Glucose 6-phosphate regulates Ca2+ steady state in endoplasmic reticulum of islets. A possible link in glucose-induced insulin secretion

Glucose stimulation of islets is coupled with the rapid intracellular release of myo-inositol 1,4,5-trisphosphate (IP3) and arachidonic acid which in turn mobilize Ca2+ stored in the endoplasmic reticulum (ER). The metabolism of glucose is required for insulin secretion although the link between glucose metabolism and the cellular events resulting in insulin release is unknown. In digitonin-permeabilized islets, glucose 6-phosphate (0.5-4 mM) increased significantly the ATP-dependent Ca2+ content of the ER at a free Ca2+ concentration of 1 microM. At 0.2 microM free Ca2+, glucose 6-phosphate (2-10 mM) had a smaller effect. Glucose, phosphate, mannose 6-phosphate, and fructose 1,6-diphosphate had no effect on the ATP-dependent Ca2+ content of the ER. Glucose 1-phosphate and fructose 6-phosphate also increased ATP-dependent Ca2+ content of the ER, presumably due to conversion to glucose 6-phosphate by islet phosphoglucomutase and phosphoglucoisomerase, respectively. The glucose 6-phosphate increase in the ATP-dependent Ca2+ content of the ER was shown to be mediated by glucose 6-phosphatase localized to the ER. Both arachidonic acid (10 microM) and the Ca2+ ionophore A23187 (2 microM) mobilized Ca2+ stored in the ER by glucose 6-phosphate. However, IP3-induced (10 microM) Ca2+ release from the ER was abolished in the presence of glucose 6-phosphate (0.5-10 mM). We propose that glucose 6-phosphate could provide a regulatory link between glucose metabolism and intracellular Ca2+ regulation by augmenting Ca2+ sequestered in the ER as well as attenuating IP3-induced Ca2+ release. Thus, glucose 6-phosphate would serve as an "off" signal leading to a decrease in intracellular Ca2+ when both the free Ca2+ and glucose 6-phosphate concentrations have increased following glucose stimulus.     

Calmodulin inhibits inositol trisphosphate-induced Ca2+ mobilization from the endoplasmic reticulum of islets

IP3-induced Ca2+ release from the endoplasmic reticulum (ER) of islets is believed to be a key intracellular event in glucose-induced insulin secretion. Calmodulin was shown to increase ATP-dependent Ca2+ steady-state and inhibit by 57.2% IP3-induced Ca2+ mobilization from the ER. Conversely, the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide (W-7), induced Ca2+ release from the ER. The combination of W-7 (100 microM) and IP3 (10 microM), resulted in a greater release of Ca2+ from the ER than either W-7 or IP3 alone. W-7 was shown not to affect the structural integrity of the ER. Our results suggest that IP3-induced Ca2+ release from the ER is regulated by a calmodulin-dependent process.     

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.     

Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion

Free fatty acids in isolated pancreatic islets have been quantified by gas chromatography-mass spectrometry after stimulation with insulin secretagogues. The fuel secretagogue D-glucose has been found to induce little change in islet palmitate levels but does induce the accumulation of sufficient unesterified arachidonate by mass to achieve an increment in cellular levels of 38-75 microM. Little of this free arachidonate is released into the perifusion medium, and most remains associated with the islets. Glucose-induced hydrolysis of arachidonate from islet cell phospholipids is reflected by release of the arachidonate metabolite prostaglandin E2 (PGE2) from perifused islets. Both the depolarizing insulin secretagogue tolbutamide (which is thought to act by inducing closure of beta-cell ATP-sensitive K+ channels and the influx of extracellular Ca2+ through voltage-dependent channels) and the calcium ionophore A23187 have also been found to induce free arachidonate accumulation within and PGE2 release from islets. Surprisingly, a major fraction of glucose-induced eicosanoid release was found not to require Ca2+ influx and occurred even in Ca(2+)-free medium, in the presence of the Ca(2+)-chelating agent EGTA, and in the presence of the Ca2+ channel blockers verapamil and nifedipine. Exogenous arachidonic acid was found to amplify the insulin secretory response of perifused islets to submaximally depolarizing concentrations of KCl, and the maximally effective concentration of arachidonate was 30-40 microM. These observations suggest that glucose-induced phospholipid hydrolysis and free arachidonate accumulation in pancreatic islets are not simply epiphenomena associated with Ca2+ influx and that arachidonate accumulation may play a role in the signaling process which leads to insulin secretion.     

