Studies on the role of inositol trisphosphate in the regulation of insulin secretion from isolated rat islets of Langerhans.

Glucose (20 mM) and carbachol (1 mM) produced a rapid increase in [3H]inositol trisphosphate (InsP3) formation in isolated rat islets of Langerhans prelabelled with myo-[3H]inositol. The magnitude of the increase in InsP3 formation was similar when either agent was used alone and was additive when they were used together. In islets prelabelled with 45Ca2+ and treated with carbachol (1 mM), the rise in InsP3 correlated with a rapid, transient, release of 45Ca2+ from the cells, consistent with mobilization of 45Ca2+ from an intracellular pool. Under these conditions, however, insulin secretion was not increased. In contrast, islets prelabelled with 45Ca2+ and exposed to 20mM-glucose exhibited a delayed and decreased 45Ca2+ efflux, but released 7-8-fold more insulin than did those exposed to carbachol. Depletion of extracellular Ca2+ failed to modify the increase in InsP3 elicited by either glucose or carbachol, whereas it selectively inhibited the efflux of 45Ca2+ induced by glucose in preloaded islets. Under these conditions, however, glucose was still able to induce a small stimulation of the first phase of insulin secretion. These results demonstrate that polyphosphoinositide metabolism, Ca2+ mobilization and insulin release can all be dissociated in islet cells, and suggest that glucose and carbachol regulate these parameters by different mechanisms.     

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.     

Effects of guanosine 5'-[gamma-thio]triphosphate and thrombin on the phosphoinositide metabolism of electropermeabilized human platelets

Incubation of human platelets with myo-[3H]inositol in a low-glucose Tyrode's solution containing MnCl2 enhanced the labelling of phosphoinositides about sevenfold and greatly facilitated the measurement of [3H]inositol phosphates formed by the activation of phospholipase C. Labelled platelets were permeabilized by high-voltage electric discharges and equilibrated at 0 degree C with ATP, Ca2+ buffers and guanine nucleotides, before incubation in the absence or presence of thrombin. Incubation of these platelets with ATP in the presence or absence of Ca2+ ions led to the conversion of [3H]phosphatidylinositol to [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PtdInsP2). At a pCa of 6, addition of 100 microM GTP[gamma S] both prevented this accumulation of [3H]PtdInsP2 and stimulated its breakdown; the formation of [3H]inositol phosphates was increased ninefold. After 5 min these comprised 70% [3H]inositol monophosphate ([3H]InsP), 28% [3H]inositol bisphosphate ([3H]InsP2) and 2% [3H]inositol trisphosphate ([3H]InsP3). In shorter incubations higher percentages of [3H]InsP2 and [3H]InsP3 were found. In the absence of added Ca2+, the formation of [3H]inositol phosphates was decreased by over 90%. Incubation of permeabilized platelets with GTP[gamma S] in the presence of 10 mM Li+ decreased the accumulation of [3H]InsP and increased that of [3H]InsP2, without affecting [3H]InsP3 levels. Addition of unlabelled InsP3 decreased the intracellular hydrolysis of exogenous [32P]InsP3 but did not trap additional [3H]InsP3. These results and the time course of [3H]inositol phosphate formation suggest that GTP[gamma S] stimulated the action of phospholipase C on a pool of [3H]phosphatidylinositol 4-phosphate that was otherwise converted to [3H]PtdInsP2 and that much less hydrolysis of [3H]phosphatidylinositol to [3H]InsP or of [3H]PtdInsP2 to [3H]InsP3 occurred. At a pCa of 6, addition of thrombin (2 units/ml) to permeabilized platelets caused small increases in the formation of [3H]InsP and [3H]InsP2. This action of thrombin was enhanced twofold by 10-100 microM GTP and much more potently by 4-40 microM GTP[gamma S]. In the presence of the latter, thrombin also increased [3H]InsP3. The total formation of [3H]inositol phosphates by permeabilized platelets incubated with thrombin and GTP[gamma S] was comparable with that observed on addition of thrombin alone to intact platelets. However, HPLC of the [3H]inositol phosphates formed indicated that about 75% of the [3H]InsP accumulating in permeabilized platelets was the 4-phosphate, whereas in intact platelets stimulated by thrombin, up to 80% was the 1-phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates.

