Characterization of agonist-stimulated incorporation of myo-[3H]inositol into inositol phospholipids and [3H]inositol phosphate formation in tracheal smooth muscle.

The effects of the muscarinic agonist carbachol, histamine and bradykinin on incorporation of [3H]inositol into the phosphoinositides and the formation of [3H]InsPs were examined in bovine tracheal smooth-muscle (BTSM) slices labelled with [3H]inositol. These agonists result in substantial and dose-related increases in the incorporation of [3H]inositol into the phospholipids. Carbachol and histamine stimulated the incorporation of [3H]inositol into the phospholipids to the same degree, despite histamine being only 35% as effective as carbachol on [3H]InsP accumulation. Histamine and carbachol, at maximal concentrations, were non-additive with respect to both the stimulated incorporation of [3H]inositol and [3H]InsP formation. For carbachol this effect on incorporation was found to occur to a similar extent in PtdInsP and PtdInsP2 as well as PtdIns. The initial effect of carbachol on [3H]inositol incorporation was rapid (maximal by 10 min); however, with prolonged stimulation large secondary declines in PtdInsP and PtdInsP2 labelling were observed, with depletion of the much larger PtdIns pool only evident in the presence of Li+. Lowering buffer [Ca2+] increased the incorporation of [3H]inositol under basal conditions, but did not attenuate the subsequent agonist-stimulated incorporation effect. The large changes in specific radioactivity of the phosphoinositides, and consequently the [3H]InsP products, after carbachol stimulation resulted in the apparent failure of atropine to reverse the [3H]InsP response completely. Labelling muscle slices with [3H]inositol in the presence of carbachol or labelling for longer periods (greater than 6 h) prevented subsequent carbachol-stimulated effects on incorporation without significantly altering the dose-response relationship for carbachol-stimulated [3H]InsP formation and resulted in steady-state labelling conditions confirmed by the ability of atropine to reverse fully the [3H]InsP response to carbachol. This study demonstrates the profound effects of a number of agonists on [3H]inositol incorporation into the phospho- and polyphosphoinositides in BTSM with important consequent changes in the specific radioactivity of these lipids and the resulting [3H]InsP products. In addition, a selective depletion of PtdInsP and PtdInsP2 over PtdIns has been demonstrated with prolonged muscarinic-receptor stimulation.     

Involvement of phosphoinositide metabolism in potentiation by adrenaline of ADP-induced aggregation of rabbit platelets.

Changes in phosphoinositide metabolism were examined in washed rabbit platelets stimulated with 0.5 microM-ADP, 50 microM-adrenaline, or ADP and adrenaline in combination. Adrenaline does not stimulate platelet aggregation when used alone, but does potentiate aggregation stimulated by ADP. In platelets prelabelled with [32P]Pi and [3H]glycerol, adrenaline was found to potentiate the ADP-induced changes in platelet phospholipids, causing larger increases in the amount and labelling of phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid than was observed with ADP alone. The combination of ADP and adrenaline did not produce a greater decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) than was produced by ADP alone. In platelets prelabelled with [3H]inositol, adrenaline potentiated the increases in labelling of inositol phosphate and inositol bisphosphate stimulated by ADP; no increase in inositol trisphosphate labelling was detected with ADP alone or with the combination of ADP and adrenaline. Phentolamine, an alpha-adrenergic-receptor antagonist, blocked potentiation by adrenaline of ADP-induced changes in phosphoinositide metabolism. Propranolol and sotalol, beta-adrenergic-receptor antagonists, augmented the potentiation; this is consistent with the concept that the effect of adrenaline is mediated by beta-adrenergic receptors. The effect of adrenaline on phosphoinositide metabolism appears to be to potentiate the mechanisms by which ADP causes turnover of PIP and possibly degradation of PI, rather than the mechanism by which PIP2 is decreased.     

