Phospholipase C activity in rat kidney. Effect of deoxycholate on phosphatidylinositol turnover

Rat renal cortical and medullary slices incorporate [14C]arachidonate into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerols. The percent distribution of [14C]arachidonate among the various phospholipids is similar in renal cortex and medulla, although the total amount of radioactively labeled phospholipids is higher in the renal medulla. Subsequent incubation of prelabeled slices in the presence of deoxycholate induces a loss of radioactivity from [14C]phosphatidylinositol, with a concomitant increase in 1,2-[14C]diacylglycerol. Neutral lipids are not affected. The degradation of phosphatidylinositol to [14C]diacylglycerol indicates the presence of phospholipase C activity. Renal medulla seems to be more sensitive to deoxycholate than the renal cortex. Deoxycholate also induces slightly the disappearance of some 14C radioactivity from phosphatidylethanolamine and phosphatidylcholine, which might reflect activation of phospholipase A2. The activity of the phospholipase C could constitute the first step in the sequence of reactions that leads to the release of arachidonic acid.     

Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells

In PC12 pheochromocytoma cells whose phospholipids had been prelabelled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1-2 min. before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells: bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.     

Effect of platelet-activating factor on arachidonic acid metabolism in renal epithelial cells

We studied the effects of platelet-activating factor (PAF-acether) on phospholipase activity in renal epithelial cells. When platelet-activating factor was added to renal cells prelabeled with [3H]arachidonic acid, it induced the rapid hydrolysis of phospholipids. Up to 26% of incorporated [3H]arachidonic acid was released into the medium from renal cells. After the addition of PAF-acether, the degradation of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were observed. The amount of [3H]arachidonic acid released were comparable to the losses of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. In renal cells biosynthetically labeled by incorporation of [3H]choline into cellular phosphatidylcholine, lysophosphatidylcholine and sphingomyelin, the range of concentrations of PAF-acether-induced hydrolysis of labeled phosphatidylcholine were approximately equal to the amounts of lysophosphatidylcholine produced. We also observed a transient rise of diacylglycerol after the addition of platelet-activating factor to these cells. To test for action of phospholipase C, the accumulations of [3H]choline, [3H]inositol and [3H]ethanolamine were determined. The radioactivities in choline and ethanolamine showed little or no change. An increase in inositol was detectable within 1 min and it peaked at 3 min. These results indicate that platelet-activating factor stimulates phospholipase A2 and phosphatidylinositol-specific phospholipase C activity in renal epithelial cells. These phospholipase activities were Ca2+ dependent. Moreover, PAF-acether enhanced changes in cell-associated Ca2+. These results suggest that the increased Ca2+ permeability of cell membrane stimulates phospholipases A2 and C in renal epithelial cells. Prostaglandin biosynthesis was also enhanced in these cells by platelet-activating factor.     

Platelet-activating factor stimulates metabolism of phosphoinositides via phospholipase A2 in primary cultured rat hepatocytes

Addition of platelet-activating factor (PAF) to cells doubly labeled with [14C]glycerol plus [3H]arachidonic acid resulted in a transient decrease of [14C]glycerol-labeled phosphatidylinositol (PI) and a transient increase of [14C]glycerol-labeled lysophosphatidylinositol (LPI). [3H]Arachidonate-labeled PI, on the other hand, decreased in a time-dependent manner. The radioactivity in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and phosphatidylserine did not change significantly. The 3H/14C ratio decreased in PI in a time-dependent manner, suggesting the involvement of a phospholipase A2 activity. Although PAF also induced a gradual increase of diacylglycerol (DG), the increase of [14C]glycerol-labeled DG paralleled the loss of triacyl [14C]glycerol and the 3H/14C ratio of DG was 16 times smaller than that of PI. Thus, DG seemed not to be derived from PI. In myo- [3H]inositol-prelabeled cells, PAF induced a transient decrease of [3H]phosphatidylinositol-4,5-bis-phosphate (TPI) and [3H]phosphatidylinositol-4-phosphate (DPI) at 1 min. PAF stimulation of cultured hepatocytes prelabeled with 32Pi induced a transient decrease of [32P]polyphosphoinositides at 20 sec to 1 min. [32P]LPI appeared within 10 sec after stimulation and paralleled the loss of [32P]PI. [3H]Inositol triphosphate, [3H]inositol diphosphate, and [3H]inositol phosphate, which increased in a time-dependent manner upon stimulation with adrenaline, did not accumulate with the stimulation due to PAF. These observations indicate that PAF causes degradation of inositol phospholipids via phospholipase A2 and induces a subsequent resynthesis of these phospholipids.     