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulations in Cultured Adrenal Chromaffin Cells in Response to Bradykinin and Histamine

In previous studies it has been shown that both bradykinin and histamine increase the formation of 3H-labeled inositol phosphates in adrenal chromaffin cells prelabelled with [3H]inositol and that both these agonists stimulate release of catecholamines by a mechanism dependent on extracellular calcium. Here, we have used mass assays of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] to investigate changes in levels of these two candidates as second messengers in response to stimulation with bradykinin and histamine. Bradykinin increased the mass of Ins(1,3,4,5)P4 despite the failure in earlier studies with [3H]inositol-labelled cells to observe a bradykinin-mediated increase in content of [3H]InsP4. Bradykinin elicited a very rapid increase in level of Ins(1,4,5)P3, which was maximal at 5-10 s and then rapidly decreased to a small but sustained elevation at 2 min. The bradykinin-elicited Ins(1,3,4,5)P4 response increased to a maximum at 30-60 s and at 2 min was still elevated severalfold above basal levels. Histamine, which produced a larger overall total inositol phosphate response in [3H]inositol-loaded cells, produced significantly smaller Ins(1,4,5)P3 and Ins(1,3,4,5)P4 responses compared with bradykinin. The bradykinin stimulation of Ins(1,4,5)P3 accumulation was partially dependent on a high (1.8 mM) extracellular Ca2+ concentration, whereas the Ins(1,3,4,5)P4 response was almost completely lost when the extracellular Ca2+ concentration was reduced to 100 nM. Changes in the inositol polyphosphate second messengers are compared with the time course of bradykinin-stimulated increases in free intracellular Ca2+ concentrations and noradrenaline release.     

Rapid formation of inositol 1,4,5-trisphosphate in rat pancreatic acini stimulated by carbamylcholine

Cholinergic stimulation of inositol phosphate formation was studied in isolated rat pancreatic acini, prelabelled with myo-[2-3H]inositol. Carbamylcholine increased incorporation of radioactivity into Ins(1,4,5)P3 and InsP4 within 5 s. Increases in [3H]Ins(1,3,4)P3 were delayed with marked stimulation occurring between 10 s and 1 min. Inositol polyphosphate formation was less sensitive to carbamylcholine concentration than was stimulation of amylase release. At a low (0.3 microM) carbamylcholine concentration, no increase in inositol polyphosphate formation was detected, whereas stimulation of amylase release, which was not dependent on extracellular calcium, was observed. Ins(1,4,5)P3 was shown to release actively accumulated 45Ca2+ from isolated rough endoplasmic reticulum membranes to a similar extent as that released from rough endoplasmic reticulum following cholinergic stimulation of pancreatic acini (Richardson, A.E. et al. (1984) Biochem. Soc. Trans. 12, 1066-1067). The data is consistent with Ins(1,4,5)P3 being produced rapidly enough to release sufficient calcium from the rough endoplasmic reticulum to cause an observed increases in cytoplasmic free Ca2+.     