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)     

Rates of production and consumption of phosphatidic acid upon thrombin stimulation of human platelets

Human platelets were labelled with [32P]Pi and [3H]glycerol before gel filtration. In unstimulated cells, the specific 32P radioactivity in phosphatidic acid (PtdOH) was similar to that of phosphatidylinositol (PtdIns) but only 4% of that of the gamma-phosphate of ATP. Upon 3 min of stimulation with 0.5 U/ml of thrombin, there was a 20-fold increase in specific 32P radioactivity of PtdOH which approached that of the ATP gamma-phosphate. Based on constant rates of synthesis and removal, this thrombin-induced increase in specific 32P radioactivity in PtdOH allowed us to calculate the flux of phosphate through PtdOH upon stimulation. Synthesis and removal occurred at rates of 107 and 52 nmol min-1/10(11) cells, respectively. The specific [3H]glycerol radioactivity was similar in PtdIns, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in unstimulated platelets. In PtdOH, it was 50% of that of the inositol phospholipids. Thrombin stimulation induced no changes in the specific 3H radioactivity of the inositol phospholipids whereas specific [3H]PtdOH increased to the level of these lipids. It is concluded that PtdIns, PtdInsP and PtdInsP2 exist in a metabolic homogenous pool in human platelets.     

Evidence that the inositol phospholipids are necessary for exocytosis. Loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase C and removal of ATP.

We directly manipulated the levels of PtdIns, PtdInsP and PtdInsP2 in digitonin-treated adrenal chromaffin cells with a bacterial phospholipase C (PLC) from Bacillus thuringiensis and by removal of ATP. The PtdIns-PLC acted intracellularly to cause a large decrease in [3H]inositol- or [32P]phosphate-labelled PtdIns, but did not directly hydrolyse PtdInsP or PtdInsP2. [3H]PtdInsP and [3H]PtdInsP2 levels declined markedly, probably because of the action of phosphatases in the absence of synthesis. Removal of ATP also caused marked decreases in [3H]PtdInsP and [3H]PtdInsP2. The decrease in polyphosphoinositide levels by PtdIns-PLC treatment or ATP removal was reflected by the inhibition of the production of inositol phosphates upon subsequent activation of the endogenous PLC by Ca2(+)-dependent catecholamine secretion from permeabilized cells was strongly inhibited by PtdIns-PLC treatment and by ATP removal. Ca2(+)-dependent secretion was similarly correlated with the sum of PtdInsP and PtdInsP2 when the level of these lipids was changed by either manipulation. PtdIns-PLC inhibited only the ATP-dependent component of secretion and did not affect ATP-dependent secretion. Both PtdIns-PLC and ATP removal inhibited the late slow phase of secretion, but had little effect on the initial rapid phase. Although we found a tight correlation between polyphosphoinositide levels and secretion, endogenous phospholipase C activity (stimulated by Ca2+, guanine nucleotides and related agents) was not correlated with secretion. Additional experiments indicated that neither the products of the PtdIns-PLC reaction (diacylglycerol and InsP1) nor the inability to generate products by subsequent activation of the endogenous PLC is likely to account for the inhibition of secretion. Incubation of permeabilized cells with neomycin in the absence of ATP maintained the level of polyphosphoinositides and more than doubled subsequent Ca2(+)-dependent secretion. The data suggest that: (1) Ca2(+)-dependent secretion has a requirement for the presence of inositol phospholipids; (2) the enhancement of secretion by ATP results in part from increased polyphosphoinositide levels; and (3) the role for inositol phospholipids in secretion revealed in these experiments is independent of their being substrates for the generation of diacylglycerol and InsP3.     

Requirement of Ca2+ for the production and degradation of inositol 1,4,5-trisphosphate in macrophages

The requirement of Ca2+ for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) or the accumulation of inositol 1,4,5-trisphosphate (InsP3) in macrophages stimulated with fMet-Leu-Phe was examined using [32P]Pi or [3H]inositol-labeled cells. The dependence on Ca2+ of inositol-trisphosphate phosphatase was also examined. The application of 1 X 10(-8) M fMet-Leu-Phe caused a rapid decrease in the amount of PtdInsP2 to 70% of the control within 10 s, and the decrease was reverted to the control level by prolonged incubation. The decrease in the amount of PtdInsP2 accompanied the accumulation of phosphatidic acid and of InsP3, indicating that the loss of PtdInsP2 is due to phosphodiesteric breakdown. The dose-dependence of fMet-Leu-Phe or its analog on the hydrolysis of PtdInsP2 was much the same as that of the increase in intracellular free Ca2+ concentration in macrophages. The loss of PtdInsP2 as induced by fMet-Leu-Phe was similarly observed in macrophages treated with ionophore A23187 in the absence of external Ca2+ for 10 min. InsP3 was degraded by the particulate or cytosol fraction prepared from macrophages, and the activity of inositol-trisphosphate phosphatase in the particulate fraction was higher than that in the cytosol fraction. The enzyme in the cytosol fraction required Mg2+ for activity, and was activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6) M in the presence of 1 mM MgCl2.     

