Hormone-evoked calcium release from intracellular stores is a quantal process

Ca2+ mobilization by hormones, ionomycin, and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) were studied to determine whether Ca2+ release is a continuous or a quantal process. Hormone-mediated Ca2+ release occurs only during the first 2-4 s of stimulation. Stimulation of acini with a maximal hormone concentration following stimulation with a submaximal concentration resulted in free cytosolic Ca2+ concentration ([Ca2+]i) increase and 45Ca efflux. The peak [Ca2+]i increase induced by a maximal concentration of agonist was nearly constant when cells were prestimulated with a submaximal dose for 1-15 min. Submaximal hormone concentrations release only a fraction of intracellular 45Ca2+, after which intracellular Ca2+ content remains constant. The partially released stores remain depleted until cell stimulation is terminated, at which time the stores reload with Ca2+. For comparison, increasing concentrations of ionomycin resulted in increasing rates of Ca2+ release. Each ionomycin concentration released all the Ca2+ from intracellular stores. We therefore conclude that hormone-evoked Ca2+ release is a quantal rather than a continuous process. In permeabilized cells, increasing concentrations of Ins-1,4,5-P3 resulted in an increased fraction of Ca2+ release. No submaximal Ins-1,4,5-P3 concentration was capable of releasing all the Ins-1,4,5-P3-mobilizable Ca2+. Therefore, it appears that the quantal properties of hormone-evoked Ca2+ release reflect the quantal properties of Ins-1,4,5-P3-mediated Ca2+ release from intracellular stores.     

Inositol trisphosphate does not release Ca2+ from permeabilized cardiac myocytes and sarcoplasmic reticulum

The possibility that inositol 1,4,5-trisphosphate (IP3) may act as a Ca2+-mobilizing second messenger in cardiac muscle in a manner analogous to its actions in other cell types has been examined using saponin-permeabilized myocytes and isolated cardiac sarcoplasmic reticulum. Myocytes permeabilized in the presence of MgATP2- sequestered Ca2+ to a level of about 200 nM, similar to the cytosolic free Ca2+ concentration of intact cells, but addition of IP3 was ineffective in causing Ca2+ release from intracellular stores. Similarly, IP3 (up to 50 microM) was unable to inhibit Ca2+ uptake or cause Ca2+ release from isolated canine cardiac sarcoplasmic reticulum vesicles in the presence of either EGTA or sodium vanadate. These results indicate that IP3 is unlikely to mediate mobilization of intracellular Ca2+ stores in myocardial cells.     

Temporal relationship between inositol polyphosphate formation and increases in cytosolic Ca2+ in quiescent 3T3 cells stimulated by platelet-derived growth factor, bombesin and vasopressin.

We determined the temporal relationship between the formation of inositol phosphates and increase in cytosolic [Ca2+] elicited by bombesin, vasopressin and platelet-derived growth factor (PDGF) in quiescent Swiss 3T3 cells. These responses were measured under identical conditions. Bombesin caused a rapid increase in inositol 1,4,5-trisphosphate which coincided with the increase in cytosolic [Ca2+]. This was followed by a slower but marked increase in inositol 1,3,4-trisphosphate and inositol-bisphosphate. Vasopressin elicited a similar sequence of events. In sharp contrast, highly purified porcine PDGF induced increases in cytosolic [Ca2+] and inositol 1,4,5-trisphosphate that were temporally uncoupled: detectable inositol polyphosphate formation occurred after Ca2+ mobilization from intracellular stores. The same temporal dissociation was observed when a recombinant v-sis product was used instead of porcine PDGF. However, PDGF was as effective as bombesin in stimulating the formation of inositol phosphates after 5-10 min of incubation. The data suggest that PDGF increases cytosolic [Ca2+] via a different signal transduction pathway from that utilized by bombesin and vasopressin. These findings have important implications for understanding the signal transduction pathway activated by PDGF.     

