Actions of inositol phosphates on Ca2+ pools in guinea-pig hepatocytes.

In permeabilized hepatocytes, inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate and inositol 4,5-bisphosphate induced rapid release of Ca2+ from an ATP-dependent, non-mitochondrial vesicular pool, probably endoplasmic reticulum. The order of potency was inositol 1,4,5-trisphosphate greater than inositol 2,4,5-trisphosphate greater than inositol 4,5-bisphosphate. The Ca2+-releasing action of inositol 1,4,5-trisphosphate is not inhibited by high [Ca2+], nor is it dependent on [ATP] in the range of 50 microM-1.5 mM. These results suggest a role for inositol 1,4,5-trisphosphate as a second messenger in hormone-induced Ca2+ mobilisation, and that a specific receptor is involved in the Ca2+-release mechanism.     

Mitochondria suppress local feedback activation of inositol 1,4, 5-trisphosphate receptors by Ca2+.

The concerted action of inositol 1,4,5-trisphosphate (IP3) and Ca2+ on the IP3 receptor Ca2+ release channel (IP3R) is a fundamental step in the generation of cytosolic Ca2+ oscillations and waves, which underlie Ca2+ signaling in many cells. Mitochondria appear in close association with regions of endoplasmic reticulum (ER) enriched in IP3R and are particularly responsive to IP3-induced increases of cytosolic Ca2+ ([Ca2+]c). To determine whether feedback regulation of the IP3R by released Ca2+ is modulated by mitochondrial Ca2+ uptake, the interactions between ER and mitochondrial Ca2+ pools were examined by fluorescence imaging of compartmentalized Ca2+ indicators in permeabilized hepatocytes. IP3 decreased luminal ER Ca2+ ([Ca2+]ER), and this was paralleled by an increase in mitochondrial matrix Ca2+ ([Ca2+]m) and activation of Ca2+-sensitive mitochondrial metabolism. Remarkably, the decrease in [Ca2+]ER evoked by submaximal IP3 was enhanced when mitochondrial Ca2+ uptake was blocked with ruthenium red or uncoupler. Moreover, subcellular regions that were relatively deficient in mitochondria demonstrated greater sensitivity to IP3 than regions of the cell with a high density of mitochondria. These data demonstrate that Ca2+ uptake by the mitochondria suppresses the local positive feedback effects of Ca2+ on the IP3R, giving rise to subcellular heterogeneity in IP3 sensitivity and IP3R excitability. Thus, mitochondria can play an important role in setting the threshold for activation and establishing the subcellular pattern of IP3-dependent [Ca2+]c signaling.     

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.     

Free cytoplasmic Ca2+ concentration oscillations in thapsigargin-treated parotid acinar cells are caffeine- and ryanodine-sensitive.

The microsomal Ca-ATPase inhibitor thapsigargin induces in rat salivary acinar cells [Ca2+]i oscillations which, though similar to those activated by agonists, are independent of inositol phosphates or inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores (Foskett, J. K., Roifman, C., and Wong, D. (1991) J. Biol. Chem. 266, 2778-2782). To examine whether the oscillation mechanism resides in another, thapsigargin- and IP3-insensitive intracellular store, we examined the effects of caffeine and ryanodine, known modulators of Ca2+ release from sarcoplasmic reticulum in excitable cells. Oscillations were induced by caffeine (1-20 mM) in nonoscillating thapsigargin-treated acinar cells, which required the continued presence of caffeine, whereas caffeine was without effect or reduced oscillation amplitude in oscillating cells. Ryanodine (10-50 microM) inhibited oscillations in most of the cells. These results suggest that Ca2+ oscillations in parotid acinar cells are driven by periodic Ca2+ release from an IP3-insensitive Ca2+ store with properties similar to sarcoplasmic reticulum of excitable cells.     