The decrease in phosphatidylinositol 4,5-bisphosphate in ADP-stimulated washed rabbit platelets is not primarily due to phospholipase C activation.

Addition of 10 micron-ADP to washed rabbit platelets caused platelet shape change and aggregation without release of the contents of the amine-storage granules, and caused a transient decrease (8.8% at 10 s) in the amount of phosphatidylinositol 4,5-bisphosphate (PIP2). By 20 s the decrease in PIP2 was no longer apparent, but by 60 s the amount of PIP2 was again decreased. Addition of thrombin (1 unit/ml), which causes platelet shape change, aggregation and the release of the contents of the amine-storage granules, caused a decrease in the amount of PIP2 (8.0% at 10 s); at 60 s the amount of PIP2 was not significantly different from that in controls. In platelets prelabelled with [3H]glycerol, the specific radioactivity of PIP2 was increased at 10 s in ADP-stimulated platelets, and unchanged in thrombin-stimulated platelets. In platelets prelabelled with [3H]inositol and incubated with 20 mM-Li+ to inhibit the degradation of the inositol phosphates to inositol, there was no increase in the labelling of inositol trisphosphate (IP3) upon stimulation with ADP. In contrast, stimulation with thrombin caused a significant increase in the labelling of IP3 at 10 s. These differences in the changes in polyphosphoinositide metabolism in ADP- and thrombin-stimulated platelets are consistent with the hypothesis that the decrease in PIP2 in ADP-stimulated platelets may be due not to degradation of PIP2 by phospholipase C, but rather to a shift in the equilibrium between PIP2 and phosphatidylinositol 4-phosphate (PIP). Increases in the labelling of phosphatidic acid at 10 s and of inositol bisphosphate and inositol phosphate after 20 s are consistent with phospholipase C being stimulated through some other mechanism that leads to the degradation of PIP and phosphatidylinositol; one possibility is that ADP causes an increase in cytoplasmic Ca2+.     

How far does phospholipase C activity depend on the cell calcium concentration? A study in intact cells.

The dependence of phospholipase C activity on the cytosolic Ca2+ concentration ([Ca2+]i) was studied in intact liver cells treated with the Ca2+-mobilizing hormone vasopressin, or not so treated. Phospholipase C (PLC) activity was estimated from the formation of [3H]inositol trisphosphate (InsP3) and the degradation of [3H]phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The [Ca2+]i of the cells was clamped from 29 to 1130 nM by quin2 loading. This wide concentration range was obtained by loading the hepatocytes with a high concentration of the Ca2+ indicator in low-Ca2+ medium or by using the Ca2+ ionophore ionomycin in medium containing Ca2+. In resting cells, in which [Ca2+]i was 193 nM, treatment with 0.1 microM-vasopressin which stimulates liver PLC maximally, tripled InsP3 content and raised [Ca2+]i to 2 microM within 15 s. Lowering [Ca2+]i partially decreased cell InsP3 content as well as the ability of vasopressin to stimulate InsP3 formation maximally. At 29 nM, the lowest Ca2+ concentration obtained in isolated liver cells, basal InsP3 content was 64% of that measured in control cells. Addition of vasopressin no longer affected [Ca2+]i, but significantly increased InsP3 by 200%, although less than in the controls (300%). The maintenance of the greater part of the PLC response at constant [Ca2+]i indicated that, in the liver, InsP3 formation does not result from an increase in [Ca2+]i. The effects of lowering [Ca2+]i were reversible. When low cell [Ca2+]i was restored to a normal value, resting InsP3 content and the ability of vasopressin to stimulate InsP3 formation maximally by 300% were also restored. Raising [Ca2+]i from 193 to 1130 nM had little effect on the InsP3 content or the vasopressin-mediated increase in InsP3. In agreement with the stimulation of PLC activity by vasopressin, cell [3H]PtdInsP2 and total PtdInsP2 were degraded by application of this hormone for 15 s. In contrast, when [Ca2+]i was lowered to 29 nM, basal [3H]PtdInsP2 and total PtdInsP2 were increased by about 30%, [3H]PtdInsP2 was further increased by vasopressin, but total PtdInsP2 was not changed. These results show that, in intact hepatocytes, PLC is little affected by [Ca2+]i concentrations above 193 nM, but is partially dependent on Ca2+ below that value. They suggest that, in addition to activating PLC activity, vasopressin might stimulate PtdInsP2 synthesis, presumably via phosphatidylinositol-phosphate kinase, and that this pathway might predominate in cells with low [Ca2+]i.     

