Biomedical subjects

J N Fain

Publications and source records attributed to J N Fain.

At least 37 records · Page 2Linked to original sources

PubMed1997

Wortmannin converts insulin but not oxytocin from an antilipolytic to a lipolytic agent in the presence of forskolin.

Insulin is an important regulator of glucose transport and lipolysis in adipocytes. The present studies compared the effects of insulin in rat adipocytes with the effects of oxytocin and peroxovanadate, which mimic some effects of insulin. The antilipolytic effects of peroxovanadate and oxytocin were unaffected by 500 nmol/L wortmannin, which blocked the antilipolytic action of insulin. However, wortmannin, which is a relatively specific inhibitor of phosphatidylinositol 3-kinase, did block most of the stimulation of glucose metabolism by peroxovanadate while having little effect on that due to oxytocin. Under appropriate conditions, it was also possible to demonstrate a lipolytic action of insulin, especially with low (0.1 to 1 nmol/L) concentrations of insulin after exposure of adipocytes to 50 nmol/L wortmannin. The data provide additional support for the hypothesis that oxytocin and peroxovanadate affect adipose tissue metabolism by mechanisms distinctly different from those involved in insulin action.

Adipocytes↗

PubMed1996

Effect of K+-induced depolarization on carbachol-stimulated inositol tetrakisphosphate accumulation in rat cerebrocortical slices.

Carbachol-stimulated accumulation of labeled IP4 or of total Ins 1,3,4,5-P4 in rat brain cortical slices was maximal in buffer containing 10 mM K+. Iso-osmotic elevation of extracellular K+ to 30 mM did not affect total Ins 1,3,4,5-P4 accumulation but did enhance carbachol stimulated Ins 1,4,5-P3 accumulation. Iso-osmotically elevated K+ suppressed carbachol stimulated accumulation of labeled IP4 while enhancing accumulation of labeled inositol mono-, bis- and trisphosphates. High K+ alone increased basal accumulation of labeled inositol mono-, bis- and trisphosphates, and total Ins 1,4,5-P3, while having no significant effect on accumulation of labeled IP4 or total Ins 1,3,4,5-P4. Long-term incubation with hyper-osmotically elevated K+ potentiated carbachol-stimulated Ins 1,3,4,5-P4 accumulation at 5 min. However, hyper-osmotically elevated K+ suppressed accumulation of labeled IP4 due to carbachol. These results indicate that there is no short-term effect of iso-osmotically elevated K+ on carbachol-stimulated total Ins 1,3,4,5-P4 accumulation. Furthermore, elevating K+ above 10 mM either iso-osmotically or hyper-osmotically suppresses carbachol stimulated accumulation of labeled IP4. The results suggest that the altered Na+/K+ ratio influenced the production of inositol tetrakisphosphates and emphasize the important role of cations such as Na+, K+, and Ca2+ in the receptor-mediated inositol response. Moreover, the results underscore the unique ability of carbachol (a cholinergic agonist) to stimulate significant accumulation of inositol tetrakisphosphate.

Animals↗

PubMed1996

Inhibition by NMDA of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices.

The present studies examined the effect of NMDA on carbachol-stimulated accumulation of inositol polyphosphates, with emphasis on the accumulation of inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4), at short time periods in rat brain cortical slices. There was a stimulatory effect of NMDA on accumulation of labeled inositol mono-, bis- and trisphosphates but not on labeled inositol tetrakisphosphates. In the presence of carbachol Ins 1,3,4,5-P4 accumulation was preferentially inhibited by NMDA at early time periods (within 30 seconds after NMDA addition). Subsequently, total phosphoinositide breakdown was inhibited by NMDA. NMDA did not stimulate accumulation of total Ins 1,3,4,5-P4 but immediately inhibited carbachol stimulated accumulation of Ins 1,3,4,5-P4. The inhibitory effect of NMDA (1 mM) was not mimicked by increasing K+ in the medium from 10 to 30 mM. However 30 mM K+ reversed the inhibitory effect of 1 mM NMDA on carbachol-stimulated Ins 1,3,4,5-P4. Parallel experiments with veratridine (a sodium channel activator) suggest that the early inhibitory effects of NMDA on Ins 1,3,4,5-P4 accumulation are not due to decreases in ATP availability or elevations in intracellular Na+. These data indicate that NMDA increases inositol mono-, bis- and trisphosphate accumulation while blocking muscarinic cholinergic stimulated accumulation of Ins 1,3,4,5-P4.