The inositol 1,4,5-trisphosphate-forming agonist histamine activates a ryanodine-sensitive Ca2+ release mechanism in bovine adrenal chromaffin cells

The role of a Ca(2+)-induced Ca2+ release (CICR) mechanism in the generation of agonist-induced increases of intracellular free Ca2+ concentration ([Ca2+]i) was studied in bovine adrenal chromaffin cells. In single cells, repetitive stimulations with caffeine at 200-s intervals evoked reproducible spikes of [Ca2+]i. Ryanodine, an agent that interacts with the CICR channel of muscle, inhibited the caffeine-induced spikes of [Ca2+]i in a "use-dependent" way. High affinity binding sites for [3H]ryanodine (Kd 3.3 nM, Bmax 26 fmol/mg protein) were also detected in membranes from chromaffin cells, supporting the presence of a caffeine- and ryanodine-sensitive CICR channel. Pretreatment of single cells with caffeine + ryanodine to reduce the size of the caffeine-sensitive Ca2+ compartment inhibited a subsequent spike of [Ca2+]i evoked by histamine, a D-myo-inositol 1,4,5-trisphosphate-forming agonist. This demonstrates that a significant portion of the Ca2+ released by histamine comes from a caffeine- and ryanodine-sensitive pool. Ryanodine inhibited by 50% the size of [Ca2+]i spikes evoked by repetitive stimulation with histamine and did so in a use-dependent manner. These data suggest that, in addition to D-myoinositol 1,4,5-trisphosphate, activation of a caffeine- and ryanodine-sensitive CICR channel participates in the generation of histamine-induced release of intracellular Ca2+.     

Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells. Studies in aequorin-expressing intact and permeabilized ins-1 cells

The precise regulation of the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) is important for protein processing and signal transduction. In the pancreatic beta-cell, dysregulation of [Ca2+]er may cause impaired insulin secretion. The Ca2+-sensitive photoprotein aequorin mutated to lower its Ca2+ affinity was stably expressed in the endoplasmic reticulum (ER) of rat insulinoma INS-1 cells. The steady state [Ca2+]er was 267 +/- 9 microM. Both the Ca2+-ATPase inhibitor cyclopiazonic acid and 4-chloro-m-cresol, an activator of ryanodine receptors, caused an almost complete emptying of ER Ca2+. The inositol 1,4,5-trisphosphate generating agonists, carbachol, and ATP, reduced [Ca2+]er by 20-25%. Insulin secretagogues that raise cytosolic [Ca2+] by membrane depolarization increased [Ca2+]er in the potency order K+ > glucose > leucine, paralleling their actions in the cytosolic compartment. Glucose, which augmented [Ca2+]er by about 25%, potentiated the Ca2+-mobilizing effect of carbachol, explaining the corresponding observation in cytosolic [Ca2+]. The filling of ER Ca2+ by glucose is not directly mediated by ATP production as shown by the continuous monitoring of cytosolic ATP in luciferase expressing cells. Both glucose and K+ increase [Ca2+]er, but only the former generated whereas the latter consumed ATP. Nonetheless, drastic lowering of cellular ATP with a mitochondrial uncoupler resulted in a marked decrease in [Ca2+]er, emphasizing the requirement for mitochondrially derived ATP above a critical threshold concentration. Using alpha-toxin permeabilized cells in the presence of ATP, glucose 6-phosphate did not change [Ca2+]er, invalidating the hypothesis that glucose acts through this metabolite. Therefore, insulin secretagogues that primarily stimulate Ca2+ influx, elevate [Ca2+]er to ensure beta-cell homeostasis.     

Spontaneous [Ca2+]i fluctuations in rat chromaffin cells do not require inositol 1,4,5-trisphosphate elevations but are generated by a caffeine- and ryanodine-sensitive intracellular Ca2+ store

A considerable fraction (65%) of single rat chromaffin cells loaded with the fluorescent [Ca2+]i indicator fura-2 exhibited spontaneous rhythmic fluctuations with an average period of approximately 100 s. Parallel patch clamp experiments as well as fura-2 experiments carried out in Ca2(+)-free and other modified media in the presence of Ca2+ and Na+ channel blockers indicated an origin from intracellular stores. Appropriate concentrations of agonists (bradykinin and histamine) for receptors (B2 and H1) that trigger generation of inositol 1,4,5-trisphosphate induced increased fluctuation frequency, recruitment of silent cells, and large [Ca2+]i changes at high doses. These effects were blocked by cell pretreatment with neomycin, a drug that inhibits inositol 1,4,5-trisphosphate generation. In contrast, spontaneous fluctuations and the effects of another drug, caffeine, which also induced increased frequency and recruitment, were unaffected by neomycin. Ryanodine caused first a prolongation and then (approximately 10 min) a block of both spontaneous fluctuations and caffeine effects, where the single transients after bradykinin and histamine were maintained. Caffeine and ryanodine are known to affect selectively the process of calcium-induced Ca2+ release; this is the first demonstration of [Ca2+]i fluctuation activity arising from Ca2(+)-induced Ca2+ release in nonmuscle cells with no strict requirement for inositol 1,4,5-trisphosphate involvement.     