Accumulation of the inositol phosphates in thrombin-stimulated, washed rabbit platelets in the presence of lithium.

Experiments with washed rabbit platelets demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml), that causes maximal aggregation and partial release of amine granule contents, also causes increased accumulation of [3H]inositol-labelled inositol trisphosphate (InsP3) in the presence of 20 mM-Li+. This concentration of Li+ was found to inhibit the degradation of inositol phosphates by phosphomonoesterases. This result indicates that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is degraded early after platelet stimulation with thrombin, although in a previous study we had found no decrease in amount. In the absence of Li+, the labelling of inositol bisphosphate (InsP2) increased more rapidly than that of InsP3, consistent with rapid degradation of InsP3 by phosphomonoesterase. After 30s the increase in InsP2 was augmented by Li+. This increase in InsP2 could have been due to increased degradation of phosphatidylinositol 4-phosphate or inhibition of breakdown of InsP2 to InsP with a lesser inhibition of breakdown of InsP3 to InsP2. The effect on InsP3 and InsP2 of stimulation of the platelets with 1.0 unit of thrombin/ml was comparable with the effect of the lower concentration of thrombin. Inositol phosphate (InsP) labelling did not increase in response to 0.1 unit of thrombin/ml, but increased when the platelets were stimulated with 1.0 unit of thrombin/ml. Whether the increase in InsP was due to increased degradation of phosphatidylinositol or a greater rate of breakdown of InsP2 to InsP than InsP to inositol cannot be determined in these experiments. These results indicate that degradation of PtdIns(4,5)P2 is an early event in platelet activation by thrombin and that formation of inositol phosphates and 1,2-diacylglycerol rather than a decrease in PtdIns(4,5)P2 may be the important change.     

Distinct occurrence of phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release and inositol 1,4,5-triphosphate-induced release in ATP-dependent Ca2+-transporting platelet microsomes

The effects of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and inositol 1,4,5-triphosphate(InsP3) on the Ca2+ release from ATP-dependent Ca2+-transporting microsomes prepared from ox platelets were investigated. Under optimal conditions, both PtdInsP2 and InsP3 released Ca2+ from the microsomes in a similar dose-dependent manner. However, the maximal amount of Ca2+ released by InsP3 was almost one-fourth of that released by PtdInsP2. Neither PtdInsP2 nor InsP3 appeared to act as a Ca2+ ionophore since they showed no effect on the Ca2+ content of liposomes prepared from platelet microsomal lipids. InsP3-induced but not PtdInsP2-induced Ca2+ release was decreased with increasing extravesicular Ca2+ from 0.1 microM to 10 microM and it was completely inhibited by 10 microM Ca2+. PtdInsP2-induced but not InsP3-induced Ca2+ release was markedly inhibited by Mg2+, ruthenium red and neomycin. In addition, InsP3 could induce no additional Ca2+ release after the accumulated Ca2+ had been maximally released by PtdInsP2. These results indicate that PtdInsP2 releases Ca2+ from platelet microsomes more effectively than InsP3 by a mechanism distinct from that of InsP3-induced release, and further that InsP3-sensitive microsomes are included within the population of PtdInsP2-sensitive microsomes.     

Stimulation, by vasopressin and other agonists, of inositol-lipid breakdown and inositol phosphate accumulation in WRK 1 cells.

WRK 1 cells were labelled to equilibrium with 2-myo-[3H]inositol and stimulated with vasopressin. Within 3 s of hormone stimulation there was a marked accumulation of 3H-labelled InsP2 and InsP3 (inositol bis- and tris-phosphate), but not of InsP (inositol monophosphate). There was an associated, and rapid, depletion of 3H-labelled PtdInsP and PtdInsP2 (phosphatidylinositol mono- and bis-phosphates), but not of PtdIns (phosphatidylinositol), in these cells. Some 4% of the radioactivity in the total inositol lipid pool of WRK 1 cells was recovered in InsP2 and InsP3 after 10 s stimulation with the hormone. The selectivity of the vasopressin receptors of WRK 1 cells for a variety of vasopressin agonists and antagonists revealed these to be of the V1a subtype. There was no receptor reserve for vasopressin-stimulated inositol phosphate accumulation in WRK 1 cells. The accumulation of inositol phosphates was enhanced in the presence of Li+ions. Half-maximal accumulation of InsP, InsP2 and InsP3 in vasopressin-stimulated cells was observed with 0.9, 3.0 and 3.6 mM-Li+ respectively. Bradykinin and 5-hydroxytryptamine also provoked inositol phosphate accumulation in WRK 1 cells. The effects of sub-optimal concentrations of bradykinin and vasopressin upon inositol phosphate accumulation were additive, but those of optimal concentrations of the hormones were not.     