GTP mobilization of Ca2+ from the endoplasmic reticulum of islets. Comparison with myo-inositol 1,4,5-trisphosphate.

The effect of the guanine nucleotide GTP on Ca2+ release from the endoplasmic reticulum of digitonin-permeabilized islets was investigated. maximal and half-maximal Ca2+ release were observed at 5 microM- and 2.5 microM-GTP respectively. GTP caused a rapid release of Ca2+ from the endoplasmic reticulum, which was complete within 1 min. GTP-induced Ca2+ release was structurally specific and required the hydrolysis of GTP. The combination of maximal concentrations of GTP (10 microM) and myo-inositol 1,4,5-trisphosphate (IP3) (10 microM) resulted in an additive effect on Ca2+ release from the endoplasmic reticulum. GDP (100 microM), which inhibits GTP-induced Ca2+ release, did not affect IP3-induced Ca2+ release. Furthermore, GTP-induced Ca2+ release was not independent on submicromolar free Ca2+ concentrations, unlike IP3-induced Ca2+ release. These observations suggest that mechanistically GTP-induced Ca2+ release is different from IP3-induced Ca2+ release from the endoplasmic reticulum.     

Effect of lysophospholipids, arachidonic acid and other fatty acids on regulation of Ca2+ transport in permeabilized pancreatic islets.

The immediate reaction products of PLA2-mediated hydrolysis of phospholipids were tested for their ability to induce Ca2+ mobilization from internal stores in permeabilized ob/ob mouse pancreatic islets. Lysophospholipids and unsaturated fatty acids increased the free Ca2+ concentration in the incubation medium of permeabilized ob/ob mouse pancreatic islets. The potency of the lysophospholipids decreased in the following order: lysophosphatidylcholine = lysophosphatidylglycerol much greater than lysophosphatidylinositol greater than lysophosphatidylserine much greater than lysophosphatidylethanolamine. Arachidonic acid and palmitoleic acid had a potency comparable to lysophosphatidylinositol, while palmitic acid was ineffective. The Ca(2+)-mobilizing effect of inositol-1,4,5-trisphosphate (IP3) in permeabilized islet cells was additive to the lysophospholipid effect, indicating different sites of action. Both Ca(2+)-mobilizing effects were counteracted by the polyamine spermine, while the presence of Mg2+ shifted the Ca2+ concentrations to higher levels. Since not only an activation of a phospholipase C but also an activation of a phospholipase A2 with subsequent generation of lysophospholipids and free fatty acids is reported to occur in glucose-induced insulin secretion, the interaction of the phospholipase C reaction product IP3 with a lysophospholipid or an unsaturated fatty acid may affect the extent and duration of the rise in the free cytoplasmic Ca2+ concentration responsible for initiation of insulin secretion.     

Evidence of Ca2+ mobilizing action of arachidonic acid in human platelets

The addition of arachidonic acid induced a rapid release of 45Ca2+ from human platelet membrane vesicles which accumulated 45Ca2+ in the presence of ATP. Docosahexaenoic acid, eicosapentaenoic acid, linolenic acid and linoleic acid were less active than arachidonic acid. In contrast, oleic acid, myristic acid and palmitic acid were without effect. The thromboxane A2 analogue induced no 45Ca2+ release. The cyclooxygenase/lipoxygenase inhibitor failed to suppress arachidonic acid-induced 45Ca2+ release at the concentration which inhibited the production of lipid peroxides. These data indicate that the activity of arachidonic acid may be due to fatty acid itself and not to its metabolites. The combination of arachidonic acid and inositol 1,4,5-trisphosphate (IP3) resulted in a greater 45Ca2+ release from platelet membrane vesicles than either compound alone. When the intracellular free Ca2+ concentration ([Ca2+]i) was measured using fura-2, the thrombin-induced [Ca2+]i increase was reduced in platelets which had been treated with a phospholipase A2 inhibitor, ONO-RS-082 (2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid). These results provide evidence that arachidonic acid alone may cause Ca2+ increase and also may induce an additional Ca2+ mobilization to IP3-induced Ca2+ release in human platelets.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Studies of the Ca2+ requirements for glucose- and carbachol-induced augmentation of inositol trisphosphate and inositol tetrakisphosphate accumulation in digitonin-permeabilized islets. Evidence for a glucose recognition site in insulin secretion