Inositol phospholipid metabolism in human platelets stimulated by ADP

ADP-induced changes in inositol phospholipids, phosphatidic acid and inositol phosphates of human platelets have been studied in detail, using not only 32P labelling, but also by examining changes in amounts of the phospholipids, their labelling with [3H]glycerol and their specific radioactivities; changes in the labelling of inositol phosphates in platelets prelabelled with [3H]inositol were also measured. During the early (10 s) stage of reversible ADP-induced primary aggregation in a medium containing fibrinogen and with a concentration of Ca2+ in the physiological range (2 mM), the amounts of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol 4-phosphate (PtdInsP) decreased (by 11.2 +/- 4.9% and 11.3 +/- 5.3%, respectively) while the labelling, but not the amount, of phosphatidic acid increased. The decreases do not appear to be attributable to the action of phospholipase C because the specific radioactivity of phosphatidic acid labelling with [3H]glycerol was not significantly increased at 10 s (although the initial specific radioactivities of the inositol phospholipids and PtdCho were more than double that of phosphatidic acid), and no increases in the labelling of inositol trisphosphate (InsP3), inositol bisphosphate (InsP2) or inositol phosphate (InsP) were detectable at 10 s. Shifts in the interconversions between PtdInsP2 and PtdInsP, and PtdInsP and PtdIns may occur. By 30 to 60 s, when deaggregation was beginning, the amounts of PtdInsP2, PtdInsP and phosphatidic acid were not different from those in unstimulated platelets, but large increases in the 32P-labelling and [3H]glycerol labelling of phosphatidic acid were observed. Formation of [3H]inositol-labelled InsP3 was not detectable at any time in association with ADP-induced primary aggregation, indicating that degradation of PtdInsP2 by phospholipase C is not appreciably stimulated by ADP. These findings were compared with those obtained when platelets were aggregated by ADP in a medium without added of Ca2+ in which secondary aggregation associated with thromboxane A2 (TXA2) formation and release of granule contents occurs. At 10 s (during primary aggregation) the changes were similar in the two media. At 30 s and 60 s (during secondary aggregation in the low-Ca2+ medium), the increases in PtdInsP2, PtdInsP and phosphatidic acid in platelets suspended in the absence of added Ca2+ were larger than those in platelets suspended in the presence of 2 mM Ca2+. In the absence of added Ca2+, ADP-induced increases in the labelling of InsP3, InsP2 and InsP which were probably due to the effects of TXA2 since they were abolished by aspirin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Does the inositol tris/tetrakisphosphate pathway exist in rat heart?

Appearance of two isomers of inositol trisphosphate (InsP3) was observed when [3H]inositol prelabelled rat heart ventricles were stimulated for 10 and 30 s with noradrenaline. In contrast, inositol tetrakisphosphate (InsP4) could not be detected. However the existence of the inositol tris/tetrakisphosphate pathway was demonstrated by studying [3H]inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) metabolism in a soluble fraction of rat heart. There, [3H]Ins-1,4,5-P3 was phosphorylated to form [3H]Ins-1,3,4,5-P4. Raising [Ca2+] from 1 nM to 1 microM increased InsP3 kinase activity by 2-fold (EC50 for Ca2+ approx. 56 nM). This effect appeared to be due to an increase of the apparent Vmax of the enzyme while the apparent Km was unchanged.     

Calcium promotes the accumulation of polyphosphoinositides in intact and permeabilized bovine adrenal chromaffin cells

1. Because cellular pools of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate turn over rapidly during phospholipase C stimulation, the continuing production of inositol phosphates requires continuing synthesis from phosphatidylinositol of the polyphosphoinositides. In the present study in adrenal chromaffin cells, we examined the effects of nicotinic stimulation and depolarization in intact cells and micromolar Ca2+ in permeabilized cells on the levels of labeled polyphosphoinositides. We compared the effects to muscarinic stimulation in intact cells and GTP gamma S in permeabilized cells. 2. Nicotinic stimulation, elevated K+, and muscarinic stimulation cause similar production of inositol phosphates (D. A. Eberhard and R. W. Holz, J. Neurochem. 49:1634-1643, 1987). Nicotinic stimulation and elevated K+ but not muscarinic stimulation increased the levels of [3H]inositol-labeled phosphatidylinositol phosphate by 30-60% and [3H]phosphatidylinositol bisphosphate by 25-30%. The increase required Ca2+ in the medium, was maximal by 1-2 min, and was not preceded by an initial decrease in phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. 3. In digitonin-permeabilized cells, Ca2+ caused as much as a twofold increase in [3H]phosphatidylinositol phosphate and [3H]phosphatidylinositol bisphosphate. Similarly, Ca2+ enhanced the production of [32P]phosphatidylinositol phosphate and [32P]phosphatidylinositol bisphosphate in the presence of [gamma-32P]ATP. In contrast, GTP gamma S in permeabilized cells decreased polyphosphoinositides in the presence or absence of Ca2+. 4. The ability of Ca2+ to increase the levels of the polyphosphoinositides decayed with time after permeabilization. The effect of Ca2+ was increased when phosphoesterase and phospholipase C activities were inhibited by neomycin. 5. These observations suggest that Ca2+ specifically enhances polyphosphoinositide synthesis at the same time that it activates phospholipase C.     