The effects of bombesin on polyphosphoinositide and calcium metabolism in Swiss 3T3 cells

Bombesin, a peptide mitogen for a variety of cell types, acts as a typical Ca2+-mobilizing hormone in Swiss 3T3 fibroblasts. At its mitogenic concentrations (1-25 nM), bombesin stimulates polyphosphoinositide turnover, i.e. breakdown of phosphatidylinositol 4,5-bisphosphate and a concomitant increase in inositol phosphates in a time- and dose-dependent manner. In particular, bombesin induces an initial transient increase in inositol 1,4,5-trisphosphate concentration, followed by an increase in the concentration of inositol 1,3,4-trisphosphate. Also, within 30 s of bombesin addition, the mass of 1,2-diacylglycerol nearly doubles and remains at this level for up to 60 min. Intracellular [Ca2+] measurements with a photoprotein, aequorin, demonstrate that bombesin stimulates a transient rise in cytosolic free Ca2+ concentration. A mobilization of Ca2+ from an intracellular pool is observed as a dose-dependent, transient increase in 45Ca2+ efflux from prelabeled cells, both in the presence and absence of extracellular Ca2+. Bombesin also induces a sustained increase in Ca2+ influx rate and stimulates 3-O-methyl-D-glucose transport across the plasma membrane. These composite results indicate that the mitogenic effect of bombesin is mediated through an activation of the Ca2+ messenger system.     

Role of free cytosolic calcium in secretagogue-stimulated amylase release from dispersed acini from guinea pig pancreas

To determine the role of free cytosolic calcium ([Ca+2]i) in stimulated enzyme secretion from exocrine pancreas, we determined the effects of various pancreatic secretagogues on [Ca+2]i and amylase release in dispersed acini from the guinea pig pancreas. Cholecystokinin-octapeptide (CCK-OP), carbachol, and bombesin, but not vasoactive intestinal peptide, stimulated rapid increases in [Ca+2]i from 100 to 600-800 nM that were independent of extracellular calcium. The increases in [Ca+2]i were transient (lasting less than 5 min) and correlated with an initial rapid phase of amylase release. After 5 min, secretagogue-stimulated amylase release occurred at basal [Ca+2]i. Carbachol pretreatment of the acini abolished the effects of CCK-OP and bombesin on [Ca+2]i and the initial rapid phase of amylase release. 4 beta-phorbol 12-myristate 13-acetate (PMA) had no effect on [Ca+2]i but stimulated an increase in amylase release. The addition of CCK-OP or A23187 to PMA-stimulated acini caused an increase in [Ca+2]i and PMA-stimulated amylase release only during the first 5 min after addition of these agents. These results indicate that CCK-OP, carbachol, and bombesin release calcium from an intracellular pool, resulting in a transient increase in [Ca+2]i and that this increase in [Ca+2]i mediates enzyme secretion during the first few minutes of incubation. The results with PMA suggest that secretagogue-stimulated secretion not mediated by increased [Ca+2]i (sustained secretion) is mediated by 1,2-diacylglycerol.     

Thapsigargin defines the roles of cellular calcium in secretagogue-stimulated enzyme secretion from pancreatic acini.

In the present study we used thapsigargin (TG), an inhibitor of microsomal calcium ATPase, to evaluate the roles of free cytoplasmic calcium and intracellular stored calcium in secretagogue-stimulated enzyme secretion from rat pancreatic acini. Using microspectrofluorimetry of fura-2-loaded pancreatic acini, we found that TG caused a sustained increase in free cytoplasmic calcium by mobilizing calcium from inositol 1,4,5-trisphosphate-sensitive intracellular stores and by increasing influx of extracellular calcium. TG also caused a small increase in basal amylase secretion, inhibited the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate, and potentiated the stimulation of amylase secretion caused by 12-O-tetradecanoylphorbol-13-acetate or secretagogues that increase cyclic adenosine 3',5'-monophosphate. Bombesin, which like TG increased free cytoplasmic calcium, also potentiated the stimulation of amylase secretion caused by secretagogues that increase cyclic adenosine 3',5'-monophosphate, but did not inhibit the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate. Finally, TG inhibited the sustained phase of cholecystokinin-stimulated amylase secretion and potentiated the time course of vasoactive intestinal peptide-stimulated amylase secretion. The present findings indicate that stimulation of amylase secretion by secretagogues that increase inositol 1,4,5-trisphosphate does not depend on increased free cytoplasmic calcium per se. In contrast, TG-induced potentiation of the stimulation of secretagogues that increase cellular cyclic adenosine 3',5'-monophosphate appears to result from increased free cytoplasmic calcium per se.     

Luminal Ca2+ controls the activation of the inositol 1,4,5-trisphosphate receptor by cytosolic Ca2+.