Effects of Ca2+ on phosphoinositide breakdown in exocrine pancreas.

Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.     

Minimal requirements for calcium oscillations driven by the IP3 receptor.

Hormones and neurotransmitters that act through inositol 1,4,5-trisphosphate (IP3) can induce oscillations of cytosolic Ca2+ ([Ca2+]c), which render dynamic regulation of intracellular targets. Imaging of fluorescent Ca2+ indicators located within intracellular Ca2+ stores was used to monitor IP3 receptor channel (IP3R) function and to demonstrate that IP3-dependent oscillations of Ca2+ release and re-uptake can be reproduced in single permeabilized hepatocytes. This system was used to define the minimum essential components of the oscillation mechanism. With IP3 clamped at a submaximal concentration, coordinated cycles of IP3R activation and subsequent inactivation were observed in each cell. Cycling between these states was dependent on feedback effects of released Ca2+ and the ensuing [Ca2+]c increase, but did not require Ca2+ re-accumulation. [Ca2+]c can act at distinct stimulatory and inhibitory sites on the IP3R, but whereas the Ca2+ release phase was driven by a Ca2+-induced increase in IP3 sensitivity, Ca2+ release could be terminated by intrinsic inactivation after IP3 bound to the Ca2+-sensitized IP3R without occupation of the inhibitory Ca2+-binding site. These findings were confirmed using Sr2+, which only interacts with the stimulatory site. Moreover, vasopressin induced Sr2+ oscillations in intact cells in which intracellular Ca2+ was completely replaced with Sr2+. Thus, [Ca2+]c oscillations can be driven by a coupled process of Ca2+-induced activation and obligatory intrinsic inactivation of the Ca2+-sensitized state of the IP3R, without a requirement for occupation of the inhibitory Ca2+-binding site.     

The sigma-2 receptor agonist PB28 inhibits calcium release from the endoplasmic reticulum of SK-N-SH neuroblastoma cells

In this paper we demonstrate that PB28 abolishes the Ca2+ release through the inositol 1,4,5-trisphosphate (InsP3) receptors and ryanodine receptors in SK-N-SH cells. Sigma receptors are divided into the subtypes sigma-1 and sigma-2, which are expressed in tumor cell lines and characterized by distinct pharmacological profiles. The sigma-1 receptor has been recently cloned, whereas the sigma-2 receptor is less well characterized. The endogenous ligand(s) of both subtypes remain unclear. In isolated guinea pig ileum, PB28 inhibits the contraction induced by carbachol dose dependently and in a non-competitive manner. In SK-N-SH cells PB28 challenge does not affect the intracellular Ca2+ concentration but incubation with PB28 for 45 min abolishes the cytosolic Ca2+ increases evoked by carbachol or histamine. This effect, not sensitive to cycloheximide, is caused by direct inhibition of the InsP3 receptors, since PB28 abolishes the response elicited by InsP3 administration in permeabilized SK-N-SH cells. Finally, incubation for 45 min with PB28 also abolishes the cytosolic Ca2+ increase evoked by caffeine.     

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+.     

Cyclic ADP-ribose regulation of ryanodine receptors involved in agonist evoked cytosolic Ca2+ oscillations in pancreatic acinar cells.

We have investigated the role of the ryanodine-sensitive intracellular Ca2+ release channel (ryanodine receptor) in the cytosolic Ca2+ oscillations evoked in pancreatic acinar cells by acetylcholine (ACh) or cholecystokinin (CCK). Ryanodine abolished or markedly inhibited the agonist evoked Ca2+ spiking, but enhanced the frequency of spikes evoked by direct internal inositol trisphosphate (InsP3) application. We have also investigated the possibility that cyclic ADP-ribose (cADP-ribose), the putative second messenger controlling the ryanodine receptor, plays a role in Ca2+ oscillations. We found that cADP-ribose could itself induce repetitive Ca2+ spikes localized in the secretory pole and that these spikes were blocked by ryanodine, but also by the InsP3 receptor antagonist heparin. Our results indicate that both the ryanodine and the InsP3 receptors are involved in Ca2+ spike generation.     

Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks

In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.     

Spatial and temporal organization of calcium signalling in hepatocytes.