Adenosine Triphosphate↗

PubMed1996

Insulin sensitizes beta-agonist and forskolin-stimulated lipolysis to inhibition by 2',5'-dideoxyadenosine.

In isolated rat adipocytes incubated in the absence of insulin, 2',5'-dideoxyadenosine blocked the increase in total adenosine 3',5'-cyclic monophosphate (cAMP) accumulation due to beta 1- or beta 3-catecholamine agonists and forskolin without affecting their stimulation of lipolysis. The inhibition of cAMP accumulation by 2',5'-dideoxyadenosine was not reflected in the total cytosolic cAMP-dependent protein kinase A activity, suggesting that the inhibition of cAMP occurred in cellular compartments distinct from those involved in the regulation of bulk protein kinase A activity. However, there was a good correlation between effects of lipolytic agents on cytosolic protein kinase A activity in fat cell extracts and lipolysis. Furthermore, it was possible to see an inhibition of the increase due to beta-agonists in cAMP accumulation, protein kinase A activity, and lipolysis by 2',5'-dideoxyadenosine in the presence of insulin. These data suggest that the readily measurable accumulation of cAMP seen with catecholamines in the absence of insulin is in a compartment separate from that involved in protein kinase A activation.

Adipocytes↗

PubMed1996

Enhanced desensitization and phosphorylation of the beta 1-adrenergic receptor in rat adipocytes by peroxovanadate.

Peroxovanadate (PVN) is an insulin-like agent that inhibits the dephosphorylation of the insulin receptor kinase. PVN inhibited the lipolytic action of 0.1 microM isoproterenol by 88%, which is a relatively specific beta 1 catecholamine agonist at this concentration, but was largely ineffective against beta 3 agonists or forskolin. To determine whether PVN-mediated desensitization of the beta 1 AR was associated with enhanced phosphorylation, we immunoprecipitated the beta 1 AR from rat adipocytes that were metabolically labeled with 32PO4. Isoproterenol enhanced the net phosphorylation of the beta 1 AR by 8 +/- 2-fold over control. PVN increased the net phosphorylation of the beta 1 AR by 5 +/- 0.5-fold, and together with isoproterenol, they enhanced the phosphorylation of the beta 1 AR by 2-fold over isoproterenol alone. Phosphoamino acid analysis of the phosphorylated receptor revealed phosphate incorporation into serine that was proportional to the radioactivity incorporated into the immunoprecipitated receptor. PVN inhibited the serine/threonine phosphatase calcineurin, suggesting that inhibition of receptor dephosphorylation may play a role in the actions of PVN. Cyanogen bromide cleavage of the phosphorylated beta 1 AR generated a phosphoprotein with a molecular mass consistent with carboxyl-terminal phosphorylation. Furthermore, the magnitude of receptor phosphorylation by isoproterenol was 3-fold larger than that due to forskolin, suggesting that beta 1 AR is a substrate for the beta AR kinase that phosphorylates carboxyl-terminal residues in the beta(2) AR. Our findings suggest that PVN may be a powerful new tool with which to study the phosphorylation of other G protein-coupled receptors.

Adipocytes↗

PubMed1995

Inhibition by veratridine of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices.

The present studies examined the inhibitory effect of veratridine (a Na+ channel activator) on carbachol (a cholinergic agonist) stimulated inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of inositol 1,3,4,5-tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of inositol 1,4,5 trisphosphate accumulation. The inhibitory effect of veratridine on carbachol stimulated inositol 1,3,4,5-tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 microM veratridine on inositol 1,3,4,5-tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylyl cyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P4 formation from Ins 1,4,5-P3.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Carbachol, but not norepinephrine, NMDA, ionomycin, ouabain, or phorbol myristate acetate, increases inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices.