The metabolism of D-glucose is believed to initiate and regulate insulin secretion by islet beta-cells, although the identity of the metabolite which couples glucose metabolism to the cellular events involved in insulin secretion is unknown. An alternative hypothesis involves the presence of a glucoreceptor for which there has been no biochemical evidence. We have investigated whether glucose recognition by the beta-cell is coupled to phospholipase C. We have used digitonin-permeabilized, [3H]inositol-prelabeled islets to study glucose and carbachol activation of phospholipase C. In this model, carbachol recognition by its muscarinic receptor was coupled to phospholipase C activation. D-Glucose (but not L-glucose) also stimulated phospholipase C activity in these permeabilized islets. This effect was not due to glucose metabolism since glucose 6-phosphate did not affect phospholipase C activity and since phosphorylation of [3H]glucose was not detectable in digitonin-permeabilized islets. Glucose had no effect on the myo-inositol-1,4,5-trisphosphate-5-phosphatase or 3-kinase activities. In the absence of agonist, free Ca2+ concentrations between 0.1 and 1 microM (as determined with a Ca2+-specific electrode) did not influence phospholipase C activity. Stimulation of phospholipase C activity by either carbachol or glucose required Ca2+ in the submicromolar range and was optimal at 0.5 microM free Ca2+.myo-Inositol-1,3,4,5-tetrakisphosphate production from permeabilized islets was synergistically augmented by Ca2+ (0.5-10 microM) and glucose. Phospholipase C activity in islets is therefore not directly activated by free Ca2+ concentrations in the submicromolar range. Furthermore, glucose per se activates phospholipase C activity independently of glucose metabolism. A working hypothesis based on these findings is that glucose is recognized by a site which is coupled to phospholipase C in islets.     

Unmasking of arachidonate-induced insulin release by removal of extracellular calcium. Arachidonic acid mobilizes cellular calcium in rat islets of Langerhans

Exogenous arachidonic acid does not stimulate insulin release in Ca++-containing medium, but a potent effect was unmasked by extracellular Ca++ depletion. This secretion met several criteria of exocytotic release. It did not require the oxygenation of arachidonate or its esterification into islet membranes, but was potentiated by the presence of 16.7 mM glucose such that 33 microM arachidonate could reverse the inhibitory effects of extracellular Ca++ removal on glucose-induced insulin secretion. Arachidonic acid alone stimulated a rise in intracellular Ca++ concentrations in dispersed islet cells (measured by the fura-2 technique) equal to that induced by 16.7 mM glucose in normal medium. Arachidonic acid may be a critical coupling signal in normal islets.     

Properties of different Ca2+ pools in permeabilized rat thymocytes

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.     

Arachidonic acid inhibits thyrotropin-releasing hormone-induced elevation of cytoplasmic free calcium in GH3 pituitary cells