Treatment of hepatocytes with agonists which act via the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), results in increases of cytosolic free Ca2+ [( Ca2+]i) which are manifest as a series of discrete [Ca2+]i transients or oscillations. With increasing agonist dose [Ca2+]i oscillation frequency increases and the initial latent period decreases, but the amplitude of the [Ca2+]i oscillations remains constant. Studies of these [Ca2+]i oscillations at the subcellular level have indicated that the [Ca2+]i changes do not occur synchronously throughout the cell, but initiate at a specific subcellular domain, adjacent to a region of the plasma membrane, and then propagate through the cell as a [Ca2+]i wave. For a given ceil, the locus of [Ca2+]i wave initiation is constant for every oscillation in a series and is also identical when the cell is sequentially stimulated with different agonists or when the phospholipase C-linked G protein is activated directly using AIF4-. The kinetics of the [Ca2+]i waves indicate that a Ca(2+)-activated mechanism is involved in propagating the oscillatory [Ca2+]i increases throughout the cell, and the data appear to be most consistent with a process of Ca(2+)-induced Ca2+ release. It is proposed that the ability to propagate [Ca2+]i oscillations into regions of the cell distal to the region in which the signal transduction apparatus is localized could serve an important function in allowing all parts of the cell to respond to the stimulus.     

Caspase-3-induced truncation of type 1 inositol trisphosphate receptor accelerates apoptotic cell death and induces inositol trisphosphate-independent calcium release during apoptosis

Inositol 1,4,5-trisphosphate receptor-deficient (IP3RKO) B-lymphocytes were used to investigate the functional relevance of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) and its cleavage by caspase-3 in apoptosis. We showed that inositol 1,4,5-trisphosphate receptor-deficient cells were largely resistant to apoptosis induced by both staurosporine (STS) and B-cell receptor (BCR) stimulation. Expression of either the wild-type IP3R1 or an N-terminal deletion mutant (Delta1-225) that lacks inositol 1,4,5-trisphosphate-induced Ca2+ release activity restored sensitivity to apoptosis and the consequent rise in free cytosolic Ca2+ concentration ([Ca2+]i). Expression of caspase-3-non-cleavable mutant receptor, however, dramatically slowed down the rate of apoptosis and prevented both Ca2+ overload and secondary necrosis. Conversely, expression of the "channel-only" domain of IP3R1, a fragment of the receptor generated by caspase-3 cleavage, strongly increased the propensity of the cells to undergo apoptosis. In agreement with these observations, caspase inhibitors impeded apoptosis and the associated rise in [Ca2+]i. Both the staurosporine- and B-cell receptor-induced apoptosis and increase in [Ca2+]i could be induced in nominally Ca2+-free and serum-free culture media, suggesting that the apoptosis-related rise in [Ca2+]i was primarily because of the release from internal stores rather than of influx through the plasma membrane. Altogether, our results suggest that IP3R1 plays a pivotal role in apoptosis and that the increase in [Ca2+]i during apoptosis is mainly the consequence of IP3R1 cleavage by caspase-3. These observations also indicate that expression of a functional IP3R1 per se is not enough to generate the significant levels of cytosolic Ca2+ needed for the rapid execution of apoptosis, but a prior activation of caspase-3 and the resulting truncation of the IP3R1 are required.     

Polyunsaturated free fatty acids stimulate an increase in cytosolic Ca2+ by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in T cells through a mechanism independent of phosphoinositide turnover

Polyunsaturated free fatty acids (PUFAs) of both w-3 and w-6 series, induce a rapid increase of cytosolic free Ca2+ concentration ([Ca2+]i) in a leukemic T-cell line (JURKAT), measured by the fluorescent indicator fura-2. The early increase in [Ca2+]i was transient, falling to a sustained level which returned to base line after 10-15 min. In Ca2+-free medium, PUFAs still caused an early increase in [Ca2+]i but rapidly returned to basal. Depletion of endoplasmic reticular Ca2+ pool by addition of OKT3 (antibodies to CD3 of the T3-antigen receptor complex) to JURKAT cells (in Ca2+-free medium) abolished the PUFAs-mediated [Ca2+]i increase and vice versa. By using saponin-permeabilized JURKAT cells, the intracellular free Ca2+ released by PUFAs was found to be the non-mitochondrial, ATP-dependent sequestered Ca2+ pool which is sensitive to inositol 1,4,5-trisphosphate. However, PUFAs do not induce any apparent increase in inositol phosphates in JURKAT cells. No Ca2+ influx was detected in JURKAT cells when stimulated with PUFAs. A correlation was observed between both the carbon chain length and the number of double bonds with the ability to mobilize cytosolic free [Ca2+]i in the w-3 PUFAs. These results demonstrate that PUFAs stimulate the release of Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in the endoplasmic reticulum of JURKAT cells via a mechanism independent of inositol lipid hydrolysis.     