Ionomycin, a Ca2+ ionophore, stimulated phosphoinositide breakdown in rat brain cortical slices incubated in the presence of 1.2 mM Ca2+, but, unlike muscarinic cholinergic stimulation, it had little effect on inositol 1,3,4,5-tetrakisphosphate accumulation. However, at 2 min, the increase in inositol 1,4,5-trisphosphate due to 10 microM ionomycin was equivalent to that seen with 1 mM carbachol. Phorbol 12-myristate 13-acetate or high K+ (30 mM) increased inositol 1,4,5-trisphosphate, but not inositol 1,3,4,5-tetrakisphosphate accumulation. The stimulation of inositol 1,4,5-trisphosphate accumulation due to ionomycin, unlike that seen with carbachol, was abolished in buffer containing 0.2 mM Ca2+. The increase in inositol 1,3,4,5-tetrakisphosphate accumulation in brain slices due to 1 mM carbachol ranged from 55 to 68% of that for inositol 1,4,5-trisphosphate. Norepinephrine, NMDA, veratridine, and ouabain also increased inositol 1,4,5-trisphosphate, but had minimal effects on inositol 1,3,4,5-tetrakisphosphate accumulation. These results suggest that there is something unique about the stimulation of inositol 1,3,4,5-tetrakisphosphate accumulation by carbachol, which is also the only one of these agents that is able to activate phosphoinositidase C beta 1 in isolated rat brain membranes.

Animals↗

PubMed1993

Modulation of the phospholipase C activity in rat brain cortical membranes by simultaneous activation of distinct monoaminergic and cholinergic muscarinic receptors.

The activation of phospholipase C (PLC) was examined in membranes of rat cerebral cortex simultaneously exposed to monoaminergic receptor and muscarinic receptor agonists after the treatment of membranes with two alkylating agents, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (100 microM EEDQ) and propylbenzilylcholine (10 nM PrBCM). Treatment of membranes with PrBCM results in a selective inactivation of M3 muscarinic receptors, while treatment with EEDQ results in a relative sparing of M1 muscarinic receptors. Stimulation of PLC by GTP gamma S alone in rat cortical membranes had an apparent EC50 of about 0.4 microM, but in the presence of carbachol (1 mM) was 0.09 microM. Treatment of rat cortical membranes with EEDQ or PrBCM did not modify the concentration-response curves for GTP gamma S alone, but the ability of carbachol (1 mM) to shift the EC50 of GTP gamma S was lost in PrBCM-treated membranes. We have previously shown that dopamine, working through D1-like dopamine receptors, alters the PLC response to carbachol by preventing this shift in the apparent EC50 for GTP gamma S16. When we reproduced these experiments in EEDQ- and PrBCM-treated membranes, only in EEDQ-treated membranes was dopamine able to inhibit the PLC response to carbachol. The results indicate that the post-receptor mechanisms of PLC activation are distinct for the putative M1 as opposed to M3 muscarinic receptors in rat cortical membranes. Further, there appears to be a specific interaction between D1 and M3 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Noradrenaline stimulation unbalances the phosphoinositide cycle in rat cerebral cortical slices.

Muscarinic cholinergic and alpha 1-adrenoceptor-mediated stimulation of phosphoinositide hydrolysis in rat cerebral cortex were compared by measuring carbachol- and noradrenaline-induced accumulation of various intermediates of the phosphoinositide cycle. Unlike carbachol, noradrenaline in the presence of guanosine 5'-O-(3-thiotriphosphate) did not stimulate phospholipase C activity in brain cortical membranes. In cortical slices, the efficacy of noradrenaline to stimulate accumulation of 3H-inositol phosphates and [32P]phosphatidic acid was 2.5 to threefold that of carbachol. However, noradrenaline was less effective than carbachol in stimulating accumulation of [3H]CDP-diacylglycerol and resynthesis of phosphatidylinositol. This was not due to calcium inhibition of CTP:phosphatidate cytidyltransferase or to different lithium requirements for carbachol- and noradrenaline-stimulated accumulation of [3H]CDP-diacylglycerol. The noradrenaline-induced unbalance of the phosphoinositide cycle, which was most apparent at relatively high concentrations of calcium (2.5 mM) in the incubation buffer, was qualitatively reproduced with ionomycin. The use of the alpha 1a-subtype-selective adrenoceptor antagonists WB4101 and 5-methylurapidil revealed a single alpha 1a-like component mediating the effects of noradrenaline. Our results suggest that the primary mechanism for phospholipase C activation by brain alpha 1 adrenoceptors involves an increase in intracellular calcium concentration.

Animals↗

PubMed1993

Chronic exposure of rat fat cells to insulin enhances lipolysis and activation of partially purified hormone-sensitive lipase.