Guanosine 5''-O-Thiotriphosphate and Sodium Fluoride Activate Polyphosphoinositide Hydrolysis in Rat Cortical Membranes by Distinct Mechanisms

NaF and guanosine 5'-O-thiotriphosphate [GTP(S)] stimulated the accumulation of [3H]inositol monophosphate ([3H]InsP) in rat brain cortical membranes, with half-maximal stimulation at 2 mM and 1 microM, respectively. Calcium also increased basal [3H]InsP formation over a range of concentrations from 10(-7) to 10(-4) M. The stimulatory effect of GTP(S) (30 microM) on [3H]InsP production was insensitive to Ca2+, whereas NaF-evoked [3H]InsP formation was dependent on Ca2+ concentrations. Guanosine 5'-O-thiodiphosphate significantly attenuated GTP(S)- but not NaF-stimulated [3H]InsP production. Coincubation of GTP(S) (30 microM) and submaximal concentrations of NaF (1 or 3 mM) stimulated [3H]InsP formation to a degree that was nearly additive with that produced by either drug alone. However, the resultant accumulation of [3H]InsP in the presence of maximally effective concentrations of GTP(S) and NaF was not different from that produced by NaF alone. Incubation of cortical membranes with GTP(S) and NaF for 1 min stimulated the accumulation of [3H]inositol bisphosphate (InsP2) but not [3H]InsP. [3H]InsP2 production elicited by GTP(S) was markedly enhanced by the muscarinic cholinergic agonist carbachol. In contrast, NaF-stimulated [3H]InsP2 formation was not potentiated by carbachol. Our findings of different characteristics of GTP(S) and fluoride activation of polyphosphoinositide (PPI) hydrolysis suggest that separate regulatory mechanisms are involved in these two modes of stimulation in brain membranes. Activation of PPI hydrolysis by fluoride may be mediated by a direct stimulation of PPI phosphodiesterase or by activating a putative guanine nucleotide regulatory protein at a location distinct from the GTP-binding site.     

Changes in free cytosolic calcium and accumulation of inositol phosphates in isolated hepatocytes by [Leu]enkephalin.

Isolated hepatocytes from fed rats were used to study the effects of the opioid peptide [Leu]enkephalin on intracellular free cytosolic Ca2+ ([Ca2+]i) and inositol phosphate production. By measuring the fluorescence of the intracellular Ca2+-selective indicator quin-2, [Leu]enkephalin was found to increase [Ca2+]i rapidly from a resting value of 0.219 microM to 0.55 microM. The magnitude of this response was comparable with that produced by maximally stimulating concentrations of either vasopressin (100 nM) or phenylephrine (10 microM). The opioid-peptide-mediated increase in [Ca2+]i showed a dose-dependency comparable with the activation of phosphorylase, but it preceded the increase in phosphorylase alpha activity. Addition of [Leu]enkephalin to hepatocytes prelabelled with myo-[2-3H(n)]inositol resulted in a significant stimulation of inositol phosphate production. At 10 min after hormone addition, there were increases in the concentrations of inositol mono-, bis- and tris-phosphate fractions of 12-, 9- and 14-fold respectively. No effect was apparent on the glycerophosphoinositol fraction. The effect of 10 microM-[Leu]enkephalin on inositol phosphate production was significantly greater than that obtained with 10 microM-phenylephrine, but marginally smaller than that induced by 100 nM-vasopressin. However, at these concentrations all three agonists gave a comparable increase in [Ca2+]i and activation of phosphorylase a. These data provide evidence for [Leu]enkephalin acting via a mechanism involving a mobilization of Ca2+ as a result of increased phosphatidylinositol turnover.     