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.     

A single-pool model for intracellular calcium oscillations and waves in the Xenopus laevis oocyte.

We construct a minimal model of cytosolic free Ca2+ oscillations based on Ca2+ release via the inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channel (IP3R) of a single intracellular Ca2+ pool. The model relies on experimental evidence that the cytosolic free calcium concentration ([Ca2+]c) modulates the IP3R in a biphasic manner, with Ca2+ release inhibited by low and high [Ca2+]c and facilitated by intermediate [Ca2+]c, and that channel inactivation occurs on a slower time scale than activation. The model produces [Ca2+]c oscillations at constant [IP3] and reproduces a number of crucial experiments. The two-dimensional spatial model with IP3 dynamics, cytosolic diffusion of IP3 (Dp = 300 microns 2 s-1), and cytosolic diffusion of Ca2+ (Dc = 20 microns 2 s-1) produces circular, planar, and spiral waves of Ca2+ with speeds of 7-15 microns.s-1, which annihilate upon collision. Increasing extracellular [Ca2+] influx increases wave speed and baseline [Ca2+]c. A [Ca2+]c-dependent Ca2+ diffusion coefficient does not alter the qualitative behavior of the model. An important model prediction is that channel inactivation must occur on a slower time scale than activation in order for waves to propagate. The model serves to capture the essential macroscopic mechanisms that are involved in the production of intracellular Ca2+ oscillations and traveling waves in the Xenopus laevis oocyte.     

Isomers of inositol trisphosphate in exocrine pancreas.

In rat pancreatic acinar cells, the Ca2+-mobilizing receptor-agonist, caerulein, at both maximal and submaximal concentrations, stimulated a rapid, transient, increase in [3H]inositol 1,4,5-trisphosphate [(1,4,5)IP3], followed by a slower, sustained, increase in [3H]inositol 1,3,4-trisphosphate [(1,3,4)IP3]. Neither activation of protein kinase C by phorbol dibutyrate nor prevention of the caerulein-stimulated elevation of cytosolic [Ca2+] significantly affected the pattern of formation of the two isomers of IP3. Although carbachol evoked an increase in cytosolic [Ca2+], it did not significantly stimulate [3H](1,4,5)IP3 accumulation, but did promote [3H](1,3,4)IP3 accumulation. Moreover, both carbachol and caerulein maintained hormone-sensitive intracellular Ca2+ pools in a Ca2+-depleted state after [3H](1,4,5)IP3 had returned to basal concentrations. One interpretation of these findings is that total cellular concentrations of [3H](1,4,5)IP3 may not accurately reflect the concentration of this putative mediator in biologically relevant compartments.     

Ryanodine receptor adaptation and Ca2+(-)induced Ca2+ release-dependent Ca2+ oscillations.

A simplified mechanism that mimics "adaptation" of the ryanodine receptor (RyR) has been developed and its significance for Ca2+(-)induced Ca2+ release and Ca2+ oscillations investigated. For parameters that reproduce experimental data for the RyR from cardiac cells, adaptation of the RyR in combination with sarco/endoplasmic reticulum Ca2+ ATPase Ca2+ pumps in the internal stores can give rise to either low [Cai2+] steady states or Ca2+ oscillations coexisting with unphysiologically high [Cai2+] steady states. In this closed-cell-type model rapid, adaptation-dependent Ca2+ oscillations occur only in limited ranges of parameters. In the presence of Ca2+ influx and efflux from outside the cell (open-cell model) Ca2+ oscillations occur for a wide range of physiological parameter values and have a period that is determined by the rate of Ca2+ refilling of the stores. Although the rate of adaptation of the RyR has a role in determining the shape and the period of the Ca2+ spike, it is not essential for their existence. This is in marked contrast with what is observed for the inositol 1,4,5-trisphosphate receptor for which the biphasic activation and inhibition of its activity by Ca2+ are sufficient to produce oscillations. Results for this model are compared with those based on Ca2+(-)induced Ca2+ release alone in the bullfrog sympathetic neuron. This kinetic model should be suitable for analyzing phenomena associated with "Ca2+ sparks," including their merger into Ca2+ waves in cardiac myocytes.