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)     

Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes.

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)     

ACh and 5-HT induced changes in the concentration of cytosolic inositol trisphosphate (InsP3) and inositol bisphosphate (InsP2) in the ABRM of Mytilus edulis L.

1. For determination of the phosphoinositides and inositol phosphates present in anterior byssus retractor muscle (ABRM) of Mytilus edulis fiber bundles of this muscle were incubated with [3H]-inositol. Close-to-equilibrium labelling was achieved after 14-17 hr of incubation. 2. The phosphoinositides formed during incubation were identified as phosphatidylinositolphosphates by thin layer chromatography and as glycerophosphoryl esters by anion-exchange chromatography after deacylation. Besides PtdIns, PtdInsP and PtdInsP2 two labelled products are formed, which could not be identified. 3. Inositol phosphates were separated by anion-exchange chromatography. InsP, InsP2 and InsP3 are present, while InsP4 seemed to be absent. 4. Incubation of pre-labelled fibers with ACh induces the accumulation of InsP3 and InsP2 immediately. While 5-Ht accomplishes the accumulation after a lag time of 25 sec. The concentration of cytosolic InsP does not change.     

Possible mechanisms of the potentiation of blood-platelet activation by adrenaline.

Vasopressin and adrenaline in combination exert synergistic effects on platelet activity. This study investigated the effects of sub-threshold concentrations of adrenaline (0.1-1 microM) on vasopressin (10 nM-1 microM)-induced platelet aggregation, ATP secretion, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Potentiation of vasopressin-induced aggregation and ATP secretion by adrenaline was accompanied by enhanced elevation of [Ca2+]i and [32P]phosphatidic acid formation. The stimulatory effects of adrenaline on vasopressin-induced platelet activation were mimicked by the combination of the Ca2+ ionophore, ionomycin, and the protein kinase C activator, phorbol 12-myristate 13-acetate, but not by either of these agents alone. These results suggest that the potentiation of vasopressin-induced platelet activation by adrenaline is mediated via enhancement of inositol phospholipid hydrolysis and elevation of [Ca2+]i.     

Inositol trisphosphate, inositol phospholipid metabolism, and germinal vesicle breakdown in surf clam oocytes

Others have reported that microinjection of inositol 1,4,5-trisphosphate (InsP3) releases stored intracellular Ca2+ and causes fertilization envelope elevation, part of the activation process normally initiated by fertilization in deuterostome eggs. In the protostome, Spisula solidissima, germinal vesicle breakdown (GVBD) is the first visible response of the egg to fertilization. To test the effects of InsP3 on egg activation in this organism, we microinjected the compound into oocytes. Microinjection of 0.4-7.0 x 10(-21) moles of InsP3 (equivalent to 5-80 pM if distributed throughout the cell) elicited GVBD in a dose-dependent manner, demonstrating that increased oocyte InsP3 can mimic part of the activation process in this protostome. Synthesis of InsP3 occurs in vivo when phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is hydrolyzed by phospholipase C. To determine whether stimulus-induced synthesis of InsP3 occurs after fertilization of Spisula oocytes, we labeled oocyte lipids with [32P]orthophosphate and measured the radioactivity in phospholipids after insemination. Fertilization resulted in a rapid, transient loss of radioactivity from PtdInsP2. Because the radioactivity in phosphatidylinositol 4-phosphate and other phospholipids did not change, the loss of radioactivity from PtdInsP2 is most likely due to its hydrolysis, yielding InsP3 and diacylglycerol. The latter compound activates protein kinase C which has also been shown to be involved in regulating Spisula oocyte GVBD. Since both of these compounds appear to be early products of fertilization, they could coordinately activate Ca2+- and protein kinase C-dependent processes involved in Spisula oocyte GVBD. These data indicate that egg activation in this protostome includes pathways similar to those found in deuterostome eggs and in other eukaryotic cells.     

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.     