Regulation of diacylglycerol levels in carbachol-stimulated pancreatic acinar cells: relationship to the breakdown of phosphatidylcholine and metabolism to phosphatidic acid.

Rat pancreatic acinar cells prelabeled with [14C]palmitic acid and then exposed to carbachol (CCh) exhibited a time-dependent increase in 1,2-[14C]diacylglycerol ([14C]DAG) levels, which was first detected at 2 min and then continued to rise in a linear manner. There was a concomitant increase in [14C]phosphatidic acid, which plateaued after 2 min and then remained at steady-state levels. CCh also promoted the release of phosphocholine, but not choline, within 60 s and caused a decrease in [14C]phosphatidylcholine in cells prelabeled with [14C]glycerol after 15 min. The inability to detect a rise in [14C]phosphatidylethanol accumulation and a fall in [14C]phosphatidate levels in [14C]palmitate prelabeled cells after exposure to CCh plus ethanol documented the absence of a phospholipase D-mediated pathway. The rapid phosphorylation of diglyceride in homogenates from unstimulated and carbachol-treated cells increased with increasing concentrations of exogenous substrate, thereby affirming that carbachol stimulates the phosphorylation of DAG by promoting the accumulation of the diglyceride. These collective findings provide evidence for the existence of an integrative control mechanism for regulating endogenous DAG levels during pancreatic acinar cell activation involving phosphatidylcholine-specific phospholipase C and DAG kinase.     

Generation of Arachidonic Acid and Diacylglycerol Second Messengers from Polyphosphoinositides in Ischemic Fetal Brain

Intracerebral administration of [3H]arachidonic acid ([3H]ArA) into 19-20-day-old rat embryos, resulted in a rapid incorporation of label into brain lipids. One hour after injection, 55.6 +/- 8.2, 18.0 +/- 3.4, and 13.7 +/- 1.3% of the total radioactivity was associated with phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, respectively. Approximately 10% of radioactivity was found acylated in neutral lipids of which free ArA comprised only 1.5 +/- 0.2% of the total radioactivity. Complete restriction of the maternal-fetal circulation for < or = 40 min did not affect the rate of [3H]ArA incorporation (t1/2 = 2 min) into fetal brain lipids, suggesting an effective acylation mechanism that proceeds irrespective of the impaired blood flow. After a short restriction period (5 min), the radioactivity in diacylglycerol was elevated by 50%. After a longer restriction period (20 min), the radioactivity in the free fatty acid and diacylglycerol fractions increased to values of 130 and 87%, respectively. Polyphosphoinositides prelabeled with either [3H]ArA or 32P were rapidly degraded after 5 min of ischemia. After 20 min, the decrease in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate radioactivity was 47 and 70%, respectively. Double labeling of phospholipids with [14C]palmitic acid and [3H]ArA indicated a preferential loss of [3H]ArA within the polyphosphoinositide species after 20 min, but not after 5 min of ischemia. The specific activity of [14C]palmitate remained unchanged. The current data suggest phospholipase C-mediated diacylglycerol formation at the beginning of the insult followed by a phospholipase A2-mediated ArA liberation at a later time, both enzymes presumably acting preferentially on polyphosphoinositide species.     