The activity of adipose tissue hormone-sensitive lipase in animals with hyperinsulinemia has been reported to be increased compared with that in control animals. We examined whether this results from a direct effect of insulin on the tissue and whether it is accompanied by alteration in the regulation of lipolysis. When rat epididymal fat pads are incubated in culture medium with bovine serum albumin for 2-4 h with 2 ng/ml or 50 microU/ml of insulin, hormone-sensitive lipase activity in the postmicrosomal supernatant fraction after acid precipitation and activation with ATP-Mg2+ increases significantly compared with preparations from tissues incubated with the vehicle. The specific activities of hormone-sensitive lipase in sonicates of adipocytes after primary culture with insulin at concentrations from 10 to 4000 ng/ml (250 microU to 100 mU/ml) increase in an insulin-dose-related manner. Lipolysis in response to 10(-7) M isoproterenol also increases in an insulin-dose-dependent manner. Enhancement of isoproterenol-mediated lipolysis is not attributable to a difference in the triglyceride content of the cells. Lipolysis caused by the beta-agonist could be completely blocked by the simultaneous presence of insulin in both control and insulin-treated cells reflecting normal responsiveness of both types of cells to the acute effect of insulin. Although an increase in lipolysis is seen with norepinephrine and growth hormone after insulin treatment, other lipolytic agents such as ACTH, thyrotropin, and glucagon evoke similar responses in insulin-treated and control cells. The simultaneous presence of growth hormone and insulin during the 16-h culture results in additive effects on the subsequent response of the cells to 10(-7) M isoproterenol compared with the responses of the cells cultured with each hormone alone. beta-Agonist-mediated cAMP accumulation in the presence of Ro-20.1724, a specific phosphodiesterase inhibitor, is significantly higher in cells cultured in the presence of insulin than in control cells. Forskolin (1-25 microM) increases the lipolytic responses of insulin-treated cells compared with control cells, but the maximal response of the insulin-treated cells to forskolin is lower than that to isoproterenol. We conclude that changes produced by chronic insulin treatment involve more than one site along the lipolytic cascade.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1993

Differential effects of alkylating agents on the multiple muscarinic receptor subtypes linked to activation of phospholipase C by carbachol in rat brain cortical membranes.

Muscarinic cholinergic receptor function in rat brain cortex was characterized by performing binding assays with [3H](-)quinuclidinyl benzilate ([3H]QNB) in parallel with assays of phospholipase C (PLC) activation by carbachol using membrane preparations and exogenous [3H]-phosphatidylinositol 4,5-bisphosphate ([3H]PIP2). Competitive binding studies revealed high- and low-affinity binding sites for the receptor antagonists, pirenzepine, methoctramine and the p-fluoro analog of hexahydro-sila-difenidol (p-F-HHSiD). Carbachol-stimulated [3H]-phosphatidylinositol 4,5-biphosphate breakdown was specifically inhibited by pirenzepine and p-F-HHiSD. The inhibition curves for these antagonists were best described by interactions at two sites. There was quantitative agreement between the antagonist affinity constants and the proportion of high- and low-affinity sites derived in functional and binding studies. The characteristics of the putative subtypes of muscarinic receptors and their stimulation of phospholipase C was examined after treatment with two alkylating agents, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline and propylbenzilylcholine mustard. Loss of receptors was closely correlated with loss of PLC activation by carbachol, without alteration of the EC50 value (21 microM) of this agonist, clearly demonstrating a lack of receptor reserve. When both alkylating treatments were adjusted to induce a decrease of 60% in the maximal number of [3H]QNB binding sites, a similar (60%) reduction in the maximal effect of carbachol on PLC activation was found. However, the characteristics of the remaining receptors after the treatment with the two alkylating agents differ markedly as determined by competition of pirenzepine, p-F-HHSiD and methoctramine for [3H]QNB binding, and for inhibition of carbachol-stimulated phospholipase C by pirenzepine and p-F-HHSiD.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗

PubMed1992

Alpha 1-adrenergic receptor-mediated activation of phospholipase D in rat cerebral cortex.