The role of alpha 1-adrenergic stimulation in inositol phosphate metabolism during post-ischaemic reperfusion.

The aim of this study was to elucidate the mechanism of enhanced inositol phosphate metabolism during reperfusion. Inositol phosphate stores were prelabelled by perfusing isolated rat hearts for 1 h with [3H]inositol (1.5 microCi/ml). LiCl (10 mM) and prazosin (0.3 microM) were subsequently added 15 min before (i) 20 min control perfusion; (ii) 20 min normothermic ischaemic cardiac arrest (NICA); (iii) 20 min NICA followed by 1 min reperfusion. The ventricles were freeze-clamped before determination of isotopical incorporation of [3H]inositol into the inositol phosphates (Dowex anion exchange chromatography) and InsP3 levels (Amersham InsP3 assay system). In addition, noradrenaline release into the perfusate was also assessed (HPLC and electrochemical detection). The results showed: (i) increased noradrenaline release into the perfusate immediately after the onset of reperfusion; (ii) significant depression of [3H]inositol incorporation into inositol phosphates and InsP3 levels after 20 min NICA; (iii) reperfusion caused an immediate significant increase in isotopical incorporation of [3H]inositol into inositol phosphates as well as InsP3 levels; (iv) the alpha 1-adrenergic blocker, prazosin (0.3 microM), completely inhibited the reperfusion-induced increase in inositol phosphate metabolism. These observations suggested that increased alpha 1-adrenergic receptor stimulation by noradrenaline might be responsible for the stimulation of ventricular inositol phosphate metabolism during postischaemic reperfusion.     

Two modes of regulation of the phospholipase C-linked substance-P receptor in rat parotid acinar cells.

In rat parotid acinar cells prelabelled with [3H]inositol, substance P (100 nM) induced the formation of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3 reached a maximum 7 s after substance P stimulation, and thereafter decreased and reached a stable value at 60 s. When the cells were exposed to substance P for 10, 30, 60, or 300 s, washed, and re-exposed to this peptide, the formation of [3H]inositol trisphosphate (InsP3) was attenuated in a time-dependent manner. In the cells pretreated as described above, the number of [3H]substance-P-binding sites (Bmax) was also decreased. Possible role(s) of Ca2+ and protein kinase (protein kinase C) control mechanisms in regulating substance P responses were investigated. Desensitization of substance P-induced InsP3 was not affected by the Ca2+ ionophore ionomycin, nor was it dependent on Ca2+ mobilization. On the other hand, in the presence of 4 beta-phorbol 12,13-dibutyrate (PDBu) and 12-O-tetradecanoyl-4 beta-phorbol 13-acetate, known activators of protein kinase C, substance P-induced InsP3 formation was inhibited. However, PDBu had no effect on [3H]substance P binding, whether present during the assay or when cells were pretreated. The persistent desensitization of InsP3 formation induced by substance P was not affected by PDBu. These results suggest that the persistent desensitization of InsP3 formation induced by substance P is a homologous process involving down-regulation of the substance P receptor; the mechanism does not appear to involve, or to be affected by, the Ca2+ or protein kinase C signalling systems. Protein kinase C activation can, however, inhibit substance P-induced InsP3 formation, which may indicate the presence of a negative-feedback control on the substance P pathway.     