Carbachol and Bradykinin Increase the Production of Diacylglycerol from Sources Other than Inositol-Containing Phospholipids in PC12 Cells

Both carbachol and bradykinin increased diacylglycerol formation in PC12 pheochromocytoma cells. The effect of carbachol was apparent only in cells that had been treated with nerve growth factor. Incubation of the cells in Ca2(+)-free medium attenuated carbachol-stimulated diacylglycerol formation but did not reduce the response to bradykinin. Pretreatment of the cells with pertussis toxin did not affect either carbachol- or bradykinin-stimulated diacylglycerol formation; therefore, the inhibitory guanine nucleotide Gi probably does not mediate this response. The time course of carbachol-stimulated diacylglycerol accumulation did not coincide with the time course of inositol 1,4,5-trisphosphate (IP3) production. IP3 was elevated at the earliest time measured, 15 s, and then slowly declined so that by 5 min IP3 levels were only 50% of maximal. Diacylglycerol levels, in contrast, were not elevated for the first 2 min and then peaked at 5 min. These data indicate that hydrolysis of phosphatidylinositol 4,5-bisphosphate was not the major source of the diacylglycerol peak at 5 min. To investigate the source of diacylglycerol, I examined the fatty acid composition of the diacylglycerol by prelabeling the cells with [3H]palmitic acid and [14C]stearic acid. The 14C/3H ratio in diacylglycerol should reflect the phospholipid(s) from which it is derived. The 14C/3H ratio of the increment in diacylglycerol produced by carbachol and bradykinin was intermediate between the 14C/3H ratios of phosphatidylcholine and phosphatidylinositol. The 14C/3H ratio in triacylglycerol was similar to that of phosphatidylcholine. These data indicate that carbachol and bradykinin stimulate the formation of diacylglycerol from sources other than inositol-containing phospholipids; phosphatidylcholine and triacylglycerol are two possible sources of this diacylglycerol.     

Secretogogue-stimulated phosphatidylinositol breakdown in the exocrine pancreas liberates arachidonic acid, stearic acid, and glycerol by sequential actions of phospholipase C and diglyceride lipase

When mouse pancreatic "minilobules" prelabeled with either [14C]arachidonic acid (AA), [14C]stearic acid (SA), or [3H]glycerol were stimulated with the secretogogue, caerulein, there was a 60-70% loss in radioactivity in phosphatidylinositol (PI) at 30 min. This loss was accompanied by the formation of [14C] phosphatidic acid (PA), [14C]diacylglycerol (DG), [14C] triacylglycerol (TG), and free [14C]AA, [14C]SA, and [3H]glycerol. The loss in radioactive PI was the same as the loss in chemically measured PI-phosphorus. Thirty to fifty per cent of the caerulein-induced loss of prelabeled PI could be accounted for as free [14C]AA, [14C]SA, or [3H]glycerol. Increased incorporation of fatty acid or glycerol residues into DG, PA, and TG accounted for the balance of the loss in PI. The specific DG-lipase inhibitor, RHC 80267, markedly inhibited the caerulein-stimulated release of [14C]AA, [14C]SA, and [3H]glycerol and roughly doubled the caerulein-induced increment in [14C]AA-, [14C]SA-, or [3H]glycerol-labeled DG, showing that the source of the caerulein-induced increment in fatty acids and glycerol was DG. When the PI was prelabeled with either [32P] orthophosphate, [3H]myoinositol, or [3H]glycerol, only 1% or less of the radioactivity in PI was in lysophosphatidylinositol (LPI), and there was no increase in radioactivity in LPI on stimulation with caerulein. These observations, taken together, argue strongly for a phospholipase C-catalyzed breakdown of PI followed by DG-lipase and argue against any significant involvement of phospholipase A2 in PI degradation in mouse pancreas. The formation of substantial amounts of free [14C]AA on stimulation supports the view that, among other things, the phosphoinositide effect in the exocrine pancreas serves to generate arachidonate (and its metabolites). The release of appreciable amounts of free fatty acids and glycerol shows that a significant portion of the DG formed as a result of caerulein-stimulated PI breakdown is not conserved in the phosphoinositide cycle.     