We have shown that arachidonic acid stimulates 45Ca2+ efflux from prelabeled rat pituitary mammotropic (GH3) cells resuspended in "Ca2+-free" medium (Kolesnick, R. N., Mussachio, I., Thaw, C., and Gershengorn, M. C. (1984) Am. J. Physiol. 246, E458-E462). In this study, we further characterize the effects of arachidonic acid on Ca2+ homeostasis in GH3 cells and demonstrate its antagonism of changes induced by thyrotropin-releasing hormone (TRH). At below 5 microM, arachidonic acid stimulated intracellular for extracellular Ca2+ exchange without affecting cell Ca2+ content. Above 5 microM, arachidonic acid decreased membrane-bound Ca2+, as monitored by chlortetracycline, and decreased total cell 45Ca2+ content by depleting nonmitochondrial and mitochondrial pools. However, arachidonic acid did not elevate cytoplasmic free Ca2+ concentration ([Ca2+]i). Arachidonic acid inhibited TRH-induced 45Ca2+ efflux, loss of membrane-bound Ca2+, mobilization of nonmitochondrial Ca2+, and elevation of [Ca2+]i. Arachidonic acid also lowered elevated [Ca2+]i caused by release of mitochondrial Ca2+ with an uncoupler or by influx of extracellular Ca2+ stimulated with K+ depolarization. Hence, arachidonic acid stimulates Ca2+ extrusion from and depletes Ca2+ stores within GH3 cells. We suggest that arachidonic acid may be an important regulator of cellular Ca2+ homeostasis which may inhibit TRH-induced elevation of [Ca2+]i.     

Inositol 1,4,5-trisphosphate mobilizes glucose-incorporated calcium from pancreatic islets

Mobilization of intracellular calcium from beta-cell-rich pancreatic islets of ob/ob-mice was studied by measuring unidirectional 45Ca efflux at 37 degrees and 18 degrees C during perifusion with a K+-rich medium deficient in Ca2+ and Na+. Addition of 100 microM carbachol induced a prominent peak of Ca2+ efflux from islets preexposed to glucose. After cell permeabilization with digitonin D-myo-inositol 1,4,5-trisphosphate (IP3) caused glucose-dependent mobilization of calcium. In demonstrating that not only carbachol but also IP3 can mobilize calcium incorporated in response to glucose, the present data suggests that the endoplasmic reticulum participates in glucose-induced lowering of cytoplasmic Ca2+ activity in the pancreatic beta-cells.     

Modulation of platelet-activating-factor-induced calcium influx and intracellular calcium release in platelets by phorbol esters.

1. The rate of 45Ca2+ efflux from prelabelled rat islets of Langerhans was stimulated by carbachol in a dose-dependent manner. 2. Significant stimulation occurred in the presence of 0.2 microM-carbachol; the response was half-maximal at 3-5 microM and was maximal at 20 microM. 3. Stimulation of 45Ca2+ efflux by carbachol was not dependent on the presence of extracellular Ca2+ and was enhanced in Ca2+-depleted medium. 4. Stimulation of 45Ca2+ efflux by 5 microM-carbachol occurred independently of any change in [3H]arachidonic acid release in prelabelled islets, and probably reflected generation of inositol trisphosphate in the cells. 5. The amphipathic peptide melittin failed to increase islet-cell 45Ca2+ efflux at a concentration of 1 microgram/ml, and caused only a modest increase at 10 micrograms/ml. 6. Despite its failure to increase 45Ca2+ efflux, melittin at 1 microgram/ml caused a marked enhancement of 3H release from islets that had been prelabelled with [3H]arachidonic acid. 7. The stimulation of 3H efflux caused by melittin correlated with a dose-dependent increase in the unesterified [3H]arachidonic acid content of prelabelled islets and with a corresponding decrease in the extent of labelling of islet phospholipids. 8. Combined addition of melittin (1 microgram/ml) and 5 microM-carbachol to perifused islets failed to augment 45Ca2+ efflux relative to that elicited by carbachol alone. 9. The data indicate that melittin promotes an increase in arachidonic acid availability in intact rat islets. They do not, however, support the proposal that this can either directly reproduce or subsequently modify the extent of intracellular Ca2+ mobilization induced by agents that cause an increase in inositol trisphosphate.     