Phospholipase Cdelta(1) does not mediate Ca(2+) responses in neonatal rat cardiomyocytes

Phospholipase C (PLC) activation in neonatal rat ventricular cardiomyocytes (NRVM) generates inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3)) in response to elevations in Ca(2+) or inositol(1,4)bisphosphate in response to G protein stimulation. Overexpression of PLCdelta(1) increased total [(3)H]inositol phosphate (InsP) content and elevated [(3)H]Ins(1,4,5)P(3), but failed to increase [(3)H]InsP responses to the Ca(2+) ionophore A23187. Antisense PLCdelta(1) expression reduced endogenous PLCdelta(1) content but did not decrease the A23187 response. In permeabilized NRVM, [(3)H]InsP responses to elevated Ca(2+) were not inhibited by Ins(1,4,5)P(3), even at concentrations 1000-fold greater than required for selective inhibition of PLCdelta(1). Taken together these data provide evidence that PLCdelta(1) does not mediate the InsP response to elevated Ca(2+) in NRVM.     

Increased Intracellular Calcium Stimulates 3H-Inositol Polyphosphate Accumulation in Rat Cerebral Cortical Slices

Agents that increase the intracellular Ca2+ concentration have been examined for their ability to stimulate 3H-inositol polyphosphate accumulation in rat cerebral cortex slices. Elevated extracellular K+ levels, the alkaloid sodium channel activator veratrine, the calcium ionophore ionomycin, and the marine toxin maitotoxin were all able to stimulate phosphoinositide metabolism. Certain features appear common to the agents studied. Thus, although [3H]inositol monophosphate, [3H]inositol bisphosphate ([3H]InsP2), and [3H]inositol trisphosphate were all stimulated, a proportionally greater effect was observed on [3H]InsP2 in comparison to stimulation by the muscarinic receptor agonist carbachol. However, only an elevated K+ level stimulated [3H]inositol tetrakisphosphate ([3H]InsP4) accumulation alone or produced marked synergy with carbachol on the formation of this polyphosphate. The results suggest that agents that elevate the cytoplasmic Ca2+ concentration in cerebral cells can increase the hydrolysis of membrane polyphosphoinositides. The pattern of the response differs from that produced by muscarinic receptor agonists and indicate that Ca2(+)-dependent hydrolysis may involve different pools of lipids, phosphoinositidase C enzymes, or both. However, clear differences in the ability of these agents to stimulate InsP4, alone or in the presence of muscarinic agonist, suggest that factors other than a simple elevated intracellular Ca2+ concentration are implicated.     

Inositol phosphate formation in fMet-Leu-Phe-stimulated human neutrophils does not require an increase in the cytosolic free Ca2+ concentration.

The accumulation of inositol phosphates in myo-[3H]inositol-labelled human neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe was measured. The challenge with the chemotactic peptide caused the generation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3). The formation of the three inositol phosphates followed a differential time course: InsP3 accumulated very rapidly and transiently, whereas InsP increased steadily for more than 2 min. Inositol phosphate formation was only partially decreased by procedures which prevented the fMet-Leu-Phe-dependent increase of cytosolic free Ca2+ concentration.     

Leukotriene B4 stimulation of phagocytes results in the formation of inositol 1,4,5-trisphosphate. A second messenger for Ca2+ mobilization.

Inositol trisphosphate (InsP3) production and cytosolic free Ca2+ ([Ca2+]i) elevations induced by leukotriene B4 (LTB4)-receptor activation were studied in the human promyelocytic-leukaemia cell line HL60, induced to differentiate by retinoic acid. The myeloid-differentiated HL60 cells respond to LTB4 by raising their [Ca2+]i with a dose-response relationship similar to that shown by normal human neutrophils. The observations of the LTB4 transduction mechanism were compared with those of the transduction mechanism of the chemotactic peptide fMet-Leu-Phe in HL60 cells differentiated with dimethyl sulphoxide. Both LTB4 and fMet-Leu-Phe triggered a rapid (less than 5 s) elevation of [Ca2+]i, which occurred in parallel with the InsP3 production from myo-[3H]inositol-labelled cells. The threshold concentrations of the agonists, for InsP3 production, were found at 10(-9) M, a slightly higher concentration than that required to detect [Ca2+]i elevations. No significant changes were noted in the phosphoinositide levels upon stimulation with LTB4. Exposure to Bordetella pertussis toxin before LTB4 stimulation abolished both the increased formation of InsP3 and the rise of [Ca2+]i. LTB4 and fMet-Leu-Phe elicited elevations of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with no detectable lag time, followed by slower and more sustained inositol 1,3,4-trisphosphate elevations. Stimulation with various leukotriene analogues revealed a good correlation between both total InsP3 as well as Ins(1,4,5)P3 formation and elevations of [Ca2+]1. Thus LTB4 receptor activation results in an increased production of Ins(1,4,5)P3 via a transduction mechanism also involving a nucleotide regulatory protein, as previously described for the fMet-Leu-Phe transduction mechanism.     