Tumor necrosis factor-alpha stimulates phosphatidylinositol breakdown by phospholipase C to coordinately increase the levels of diacylglycerol, free arachidonic acid and prostaglandins in an osteoblast (MC3T3-E1) cell line

The effects of (human recombinant) tumor necrosis factor-alpha on phosphatidylinositol breakdown, release of 1,2-diacylglycerols, mobilization of arachidonate from diacylglycerol and prostaglandin synthesis were examined in a model osteoblast cell line (MC3T3-E1). Tumor necrosis factor-alpha (10 nM) caused a specific (30%) decrease in the mass of phosphatidylinositol (and no other phospholipids) within 30 min of exposure. Tumor necrosis factor-alpha doubled the rate of incorporation of [32P]orthophosphoric acid into phosphatidylinositol, indicating that the turnover of inositol phosphate was enhanced, and increased the content of diacylglycerol in parallel with phosphatidylinositol breakdown. The cytokine (10-50 nM; 4 h) also promoted a specific release of 24-34% of the [3H]arachidonate from prelabeled phosphatidylinositol, a release of 80% of the 3H-fatty acid from the diacylglycerol pool, and a 30-fold increase in the synthesis of prostaglandin E2. The tumor necrosis factor-alpha induced liberation of [3H]arachidonate from diacylglycerol, cellular arachidonate release and the synthesis of prostaglandin E2 were each blocked by an inhibitor of diacylglycerol lipase, the compound RHC 80267 (30 microM). Therefore, we conclude that, in the MC3T3-E1 cell line, tumor necrosis factor-alpha activates a phosphatidylinositol-specific phospholipase C (phosphatidylinositol inositolphosphohydrolase; EC 3.1.4.3) to release diacylglycerol, and increases the metabolism of diacylglycerol to liberate arachidonate for prostaglandin synthesis.     

Acute effect of thyrotropin on phosphatidylinositol degradation and transient accumulation of diacylglycerol in isolated thyroid follicles

Pig thyroid follicles isolated by collagenase treatment were incubated with or without thyrotropin. 1) Within 30 seconds the intracellular diacylglycerol content was doubled but then returned to the initial level after a further 30 sec incubation. 2) In the follicles prelabeled with [³²P]-phosphate or [¹⁴C]-arachidonate, thyrotropin-dependent degradation was observed in phosphatidylinositol. 3) Diacylglycerol containing [¹⁴C]-arachidonate was increased in parallel with the accumulation of total diacylglycerol mentioned in 1). The physiological role of the rapid thyrotropin action was discussed in relation to phosphatidylinositol turnover and possible production of free arachidonate which was known to be a precursor of prostaglandin.     

Stimulation of phosphatidic acid production in platelets precedes the formation of arachidonate and parallels the release of serotonin.

Thrombin rapidly induces the formation of labeled phosphatidic acid from platelets prelabeled with [17C]arachidonate or 32PO34- and specifically decreases by 50--75% the content of phosphatidylinositol. Ionophore A23187 also stimulates phosphatidate labeling, but less effectively than thrombin. This effect on phosphatidic acid is blocked by increasing the levels of cyclic AMP by preincubation with dibutyryl cyclic AMP, cyclic AMP-phosphodiesterase inhibitors or prostacyclin. Indomethacin and eicosatetraynoic acid do not alter the production of phosphatidate, indicating independence from cyclooxygenase or lipoxygenase products. Increased turnover of [14C]- or [32P]phosphatidate occurs within 2--5 s after platelet activation by thrombin and is observed before endogenous, 14C-labeled arachidonate can be detected. The rate of phosphatidate formation parallels the induced rate of serotonin release. Release of [3H]serotonin is not affected by eicosatetraynoic acid. Phosphatidate production reflects the generation of diacylglycerol by C-type phospholipase degradation of phosphatidylinositol. Diacylglycerol and phosphatidic acid may participate in the membrane modification related to the early changes in platelet shape, release reactions or aggregation which occur on stimulation.     

Ca2+ ionophores affect phosphoinositide metabolism differently than thyrotropin-releasing hormone in GH3 pituitary cells

