced by 8-bromo-cAMP and the catalytic subunit of cAMP-dependent kinase. Cyclic AMP-dependent kinase shifted the dose-response curve for inositol 1,4,5-trisphosphate-mediated Ca2+ release to the left by a factor of 4 and increased the total amount of Ca2+ released by 25%. These results indicate that increased sensitivity of the intracellular Ca2+ releasing organelle to inositol 1,4,5-trisphosphate is responsible for synergism between phospholipase C- and adenylylcyclase-linked hormones in the liver.     

Inositol lipids and cell stimulation in mammalian salivary gland

The rat parotid salivary gland shows marked alterations in phospholipid metabolism when stimulated by certain agonists. These agonists are those which cause cellular Ca mobilization by activation of muscarinic, alpha-adrenergic or peptidergic (substance P) receptors. The phospholipid changes apparently reflect the activation of a phosphoinositide-phosphatidic acid cycle, the precise pathways of which are not known with certainty. The observed effects include (1) an increased labelling by 32PO4 of phosphatidylinositol and phosphatidic acid, (2) net synthesis of phosphatidic acid, (3) net breakdown of phosphatidylinositol and phosphatidylinositol-4,5-bisphosphate. These effects apparently do not require the presence of extracellular Ca or the release of internal Ca and cannot be produced by the artificial introduction of Ca into the cytosol with Ca ionophores. These findings are consistent with the view that a receptor-mediated alteration in phosphoinositide metabolism represents an early step in the stimulus-response pathway in the parotid acinar cell. It has been suggested that phosphatidic acid synthesis might be of central importance in mediating Ca influx and that PIP2 breakdown might play a role in activation of Ca release. Evidence for these latter ideas is for the present largely circumstantial.     

Methods for studying store-operated calcium entry

Activation of surface membrane receptors coupled to phospholipase C results in the generation of cytoplasmic Ca2+ signals comprised of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. A primary mechanism for this Ca2+ entry process is attributed to store-operated Ca2+ entry, a process that is activated by depletion of Ca2+ ions from an intracellular store by inositol 1,4,5-trisphosphate. Our understanding of the mechanisms underlying both Ca2+ release and store-operated Ca2+ entry have evolved from experimental approaches that include the use of fluorescent Ca2+ indicators and electrophysiological techniques. Pharmacological manipulation of this Ca2+ signaling process has been somewhat limited; but recent identification of key molecular players, STIM and Orai family proteins, has provided new approaches. Here we describe practical methods involving fluorescent Ca2+ indicators and electrophysiological approaches for dissecting the observed intracellular Ca2+ signal to reveal characteristics of store-operated Ca2+ entry, highlighting the advantages, and limitations, of these approaches.     

Mutual antagonism of calcium entry by capacitative and arachidonic acid-mediated calcium entry pathways

In nonexcitable cells, the predominant mechanism for regulated entry of Ca(2+) is capacitative calcium entry, whereby depletion of intracellular Ca(2+) stores signals the activation of plasma membrane calcium channels. A number of other regulated Ca(2+) entry pathways occur in specific cell types, however, and it is not know to what degree the different pathways interact when present in the same cell. In this study, we have examined the interaction between capacitative calcium entry and arachidonic acid-activated calcium entry, which co-exist in HEK293 cells. These two pathways exhibit mutual antagonism. That is, capacitative calcium entry is potently inhibited by arachidonic acid, and arachidonic acid-activated entry is inhibited by the pre-activation of capacitative calcium entry with thapsigargin. In the latter case, the inhibition does not seem to result from a direct action of thapsigargin, inhibition of endoplasmic reticulum Ca(2+) pumps, depletion of Ca(2+) stores, or entry of Ca(2+) through capacitative calcium entry channels. Rather, it seems that a discrete step in the pathway signaling capacitative calcium entry interacts with and inhibits the arachidonic acid pathway. The findings reveal a novel process of mutual antagonism between two distinct calcium entry pathways. This mutual antagonism may provide an important protective mechanism for the cell, guarding against toxic Ca(2+) overload.     