Mobilization of cellular Ca2+ by lysophospholipids in rat islets of Langerhans

To determine whether lysophospholipids mobilize cellular Ca2+, intact rat islets were prelabelled with 45Ca2+ and subjected to three maneuvers designed to simulate the physiologic accumulation of lysophospholipids: (1) exogenous provision; (2) addition of porcine pancreatic phospholipase A2; and (3) provision of p-hydroxymercuribenzoic acid, which impedes both the reacylation and hydrolysis of endogenous lysophospholipids, leading to their accumulation in islets. Each maneuver provoked 45Ca2+ efflux at concentrations nearly identical to those previously reported to induce insulin release in the absence of toxic effects on the islets. Lysophosphatidylcholine (lysoPC) and lysophosphatidylinositol were active, whereas the ethanolamine and serine derivatives, and lysophosphatidic acid, were much less effective. The effects of lysoPC were reversible; they also were reduced by lanthanum or gentamicin (which are probes of superficial, plasma membrane-bound stores of Ca2+) or by prior depletion of membrane-bound cellular Ca2+ stores using ionomycin, but not by removal of extracellular Ca2+ or Na+. The effects of lysoPC, phospholipase A2 and p-hydroxymercuribenzoic acid were largely independent of any hydrolysis to, or accumulation of, free fatty acids as assessed by resistance to dantrolene or trifluoperazine (which selectively reduce arachidonic acid-induced 45Ca2+ efflux and insulin release). Thus, lysophospholipids are a newly recognized class of lipid mediators which may promote insulin release at least in part via mobilization of a pool(s) of Ca2+ ('trigger Ca2+') bound in the plasma membrane and possibly in other cellular membranes.     

Parallel effects of arachidonic acid on insulin secretion, calmodulin-dependent protein kinase activity and protein kinase C activity in pancreatic islets.

A potential role of arachidonic acid in the modulation of insulin secretion was investigated by measuring its effects on calmodulin-dependent protein kinase and protein kinase C in islet subcellular fractions. The results were interpreted in the light of arachidonic acid effects on insulin secretion from intact islets. Arachidonic acid could replace phosphatidylserine in activation of cytosolic protein kinase C (K0.5 of 10 microM) and maximum activation was observed at 50 microM arachidonate. Arachidonic acid did not affect the Ca2+ requirement of the phosphatidylserine-stimulated activity. Arachidonic acid (200 microM) inhibited (greater than 90%) calmodulin-dependent protein kinase activity (K0.5 = 50-100 microM) but modestly increased basal phosphorylation activity (no added calcium or calmodulin). Arachidonic acid inhibited glucose-sensitive insulin secretion from islets (K0.5 = 24 microM) measured in static secretion assays. Maximum inhibition (approximately 70%) was achieved at 50-100 microM arachidonic acid. Basal insulin secretion (3 mM glucose) was modestly stimulated by 100 microM arachidonic acid but in a non-saturable manner. In perifusion secretion studies, arachidonic acid (20 microM) had no effect on the first phase of glucose-induced secretion but nearly completely suppressed second phase secretion. At basal glucose (4 mM), arachidonic acid induced a modest but reproducible biphasic insulin secretion response which mimicked glucose-sensitive secretion. However, phosphorylation of an 80 kD protein substrate of protein kinase C was not increased when intact islets were incubated with arachidonic acid, suggesting that the small increases in insulin secretion seen with arachidonic acid were not mediated by protein kinase C. These data suggest that arachidonic acid generated by exposure of islets to glucose may influence insulin secretion by inhibiting the activity of calmodulin-dependent protein kinase but probably has little effect on protein kinase C activity.     

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 microM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) could not replace GTP but prevented the action of GTP. The effects of GTP and GTP gamma S were reversible. Neither GTP nor GTP gamma S induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 microM free Ca2+, a half-maximal Ca2+ no Ca2+ release was observed with 0.1 microM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 microM) were required to evoke Ca2+ release. At 8 microM free Ca2+, even 0.25 microM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 microM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.     