Thyrotropin-releasing hormone (TRH) stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) by a phospholipase C (or phosphodiesterase) and elevates cytoplasmic-free Ca2+ concentration ([Ca2+]i) in GH3 pituitary cells. To explore whether hydrolysis of PtdIns-4,5-P2 is secondary to the elevation of [Ca2+]i, we studied the effects of Ca2+ ionophores, A23187 and ionomycin. In cells prelabeled with [3H]myoinositol, A23187 caused a rapid decrease in the levels of [3H]PtdIns-4,5-P2, [3H]PtdIns-4-P, and [3H]PtdIns to 88 +/- 2%, 88 +/- 4%, and 86 +/- 1% of control, respectively, and increased [3H]inositol bisphosphate to 200 +/- 20% at 0.5 min. There was no increase in [3H] Ins-P3; the lack of a measurable increase in [3H]Ins-P3 was not due to its rapid dephosphorylation. In cells prelabeled with [14C]stearic acid, A23187 increased [14C]diacylglycerol and [14C]phosphatidic acid to 166 +/- 20% and 174 +/- 17% of control, respectively. In cells prelabeled with [3H]arachidonic acid, A23187, but not TRH, increased unesterified [3H]arachidonic acid to 166 +/- 8% of control. Similar effects were observed with ionomycin. Hence, Ca2+ ionophores stimulate phosphodiesteratic hydrolysis of PtdIns-4-P but not of PtdIns-4,5-P2 and elevate the level of unesterified arachidonic acid in GH3 cells. These data demonstrate that Ca2+ ionophores affect phosphoinositide metabolism differently than TRH and suggest that TRH stimulation of PtdIns-4,5-P2 hydrolysis is not secondary to the elevation of [Ca2+]i.     

Vasopressin-induced polyphosphoinositide and phosphatidylcholine degradation in fibroblasts. Temporal relationship for formation of phospholipase C and phospholipase D hydrolysis products

Cultured fibroblasts (REF52 cells) were employed to investigate phospholipid degradation in response to vasopressin (VP) treatment. There have been few studies in fibroblasts which characterize the pattern and relationship of phosphatidylinositol 4,5-bisphosphate (PIP2) and non-phosphoinositide hydrolysis elicited by VP. Here we demonstrate that VP-induced PIP2 hydrolysis is closely accompanied by phosphatidylcholine (PC) degradation by phospholipase D. Cells prelabeled with [3H]arachidonic acid showed rapid formation and diminution of [3H]diacylglycerol (DG) (5-15s) when treated with VP; this was accompanied by a reduction in polyphosphoinositide radioactivity. Radiolabeled inositol trisphosphate was generated with a similar time frame. In cells prelabeled with [3H]myristic acid, which is predominantly incorporated into cellular PC, VP elicited the generation of [3H]myristoyl phosphatidate (PA) as early as 15 s, in the absence of an increase in labeled DG. In the presence of ethanol the pattern of [3H]myristoyl phosphatidylethanol (PEt) formation coincided with [3H]myristoyl-PA formation in the absence of ethanol. PEt was similarly formed, in response to VP treatment, in cells prelabeled with 1-O-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine. The formation of PC-derived [3H]myristoyl-DG was characterized by a lag period of approximately 1 min, after which DG increased steadily over a 10-min period. Biphasic formation of DG was observed in cells prelabeled with [3H]arachidonic acid, and the formation of [3H]PA occurred in an uninterrupted fashion. Two protein kinase C agonists, phorbol diester and dioctanoylglycerol, elicited the formation of [3H]myristoyl-PEt. The inclusion of staurosporine, a protein kinase C inhibitor, blocked VP-induced [3H]myristoyl-PEt formation by 88%. These data demonstrate that VP elicits the coordinated hydrolysis of PIP2 by phospholipase C and PC hydrolysis by phospholipase D. This event results in the prolonged generation of PA and biphasic formation of DG. From the time courses shown, we hypothesize that the early generation of PA, heretofore ascribed to products of the polyphosphoinositide cycle, are in part derived from PC by phospholipase D.     

Evidence that phospholipid turnover is the signal transducing system coupled to serotonin-S2 receptor sites

Upon stimulation with serotonin of washed human platelets prelabeled with [32P]orthophosphate, we found an approximately 250% increase in [32P]phosphatidic acid (PA) formation, a small decrease in [32P]phosphatidylinositol 4,5-bisphosphate, and a concomitant increase in [32P]phosphatidylinositol 4-phosphate. Using [3H]arachidonate for prelabeling, [3H]diacylglycerol accumulated transiently at 10 s after addition of the agonist, [3H]PA increased but to a lower extent compared to 32P-labeled lipid, and the formation of both [3H]polyphosphoinositides increased. The serotonin-induced dose-dependent changes in [32P]PA correlate with its effect on the changes in slope of aggregation of platelets. The potency of 13 drugs to antagonize the serotonin-induced PA formation closely corresponds to both their potency to inhibit platelet aggregation and their binding affinity for serotonin-S2 receptor sites. It is suggested that at least part of the signal transducing system following activation of the serotonin-S2 receptors involves phospholipase C catalyzed inositol lipid breakdown yielding diacylglycerol which is subsequently phosphorylated to PA.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