Luminal Ca2+ controls the sensitivity of the intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Ins(1,4,5)P3-induced Ca2+ release is also controlled by cytosolic Ca2+; low concentrations of Ca2+ stimulate the release. The aim of this work was to investigate whether luminal Ca2+ would affect the stimulation of the Ins(1,4,5)P3 receptor by cytosolic Ca2+ in permeabilized A7r5 smooth muscle cells. We also report that the Ins(1,4,5)P3 receptor in A7r5 cells is activated by low concentrations of cytosolic Ca2+. Cytoplasmic Ca2+ increases the Ins(1,4,5)P3 sensitivity without affecting the cooperativity. The increase in Ins(1,4,5)P3 sensitivity becomes relatively more pronounced when the Ca2+ content of the stores decreases. This modulatory effect of luminal Ca2+ on the responsiveness to cytosolic Ca2+ is an intrinsic property of the Ins(1,4,5)P3 receptor.     

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 1,2-cyclic 4,5-trisphosphate is an order of magnitude less potent than inositol 1,4,5-trisphosphate in mobilizing intracellular stores of calcium in mouse pancreatic acinar cells

Semi-synthetic inositol 1,2-cyclic 4,5-trisphosphate is 1/16th as potent as inositol 1,4,5-trisphosphate in releasing Ca2+ from intracellular stores in permeabilized mouse pancreatic acinar cells. Competitive displacement studies in mouse pancreatic microsomes show that the affinity of inositol 1,2-cyclic 4,5-trisphosphate is 1/20th of that of inositol 1,4,5-trisphosphate at the latter's receptor, indicating that the lower potency of inositol 1,2-cyclic 4,5-trisphosphate in releasing Ca2+ can be accounted for by a weaker affinity at the receptor. These results suggest that inositol 1,2-cyclic 4,5-trisphosphate is unlikely to play any significant role in Ca2+ mobilization, at least in mouse pancreatic acinar cells.     

Measurement of cytosolic free Ca2+ in individual pancreatic acini

The kinetics of changes in cytosolic free Ca2+ ([Ca2+]i) were determined in individual rat pancreatic acini by microfluorimetry. Three major findings are reported. First, at maximal stimulatory concentrations for amylase release, both caerulein and bombesin induced an initial rise in [Ca2+]i followed by prolonged secondary oscillations of smaller amplitude. The latter effect was not observed with supramaximal doses of caerulein. Second, these cyclic changes were dependent, at least in part, on extracellular Ca2+. Finally, comparison of the threshold doses for [Ca2+]i mobilization and enzyme discharge demonstrated that pathways independent of an elevation of [Ca2+]i control the secretory activity of pancreatic acini at low, picomolar agonist concentrations.     

How do inositol phosphates regulate calcium signaling?

Activation of a variety of cell surface receptors results in the phospholipase C-catalyzed hydrolysis of the minor plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate, with concomitant formation of inositol 1,4,5-trisphosphate and diacylglycerol. There is strong evidence that inositol 1,4,5-trisphosphate stimulates Ca2+ release from intracellular stores. The Ca2+-releasing actions of inositol 1,4,5-trisphosphate are terminated by its metabolism through two distinct pathways. Inositol 1,4,5-trisphosphate is dephosphorylated by a 5-phosphatase to inositol 1,4-bisphosphate; alternatively, inositol 1,4,5-trisphosphate can also be phosphorylated to inositol 1,3,4,5-tetrakisphosphate by a 3-kinase. Although the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known; the proposal that inositol 1,4,5-trisphosphate secondarily elicits Ca2+ entry by emptying an intracellular Ca2+ pool is considered.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.     

Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes

To investigate the mechanisms by which inositol phosphates regulate cytosolic free Ca2+ concentration ([Ca2+]c), we injected Xenopus oocytes with inositol phosphates and measured Ca2+-activated Cl- currents as an assay of [Ca2+]c. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) injection (0.1-10.0 pmol) induced an initial transient Cl- current (I1) followed by a second more prolonged Cl- current (I2). Both currents were Ca2+-dependent, but the source of Ca2+ was different. Release of intracellular Ca2+ stores produced I1, whereas influx of extracellular Ca2+ produced I2; Ca2+-free bathing media and inorganic calcium channel blockers (Mn2+, Co2+) did not alter I1 but completely and reversibly inhibited I2. Injection of the Ins(1,4,5)P3 metabolite, inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (0.2-10.0 pmol) generated a Ca2+-dependent Cl- current with superimposed current oscillations that resulted from release of intracellular Ca2+, not Ca2+ influx. Injection of the Ins(1,3,4,5)P4 metabolite, inositol 1,3,4-trisphosphate (10.0 pmol), or the synthetic inositol trisphosphate isomer, inositol 2,4,5-trisphosphate (1.0-10.0 pmol), mimicked the effect of Ins(1,4,5)P3, stimulating an I1 resulting from release of intracellular Ca2+ and an I2 resulting from influx of extracellular Ca2+. The results indicate that several inositol trisphosphate isomers stimulate both release of intracellular Ca2+ and influx of extracellular Ca2+. Ins(1,3,4,5)P4 also stimulated release of intracellular Ca2+, but it was neither sufficient nor required for Ca2+ influx.     