Endothelial inositol phosphate generation and prostacyclin production in response to G-protein activation by AlF4-.

In order to elucidate the role of guanine-nucleotide-binding proteins (G-proteins) in endothelial prostacyclin (PGI2) production, human umbilical vein endothelial cells, prelabelled with either [3H]inositol or [3H]arachidonic acid, were stimulated with the non-specific G-protein activator aluminium fluoride (AlF4-). AlF4- caused a dose- and time-dependent generation of inositol phosphates, release of arachidonic acid and production of PGI2. The curves for the three events were similar. When the cells were stimulated in low extracellular calcium (60 nM), they released [3H]arachidonic acid and produced PGI2, but depleting the intracellular Ca2+ stores by pretreatment with the Ca2+ ionophore A23187 totally inhibited both events, although the cells still responded when extracellular Ca2+ was added. The Ca2+ ionophore did not inhibit the generation of inositol phosphates in cells maintained at low extracellular Ca2+. Pertussis toxin pretreatment (14 h) altered neither inositol phosphate nor PGI2 production in response to AlF4-. To investigate the functional role of the diacylglycerol/protein kinase C arm of the phosphoinositide system, the cells were pretreated with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the protein kinase C inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). TPA inhibited the AlF4(-)-induced inositol phosphate generation but stimulated both the release of arachidonic acid and the production of PGI2. H7 had opposite effects both on inositol phosphate generation and on PGI2 production. These results suggest that AlF4(-)-induced PGI2 production is mediated by a pertussis-toxin-insensitive G-protein which activates the phosphoinositide second messenger system. This production of PGI2 can be modulated by protein kinase C activation, both at the level of inositol phosphate generation and at the level of arachidonic acid release.     

Platelet-activating factor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate stimulates the production of reactive oxygen intermediates in macrophages.

To investigate the relationship between inositol lipid hydrolysis and reactive oxygen-intermediate (ROI) production in macrophages we have examined the effect of platelet-activating factor (PAF) on normal bone marrow-derived macrophages. Addition of PAF to macrophages prelabelled with [3H]inositol caused a marked and rapid increase in [3H]inositol trisphosphate levels. Similarly when PAF was added to [3H]-glycerol prelabelled macrophages there was a rapid increase in 1,2-diacyl[3H]glycerol levels. These events preceded any increase in the rate of PAF-stimulated ROI production by a discernible period of several seconds. Increasing concentrations of PAF led to a markedly similar increase in both ROI production and [3H]inositol lipid hydrolysis suggesting that inositol lipid hydrolysis may lead to the generation of ROI in macrophages. Further evidence that this is the case came from experiments in which pretreatment of macrophages with phorbol esters was shown to inhibit both PAF-stimulated [3H]inositol phosphate production and ROI production to a markedly similar degree. Similarly pertussis toxin inhibited both PAF-stimulated ROI production and [3H]inositol phosphate production. Phorbol esters were shown to activate ROI production in normal bone marrow-derived macrophages whereas the Ca2+ ionophore, A23187, did not. These experiments suggest that PAF stimulates a pertussis toxin-sensitive activation of inositol lipid hydrolysis leading to the formation of inositol trisphosphate and diacylglycerol. The diacylglycerol formed can then activate protein kinase C leading to the stimulation of ROI production in normal bone marrow-derived macrophages.     

Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilization in cultured astrocytoma cells

In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.