Mechanisms of phospholipase C-regulated calcium entry

In a variety of cell types, activation of phospholipase C-linked receptors results in the generation of intracellular Ca2+ signals comprised of components of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. This entry of Ca2+ occurs by either of two general mechanisms: the release of stored Ca2+ can activate, by an unknown mechanism, store-operated channels in the plasma membrane, a process known as capacitative calcium entry. Alternatively, second messengers generated at the plasma membrane can activate Ca2+ channels more directly, a non-capacitative calcium entry process. This review summarizes current knowledge of the underlying signaling mechanisms and the nature of the channel molecules responsible for these two general categories of regulated Ca2+ entry.     

Signaling mechanism for receptor-activated canonical transient receptor potential 3 (TRPC3) channels

Canonical transient receptor potential 3 (TRPC3) is a receptor-activated, calcium permeant, non-selective cation channel. TRPC3 has been shown to interact physically with the N-terminal domain of the inositol 1,4,5-trisphosphate receptor, consistent with a "conformational coupling" mechanism for its activation. Here we show that low concentrations of agonists that fail to produce levels of inositol 1,4,5-trisphosphate sufficient to induce Ca(2+) release from intracellular stores substantially activate TRPC3. By several experimental approaches, we demonstrate that neither inositol 1,4,5-trisphosphate nor G proteins are required for TRPC3 activation. However, diacylglycerols were sufficient to activate TRPC3 in a protein kinase C-independent manner. Surface receptor agonists and exogenously applied diacylglycerols were not additive in activating TRPC3. In addition, inhibition of metabolism of diacylglycerol slowed the reversal of receptor-dependent TRPC3 activation. We conclude that receptor-mediated activation of phospholipase C in intact cells activates TRPC3 via diacylglycerol production, independently of G proteins, protein kinase C, or inositol 1,4,5-trisphosphate.     

Na-H exchange-dependent increase in intracellular pH times G2/M entry and transition

It is well established that activation of the Na-H exchanger NHE1 and increases in intracellular pH (pHi) are early and universal responses to mitogens and have permissive effects in promoting cell proliferation. Despite this evidence, a specific role for NHE1 or pHi in cell cycle progression remains undetermined. We now show that NHE1 activity and pHi regulate the timing of G2/M entry and transition. Prior to G2/M entry there is a rapid and transient increase in NHE1 activity and pHi, but in fibroblasts expressing a mutant NHE1 that lacks ion translocation activity, this increase in pHi is attenuated, S phase is delayed, and G2/M transition is impaired. In the absence of ion translocation by NHE1, expression of cyclin B1 and the kinase activity of Cdc2 are decreased and Wee1 kinase expression increases. Increasing pHi in the absence of NHE1 activity, however, is sufficient to restore Cdc2 activity and cyclin B1 expression and to promote G2/M entry and transition. These data indicate that a transient increase in pHi induced by NHE1 promotes the timing of G2/M, and they suggest that increases in pHi at the completion of S phase may constitute a previously unrecognized checkpoint for progression to G2 and mitosis.     

Inositol lipids and TRPC channel activation

The original hypothesis put forth by Bob Michell in his seminal 1975 review held that inositol lipid breakdown was involved in the activation of plasma membrane calcium channels or 'gates'. Subsequently, it was demonstrated that while the interposition of inositol lipid breakdown upstream of calcium signalling was correct, it was predominantly the release of Ca2+ that was activated, through the formation of Ins(1,4,5)P3. Ca2+ entry across the plasma membrane involved a secondary mechanism signalled in an unknown manner by depletion of intracellular Ca2+ stores. In recent years, however, additional non-store-operated mechanisms for Ca2+ entry have emerged. In many instances, these pathways involve homologues of the Drosophila trp (transient receptor potential) gene. In mammalian systems there are seven members of the TRP superfamily, designated TRPC1-TRPC7, which appear to be reasonably close structural and functional homologues of Drosophila TRP. Although these channels can sometimes function as store-operated channels, in the majority of instances they function as channels more directly linked to phospholipase C activity. Three members of this family, TRPC3, 6 and 7, are activated by the phosphoinositide breakdown product, diacylglycerol. Two others, TRPC4 and 5, are also activated as a consequence of phospholipase C activity, although the precise substrate or product molecules involved are still unclear. Thus the TRPCs represent a family of ion channels that are directly activated by inositol lipid breakdown, confirming Bob Michell's original prediction 30 years ago.