Molecular species of phosphatidylinositol, phosphatidic acid and diacylglycerol in a phytohemagglutinin-stimulated T-cell leukemia line

Addition of phytohemagglutinin to JURKAT cells, a human T-cell leukemia line, induced a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (and may also be phosphatidylinositol 4-phosphate) and an accumulation of phosphatidic acid. The accumulation and disappearance of the various molecular species of phosphatidic acid, diacylglycerol and phosphatidylinositol (PtdIns) in response to phytohemagglutinin was studied in JURKAT cells. The cells were prelabeled with [2-3H]glycerol for 2 days and 3H-labeled lipids were isolated from the cells after incubation for 2 min at 37 degrees C in the absence or in the presence of phytohemagglutinin. The isolated 3H-labeled lipids were separated into individual molecular species by reverse-phase HPLC after conversion to their 1,2-[3H]diacylglycerol acetate derivatives either by acetolysis or by acetylation. Stimulation with phytohemagglutinin induced a 2-fold increase in [3H]phosphatidic acid. The molecular species of the accumulated [3H]phosphatidic acid consisted of polyenoic species, which were almost absent in the [3H]phosphatidic acid of the unstimulated cells. Stearoylarachidonoyl species of [3H]phosphatidic acid accumulated most prominently. Although an accumulation of [3H]diacylglycerol was hardly measurable in the phytohemagglutinin-stimulated cells, the HPLC analysis of the molecular species of [3H]diacylglycerol showed a 2-fold increase in the stearoylarachidonoyl species in the stimulated cells. Stimulation with phytohemagglutinin had almost no effect on the composition of molecular species of [3H]PtdIns. The stearoylarachidonyl species is the most abundant molecular species of PtdIns in JURKAT cells. These results suggest that the [3H]diacylglycerol moiety of [3']phosphatidic acid originates from inositol lipid(s). The results also suggest a rapid and preferential phosphorylation of the diacylglycerol formed by receptor-stimulated hydrolysis of inositol lipid(s).     

Stimulus-responsive and rapid formation of inositol pentakisphosphate in cultured adrenal chromaffin cells

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with high K+ (56 mM) and nicotine (10 microM), a large and transient increase in [3H]inositol 1,3,4,5,6-pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s and then declined to the basal level at 2 min. The time course of accumulation of InsP5 was parallel to that of [3H]inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Angiotensin II (Ang II) (10 microM) rapidly accumulated InsP5, but the level was sustained for 2 min. With a slower time course and a lesser amount than InsP5, high K+, nicotine, and Ang II caused an accumulation of [3H]inositol 1,3,4,5-tetrakisphosphate and [3H]inositol hexakisphosphate. Veratridine (100 microM), maitotoxin (10 ng/ml), ATP (30 microM), platelet-derived growth factor (10 ng/ml), and endothelin (10 ng/ml) also induced the InsP5 accumulation. High K+, nicotine, veratridine, and maitotoxin induced an increase in 45Ca2+ uptake, whereas Ang II, ATP, platelet-derived growth factor, and endothelin did not cause 45Ca2+ uptake. Nifedipine, a calcium channel antagonist, inhibited the high K(+)-induced InsP5 accumulation but failed to affect the Ang II-induced InsP5 accumulation. In an EGTA-containing and Ca2(+)-depleted medium, the high K(+)-induced InsP5 accumulation was completely inhibited, whereas the InsP5 accumulation induced by Ang II was not significantly inhibited. 12-O-tetradecanoylphorbol-13-acetate inhibited partially the Ang II-induced InsP5 accumulation but failed to inhibit the high K(+)-induced accumulation. In those experiments, the changes of InsP5 accumulation were closely correlated to those of Ins(1,4,5)P3. In the chromaffin cell homogenate, [3H] Ins(1,4,5)P3 was converted eventually to [3H]InsP5 through [3H]inositol 1,3,4,6-tetrakisphosphate. Taken together, the above results suggest that InsP5 is rapidly formed by a variety of stimulants and that the formation of InsP5 may occur through two mechanisms, i.e. Ca2+ uptake-dependent and Ca2+ uptake-independent ones in cultured adrenal chromaffin cells.     

Stimulatory and Inhibitory Effects of N-Methyl-D-Aspartate on 3H-Inositol Polyphosphate Accumulation in Rat Cortical Slices

The actions of the excitatory amino acid N-methyl-D-aspartate (NMDA) on the accumulation of 3H-inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose-dependent accumulation of only [3H]inositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1,3,4,5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol-induced accumulation of 3H-inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 microM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+ resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 microM NMDA on carbachol responses. These studies, therefore, reveal Ca(2+)-dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.