Mechanism of Ca2+ wave propagation in pancreatic acinar cells.

An increase in cytosolic Ca2+ often begins as a Ca2+ wave, and this wave is thought to result from sequential activation of Ca(2+)-sensitive Ca2+ stores across the cell. We tested that hypothesis in pancreatic acinar cells, and since Ca2+ waves may regulate acinar Cl- secretion, we examined whether such waves also are important for amylase secretion. Ca2+ wave speed and direction was determined in individual cells within rat pancreatic acini using confocal line scanning microscopy. Both acetylcholine (ACh) and cholecystokinin-8 induced rapid Ca2+ waves which usually travelled in an apical-to-basal direction. Both caffeine and ryanodine, at concentrations that inhibit Ca(2+)-induced Ca2+ release (CICR), markedly slowed the speed of these waves. Amylase secretion was increased over 3-fold in response to ACh stimulation, and this increase was preserved in the presence of ryanodine. These results indicate that 1) stimulation of either muscarinic or cholecystokinin-8 receptors induces apical-to-basal Ca2+ waves in pancreatic acinar cells, 2) the speed of such waves is dependent upon mobilization of caffeine- and ryanodine-sensitive Ca2+ stores, and 3) ACh-induced amylase secretion is not inhibited by ryanodine. These observations provide direct evidence that Ca(2+)-induced Ca2+ release is important for propagation of cytosolic Ca2+ waves in pancreatic acinar cells.     

The digitonin-permeabilized pancreatic islet model. Effect of myo-inositol 1,4,5-trisphosphate on Ca2+ mobilization.

Glucose-induced insulin secretion is thought to be mediated by submicromolar increases in intracellular Ca2+, although the intracellular processes are not well understood. We have used the previously characterized digitonin-permeabilized insulin-secreting pancreatic islet model to study the role of myo-inositol 1,4,5-trisphosphate (IP3), a putative second messenger for mobilization of intracellular Ca2+. Ca2+ efflux from the endoplasmic reticulum was studied with or without vanadate present to inhibit Ca2+ reuptake. IP3 (10 microM), at a free Ca2+ level of 0.06 microM, increased Ca2+ release by 30% and, when vanadate was present, by 50%. Maximal and half-maximal Ca2+ release was observed at 10 microM- and 2.5 microM-IP3, respectively. IP3 provoked a rapid release that was followed by slow reuptake. Reuptake was diminished in the presence of vanadate. Inositol 1,4-bisphosphate, inositol 1-phosphate and other phosphoinositide metabolites did not have any significant effect. Because increases in Ca2+ levels in the submicromolar range have been previously shown to induce insulin release in digitonin-permeabilized islets, our results are consistent with the concept of IP3 serving as a second messenger for insulin secretion.     

Factors regulating amylase secretion from chicken pancreatic acini in vitro

In mammals, cholecystokinin regulates pancreatic exocrine secretion under physiological conditions. We have shown, however, that cholecystokinin at physiological concentrations does not induce pancreatic amylase secretion in birds. Therefore, we investigated the effects of various neurotransmitters and gut hormones on the pancreatic amylase secretory response in isolated chicken pancreatic acini. Acetylcholine (half-maximal stimulation at 800 nM) and vasoactive intestinal polypeptide (half-maximal stimulation at 40 pM) produced a concentration-dependent increase in amylase secretion at physiological concentrations. The combination of acetylcholine and vasoactive intestinal polypeptide produced an additive response in amylase secretion. Sodium nitroprusside, a spontaneous nitric oxide releaser, and bombesin, induced amylase secretion at concentrations greater than 10 nM and 100 nM, respectively. Gastrin and secretin increased amylase secretion at pharmacological concentrations (10 to 100 nM). Our findings suggest that neural regulation is important for pancreatic enzyme secretion in birds and the contribution of gut hormones seems to be physiologically unimportant.     