We have investigated phospholipase D activity in rat brain cortical slices prelabeled with [32P]orthophosphoric acid. In the presence of ethanol (170 mM), norepinephrine stimulated, in a dose-dependent manner (EC50 = 2.2 microM), the accumulation of [32P]phosphatidylethanol as a result of phospholipase D activity. Norepinephrine-stimulated phospholipase D activity was completely inhibited by prazosin, a specific alpha 1-adrenergic antagonist (Ki = 2.8 nM). However, no accumulation of phosphatidylethanol was observed in the presence of the muscarinic agonist carbachol. The Ca2+ ionophore ionomycin and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also stimulated [32P]phosphatidylethanol accumulation in cortical slices, in a dose- and time-dependent manner, whereas the inactive phorbol, 4 alpha-phorbol 12,13-didecanoate, did not stimulate phospholipase D activity. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, two potent inhibitors of protein kinase C, inhibited PMA and ionomycin stimulation of phospholipase D activity, but did not affect the response to norepinephrine. Furthermore, the effects of PMA and norepinephrine were additive. Differences between PMA and norepinephrine stimulation of phospholipase D activity were also found with regard to the extracellular Ca2+ requirement and time course of phosphatidylethanol accumulation. No stimulation of phospholipase D activity by norepinephrine was observed in slices from cerebellum, a brain area with a low density of alpha 1-adrenergic receptors, while the effect of PMA was greater in the cerebellum than in cortical or hippocampal slices. These results strongly suggest that activation of phospholipase D in cortical slices by norepinephrine and PMA involve different mechanisms.

Animals↗

PubMed1992

Ethanol is a potent stimulator of phosphatidylcholine breakdown in cultured rat hepatocytes.

Addition of ethanol (17 to 340 mM) to cultured rat hepatocytes stimulated the breakdown of phosphatidylcholine phospholipases D and C as measured by an increase in the rate of release of choline and phosphocholine into the medium. The effects of ethanol were mimicked by propanol, dimethylsulfoxide and to a lesser extent methanol. The magnitude of the stimulation seen with ethanol was equivalent to and additive to that produced by glucagon vasopressin, norepinephrine, A23187 or PMA. In contrast, ethanol (340 mM) stimulated PI-specific phospholipase C activity by less than 20%. An equivalent stimulation of PC-specific phospholipase D and C was seen with as little as 20 mM ethanol and a 100% increase was seen with 340 mM ethanol. Ethanol did not significantly affect the ability of vasopressin, norepinephrine, ATP or A23187 to stimulate PI-specific phospholipase C. It is concluded that while ethanol is only a weak stimulator of PI-specific phospholipase C, it is a potent stimulator of phosphatidylcholine breakdown in rat hepatocytes.

Adenosine Triphosphate↗

PubMed1992

Magnesium-dependent inhibition of agonist-stimulated phosphoinositide breakdown in rat cortical slices by excitatory amino acids.

The excitatory amino acid agonists kainate, N-methyl-D-aspartate (NMDA), and quisqualate inhibited ligand-stimulated phosphoinositide hydrolysis in rat cortical slices. The NMDA channel blocker MK-801 antagonized the inhibition by NMDA but had no effect on the inhibition due to kainate or quisqualate. The antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the effects of quisqualate and kainate but not the effect of NMDA. These data indicate that activation of the NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate types of ionotropic receptors has the same effect. In membranes prepared from cortical slices, there was no inhibition of carbachol-stimulated phosphoinositidase C activity by excitatory amino acids, suggesting that excitatory amino acids indirectly affect carbachol-stimulated phosphoinositide hydrolysis. The inhibition by excitatory amino acids of carbachol-stimulated phosphoinositide breakdown was dependent on extracellular Mg2+ and was abolished by procedures that increase intracellular Ca2+. Veratridine inhibition of carbachol-stimulated phosphoinositide hydrolysis was reversed by ouabain but not by other procedures that increase intracellular Ca2+. In contrast to excitatory amino acids, veratridine potentiated carbachol-stimulated phosphoinositide breakdown in the presence of 10 mM extracellular Mg2+. These data suggest that excitatory amino acids inhibit carbachol-stimulated phosphoinositide breakdown in rat cortex by lowering intracellular Ca2+ through a mechanism dependent on extracellular Mg2+.

Amino Acids↗

PubMed1992

Quisqualate-stimulated phosphatidylinositol breakdown in rat cerebellar membranes.