Bradykinin-induced activation of phospholipase A2 is independent of the activation of polyphosphoinositide-hydrolyzing phospholipase C

This study evaluates the role of phosphatidylinositol 4,5-bisphosphate (PIP2) and its metabolites as possible mediators in the activation of phospholipases A2 in porcine aortic endothelial cells. We compared the time courses of bradykinin-induced turnover of phosphoinositides and the appearance of unesterified arachidonic acid (uAA) and eicosanoids. The metabolism of phosphoinositides was examined in cells prelabeled with [3H]inositol, which has a similar distribution as the endogenous inositol lipids. At 37 degrees C, bradykinin induced a rapid rise in lysophosphatidylinositol (lyso-PI) and inositol 1,4,5-trisphosphate (IP3) as well as a decrease in PIP2. Lyso-PI formation was detected at 10 s, as early as PIP2 degradation and IP3 formation. This suggests that the activation of PIP2-hydrolyzing phospholipase C and PI-hydrolyzing phospholipase A2 are simultaneous. However, at 30 degrees C, lyso-PI formation was detected in the absence of an increase in IP3 indicating that the activation of phospholipase A2 does not require the accumulation of IP3. The time course of formation of uAA and eicosanoids were examined in [3H]arachidonic acid-prelabeled cells. The 3H radioactivity was distributed among the phospholipid classes and subclasses the same as the endogenous phospholipids. Bradykinin stimulated the intracellular accumulation of uAA, detectable at 5 s, earlier than that of 1,2-diacylglycerol and phosphatidic acid. Such immediate formation of uAA further supports the notion that activation of phospholipase A2 is a very early event during the interaction of bradykinin with porcine endothelial cells, and that PIP2 hydrolysis is not prerequisite for the initial activation of phospholipase A2.     

Production of diacylglycerol by exogenous phospholipase C stimulates CTP:phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in human neuroblastoma cells.

The involvement of endogenous diacylglycerol production in the stimulation of phosphatidylcholine synthesis by exogenous phospholipase C was examined using a neuroblastoma (LA-N-2) cell line. Phospholipase C treatment (0.1 unit/ml) of intact cells stimulated CTP:phosphocholine cytidylyltransferase activity significantly more effectively than did maximally effective concentrations of the synthetic diacylglycerol sn-1,2-dioctanoylglycerol (1 mM). When added to cells together with phospholipase C, oleic acid, but not dioctanoylglycerol, further increased cytidylyltransferase activity with respect to phospholipase C treatment alone, indicating that the enzyme was not maximally activated by the lipase. This suggests that the lack of additivity of diacylglycerol and phospholipase C reflects a common mechanism of action. The time course of activation of cytidylyltransferase by phospholipase C paralleled that of [3H]diacylglycerol production in cells prelabeled for 24 h with [3H]oleic acid. Diacylglycerol mass was similarly increased. Significant elevations of [3H]oleic acid and total fatty acids occurred later than did the increases in cytidylyltransferase activity and diacylglycerol levels. No significant reduction in total or [3H]phosphatidylcholine was elicited by this concentration of phospholipase C, but higher concentrations (0.5 unit/ml) significantly reduced phosphatidylcholine content. The stimulation of cytidylyltransferase activity by phospholipase C or dioctanoylglycerol was also associated with enhanced incorporation of [methyl-14C]choline into phosphatidylcholine. Dioctanoylglycerol was more effective than phospholipase C at stimulating the formation of [14C]phosphatidylcholine, and the effects of the two treatments were additive. However, further analysis revealed that dioctanoylglycerol served as a precursor for [14C]dioctanoylphosphatidylcholine as well as an activator of cytidylyltransferase; and when corrections were made for this effect, the apparent additivity disappeared. The results indicate that the generation of diacylglycerol by exogenous phospholipase C (and possibly the subsequent production of fatty acids via diacylglycerol metabolism) activates cytidylyltransferase activity in neuronal cells under conditions in which membrane phosphatidylcholine content is not measurably reduced.     