Basic fibroblast growth factor is a calcium-mobilizing secretagogue in rat pancreatic acini.

Basic fibroblast growth factor (bFGF) induced a marked increase in the levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and a rapid rise in cytosolic free calcium [Ca2+]i levels in rat pancreatic acini. The bFGF-mediated calcium transient was not dependent on the presence of extracellular calcium, and was abolished by pretreatment of acini with carbachol. bFGF stimulated amylase release in pancreatic acini in a monophasic, dose-dependent manner, and this effect was blocked by neutralizing anti-bFGF antibodies. At much higher concentrations, epidermal growth factor (EGF), but not insulin-like growth factor-I (IGF-I), partially mimicked some of the actions of bFGF. These findings suggest that bFGF is a previously unrecognized calcium-mobilizing pancreatic secretagogue that may participate in the regulation of pancreatic exocrine function.     

Second messenger function of inositol 1,4,5-trisphosphate. Early changes in inositol phosphates, cytosolic Ca2+, and insulin release in carbamylcholine-stimulated RINm5F cells

The second messenger function of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) was investigated in carbamylcholine-stimulated RINm5F cells by analysis of the early changes in inositol phosphates, cytosolic free Ca2+ concentration ([Ca2+]i), and insulin secretion. After a lag of 2 s, [Ca2+]i rose to a peak at 13 +/- 2 s, a response which was due mainly to mobilization from intracellular stores since it persisted even in the absence of extracellular Ca2+. The Ca2+ response had already declined toward prestimulatory levels by the time insulin secretion reached its maximal rate (2-3 min). Although the rises in inositol trisphosphate preceded those of both inositol bisphosphate and monophosphate, all three attained maximal concentrations after 1 min and remained elevated for at least 10 min. The accumulation of inositol trisphosphate was truly Ca2+-independent since it persisted under conditions in which the rise in [Ca2+]i was abolished by prior depletion of intracellular Ca2+ pools. Further analysis by high performance liquid chromatography revealed the presence of the two isomers, Ins-1,4,5-P3 and Ins-1,3,4-P3 in stimulated cells. The latter was virtually absent under nonstimulatory conditions but started to accumulate after a 5-s lag and reached maximal levels after 30 s of stimulation. Ins-1,4,5-P3 doubled within 1 s of carbamylcholine addition, reached a peak after 5 s, and, although declining thereafter, remained slightly elevated for at least 3 min. Hence, both the onset and peak of the rise of Ins-1,4,5-P3 preceded that of [Ca2+]i, which in turn preceded the peak in insulin release. These results strongly suggest that Ins-1,4,5-P3 acts as the second messenger by which carbamylcholine mobilizes intracellular Ca2+ during the initiation of insulin release.     

Calcium regulates inositol 1,3,4,5-tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A.

Lysed mouse thymocytes release [3H]inositol 1,4,5 trisphosphate from [3H]inositol-labelled phosphatidyl inositol 4,5-bisphosphate in response to GTP gamma S, and rapidly phosphorylate [3H]inositol 1,4,5-trisphosphate to [3H]inositol 1,3,4,5-tetrakisphosphate. The rate of phosphorylation is increased approximately 7-fold when the free [Ca2+] in the lysate is increased from 0.1 to 1 microM, the range in which the cytosolic free [Ca2+] increases in intact thymocytes in response to the mitogen concanavalin A. Stimulation of the intact cells with concanavalin A also results in a rapid and sustained increase in the amount of inositol 1,3,4,5-tetrakisphosphate, and a much smaller transient increase in 1,4,5-trisphosphate. Lowering [Ca2+] in the medium from 0.4 mM to 0.1 microM before addition of concanavalin A reduces accumulation of inositol 1,3,4,5-tetrakisphosphate by at least 3-fold whereas the increase in inositol 1,4,5-trisphosphate is sustained rather than transient. The data imply that in normal medium the activity of the inositol 1,4,5-trisphosphate kinase increases substantially in response to the rise in cytosolic free [Ca2+] generated by concanavalin A, accounting for both the transient accumulation of inositol 1,4,5-trisphosphate and the sustained high levels of inositol 1,3,4,5-tetrakisphosphate. Inositol 1,3,4,5-tetrakisphosphate is a strong candidate for the second messenger for Ca2+ entry across the plasma membrane. This would imply that the inositol polyphosphates regulate both Ca2+ entry and intracellular Ca2+ release, with feedback control of the inositol polyphosphate levels by Ca2+.