The effect of quisqualate, an excitatory amino acid agonist, on the breakdown of exogenously added phosphatidylinositol was investigated in a membrane preparation from the cerebellum of young rats. Quisqualate stimulated phospholipase C activity in a dose-dependent manner in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Half-maximal activation of the quisqualate response required 0.15 microM GTP gamma S and was optimal at a free Ca2+ concentration of 300 nM. Phosphoinositide breakdown was also stimulated by quisqualate using either exogenous phosphatidylinositides 4,5-bisphosphate or endogenous labeled phosphoinositides as the substrate for phospholipase C in cerebellar membranes. In the presence of guanine nucleotides, other excitatory amino acid agonists, such as L-glutamate, trans-D,L-1-aminocyclopentyl-1,3-dicarboxylic acid, and ibotenate, but not N-methyl-D-aspartate, stimulated phosphatidylinositol breakdown. However, quisqualate displayed the highest response among these excitatory amino acid agonists. These data indicate that there is a direct activation of phosphoinositide-specific phospholipase C by excitatory amino acids through a process dependent on the presence of guanine nucleotides.

Amino Acids↗

PubMed1992

Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes.

[3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]Ins was incorporated into a common lipid pool through both CMP-dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl-inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP-dependent or independent fashions were hydrolyzed by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and carbachol-stimulated phospholipase C. Unlike CMP-dependent labeling, however, CMP-independent incorporation of [3H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxycholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [3H]Ins was incorporated into lipids primarily through the PtdIns synthase-catalyzed reaction because of the presence of deoxycholate required to observe carbachol-stimulation of phospholipase C. Little direct breakdown of [3H]PtdIns was detected because production of myo-[3H]inositol 1-monophosphate was minimal and myo-[3H]inositol 1,4-bisphosphate was the predominant product. Although PtdIns labeling and 3H-polyphosphoinositide formation were unaffected by GTP gamma S and carbachol and had no or little lag period, GTP gamma S- and carbachol-stimulated appearance of 3H-Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to 3H-Ins phosphates was restricted to a narrow range of free calcium concentrations (10-300 nM). These results show the concerted activities of PtdIns synthase, PtdIns 4-kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide-dependent agonist stimulation of phospholipase C in a brain membrane system using [3H]Ins as labeled precursor.

Animals↗

PubMed1991

Activation of membrane protein kinase C by glucagon and Ca(2+)-mobilizing hormones in cultured rat hepatocytes. Role of phosphatidylinositol and phosphatidylcholine hydrolysis.

We found that glucagon stimulated membrane protein kinase C (PKC) activity and phosphatidylcholine hydrolysis in 24 h-cultured rat hepatocytes. Phorbol myristate acetate, 8-bromo cyclic AMP, vasopressin, noradrenaline and the Ca2+ ionophore A23187 also stimulated membrane PKC activity. However, only vasopressin and noradrenaline stimulated inositol phosphate accumulation, whereas all agonists stimulated the rate of release of water-soluble choline metabolites into the medium. Choline, and to a much lesser extent phosphocholine, were released, suggesting predominantly phospholipase D activation. This was supported by the finding that the accumulation of phosphatidate and diacylglycerol was enhanced by the agents in [3H]myristate-labelled hepatocytes, as was [32P]phosphatidylethanol formation. Since the time courses for the release of choline into the medium and the accumulation of phosphatidate and diacylglycerol caused by vasopressin and glucagon were similar, the more rapid activation of PKC by vasopressin probably reflects diacylglycerol formation from phosphoinositide breakdown. The inability of glucagon to stimulate inositol phosphate production was not due to the prolonged culture, since similar results were obtained in 4 h cultures. We conclude that the stimulation of membrane PKC activity by glucagon correlates with accumulation of diacylglycerol and phosphatidate derived from the hydrolysis of phosphatidylcholine.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Stimulation of phospholipase A2 activity in human platelets by trypsin and collagen.

Type I collagen enhanced human platelet phospholipase A2 activity whether added to platelet-rich plasma or washed platelets. The stimulatory effect of type I collagen on platelet membrane phospholipase A2 activity was also observed in a cell-free system utilizing platelet membranes. The release of arachidonic acid was enhanced by types I and III but not by type V collagen. The activation of platelet phospholipase A2 by type I collagen was inhibited by soybean trypsin inhibitor and mimicked by trypsin. However, type I collagen addition was not associated with any detectable changes in platelet membrane proteins while trypsin altered many proteins. These results point to acid soluble phospholipase A2 activity of platelets as an enzyme activated by type I collagen.

Blood Platelets↗