Arachidonic acid stimulates 45calcium efflux and hPL release in isolated trophoblast cells

Previous investigations from this laboratory have indicated that arachidonic acid stimulates a rapid, dose-dependent and reversible increase in hPL release which is not dependent on cyclooxygenase or lipoxygenase metabolism. To investigate further the mechanism by which arachidonic acid stimulates the release of hPL, the effect of arachidonic acid on the release of 45Ca from perifused cells prelabelled with 45CaCl was examined in an enriched cell culture population of term human syncytiotrophoblast. Arachidonic acid (10-100 microM) stimulated a dose-dependent, rapid, and reversible increase in the release of both 45Ca and hPL from the perifused placental cells. On the other hand, palmitic acid had little effect on either hPL release or 45Ca release even at concentrations as high as 100 microM. Ionophore A23187 (1-10 microM) also stimulated a dose-dependent and reversible increase in hPL release. Since arachidonic acid increases the mobilization of cellular calcium, as reflected by the increased 45calcium efflux, and since an increase in the intracellular calcium concentration appears to stimulate an increase in hPL release, these results suggest that the stimulation of hPL release by arachidonic acid may be due, at lease in part, to the effects of the fatty acid on cellular calcium mobilization.     

Recombinant human tumour necrosis factor increases cytosolic free calcium in murine fibroblasts and stimulates inositol phosphate formation in L-M and arachidonic acid release in 3T3 cells

Stimulation of murine L-M and 3T3 fibroblasts with human recombinant tumour necrosis factor (rTNF) resulted in an increase in the cytosolic free Ca2+ concentration ([Ca2+]i). In 3T3 cells rTNF also induced release and metabolization of arachidonic acid, whereas in L-M cells rTNF provoked rapid increases in the levels of inositol mono-, bis- and trisphosphates (IP1, IP2 and IP3). In these cells the Ca2+ response was also observed in Ca2+ free medium, suggesting that rTNF promotes mobilization of Ca2+ from intracellular stores. In 3T3 cells, however, Ca2+ originated from the extracellular space, since the response was abolished in medium containing 1 mM EGTA. Both rTNF-induced calcium responses were inhibited by a specific rabbit IgG antibody to rTNF but not by 1-verapamil, a blocker potential-operated calcium channels. These results suggest that increased formation of inositol phosphates, arachidonic acid release and increased cytosolic free Ca2+ are involved in the biological effects of rTNF. However, rTNF generate these signals by different mechanisms depending upon the target cell.     

Mobilization of Ca2+ stores in individual pancreatic beta-cells permeabilized or not with digitonin or alpha-toxin

The concentration of free Ca2+ in the cytoplasm and organelles of individual mouse pancreatic beta-cells was estimated with dual wavelength microfluorometry and the indicators Fura-2 and furaptra. Measuring the increase of cytoplasmic Ca2+ resulting from intracellular mobilization of the ion in ob/ob mouse beta-cells, most organelle calcium (92%) was found in acidic compartments released when combining the Ca2+ ionophore Br-A23187 with a protonophore. Only 3-4% of organelle calcium was recovered from a pool sensitive to the Ca(2+)-ATPase inhibitor thapsigargin. Organelle Ca2+ was also measured directly in furaptra-loaded beta-cells after controlled plasma membrane permeabilization. The permeabilizing agent alpha-toxin was superior to digitonin in preserving the integrity of intracellular membranes, but digitonin provided more reproducible access to intracellular sites. After permeabilization, the thapsigargin-sensitive fraction of Ca2+ detected by furaptra was as high as 90%, suggesting that the indicator essentially measures Ca2+ in endoplasmic reticulum (ER). Both alpha-toxin- and digitonin-permeabilized cells exhibited ATP-dependent uptake of Ca2+ into thapsigargin-sensitive stores with half-maximal and maximal filling at 6-11 microM and 1 mM ATP respectively. Most of the thapsigargin-sensitive Ca2+ was mobilized by inositol 1,4,5-trisphosphate (IP3), whereas caffeine, ryanodine, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate lacked effects both in beta-cells from ob/ob mice and normal NMRI mice. Mobilization of organelle Ca2+ by 4-chloro-3-methylphenol was attributed to interference with the integrity of the ER rather than to activation of ryanodine receptors. The observations emphasize the importance of IP3 for Ca2+ mobilization in pancreatic beta-cells, but question a role for ryanodine receptor agonists.