Protein kinase C-independent activation of a novel nonspecific phospholipase C pathway by phorbol myristate acetate releases arachidonic acid for prostaglandin synthesis in MC3T3-E1 osteoblasts

The effects of phorbol myristate acetate, an activator of protein kinase C, on the release of [³H]arachidonic acid and prostaglandin synthesis were studied in an osteoblast cell line (MC3T3-E1). Phorbol myristate acetate (20 uM) liberated 16 and 55% of the [³H]arachidonate in prelabeled phosphatidylinositol and phosphatidylethanolamine, respectively, and evoked a 19-fold stimulation in the synthesis of prostaglandin E₂. Phorbol myristate acetate doubled the cellular mass of 1,2-diacylglycerol and stimulated the liberation of [³H]arachidonate from the diacylglycerol pool in prelabeled cells. The diacylglycerol lipase inhibitor RHC 80267 blocked 75–80% of the phorbol ester-promoted (total) cellular liberation of [³H]arachidonic acid and production of prostaglandin E₂. In comparison, the release of [³H]arachidonate from phosphatidylethanolamine (but not phosphatidylinositol) was only partially antagonized (to the same degree) by the PLA₂ inhibitor p-bromophenacylbromide and the protein kinase C inhibitor Et-18-OMe. PMA-induced formation of diacylglycerol or synthesis of PGE₂ was not affected by the prior inhibition of protein kinase C. Therefore, we have shown a novel pathway for the liberation of arachidonic acid in osteoblasts involving the nonspecific hydrolysis of phosphatidylinositol and phosphatidylethanolamine by phospholipase C followed by the deesterification of diacylgycerol. This pathway can be activated by a phorbol ester through a protein kinase C-independent mechanism.     

Phosphoinositide phosphorylation and hydrolysis in pancreatic islet cell membrane

Membranes were isolated from dispersed rat pancreatic islet cells by attachment to Sephadex beads. When these membranes were exposed to [gamma-32P]ATP, formation of 32P-labeled phosphatidate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate was observed. Carbamylcholine, added 10 s prior to lipid extraction, caused a dose-related fall in 32P-labeled phospholipids. The effect of the cholinergic agent was suppressed by atropine, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and verapamil, and simulated, in part, by an increase in Ca2+ concentration. When the membranes were derived from islet cells prelabeled with [U-14C]arachidonate, carbamylcholine stimulation, in addition to decreasing labeled polyphosphoinositides, was accompanied by an increased production of labeled diacylglycerol, without a concomitant increase in labeled phosphatidylinositol. These results indicate that activation of a plasma membrane-associated phospholipase C directed against polyphosphoinositides represents a primary event in the functional response of the pancreatic beta cell to cholinergic agents.     

Phosphatidylinositol Metabolism During In Vitro Hypoxia

The effects of in vitro histotoxic hypoxia (0.5 mM KCN) on potassium-stimulated phosphatidylinositol turnover were determined. In rat cortical slices that were prelabeled with [2-3H]inositol, depolarization with 60 mM KCl increased [2-3H]inositol monophosphate and [2-3H]inositol bisphosphate accumulation in a Ca2+-dependent manner. At early times (10 s and 1 min), histotoxic hypoxia enhanced potassium-stimulated [2-3H]inositol monophosphate and inositol bisphosphate accumulation. Under basal conditions, hypoxia did not alter the accumulation of [2-3H]inositol phosphates. These results are consistent with the following hypothesis. The hypoxic-induced increase in cytosolic free calcium that we reported previously may lead to the early stimulation of inositol phosphates formation during hypoxia through activation of phospholipase C. The impairment of inositol phosphates formation during more prolonged hypoxia may be due to negative feedback regulation of the phosphatidylinositol cascade by protein kinase C or to a reduction in ATP levels.