Stimulation of arachidonic acid release by guanine nucleotide in saponin-permeabilized neutrophils: evidence for involvement of GTP-binding protein in phospholipase A2 activation

Addition of a guanine nucleotide analog, guanosine 5'-O-(thiotriphosphate) (GTP gamma S)(1-100 microM) induced release of [3H]arachidonic acid from [3H]arachidonate-prelabeled rabbit neutrophils permeabilized with saponin. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced arachidonate release was enhanced by GTP gamma S, Ca2+, or their combination. Ca2+ alone (up to 100 microM) did not effectively stimulate lipid turnover. However, the combination of fMLP plus GTP gamma S elicited greater than additional effects in the presence of resting level of free Ca2+. The addition of 100 microM of GTP gamma S reduced the Ca2+ requirement for arachidonic acid liberation induced by fMLP. Pretreatment of neutrophils with pertussis toxin resulted in the abolition of arachidonate release and diacylglycerol formation. Neomycin (1 mM) caused no significant reduction of arachidonate release. In contrast, about 40% of GTP gamma S-induced arachidonate release was inhibited by a diacylglycerol lipase inhibitor, RHC 80267 (30 microM). These observations indicate that liberation of arachidonic acid is mediated by phospholipase A2 and also by phospholipase C/diacylglycerol lipase pathways. Fluoride, which bypasses the receptor and directly activates G proteins, induced arachidonic acid release and diacylglycerol formation. The fluoride-induced arachidonate release also appeared to be mediated by these two pathways. The loss of [3H]arachidonate was seen in phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. These data indicate that a G protein is involved between the binding of fMLP to its receptor and activation of phospholipase A2, and also that the arachidonic acid release is mediated by both phospholipase A2 and phospholipase C/diacylglycerol lipase.     

Phospholipase C and diacylglycerol lipase activities associated with plasma membranes of chromaffin cells isolated from bovine adrenal medulla

The plasma membranes of bovine adrenal chromaffin cells were isolated and the activities of enzymes involved in arachidonic acid liberation were investigated. Only a minute activity of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) could be detected using externally added phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as substrate. When membranes were treated with exogenous phospholipase C (orthophosphoric acid diester phosphohydrolase, EC 3.1.4.1) there was a liberation of free fatty acids from the sn-2 position of PC. The enzyme responsible for this effect could be demonstrated to be a diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) localized in the plasma membrane. Using phosphatidylinositol (PI) as a substrate, it was found that an endogenous phospholipase C exists which co-purifies with the membrane preparation. The produced diacylglycerol is subsequently hydrolyzed by diacylglycerol lipase liberating arachidonic acid. The two enzymes, phospholipase C and diacylglycerol lipase were characterized. Phospholipase C was found to be calcium dependent and PI specific, showing an activity of 60 pmol/micrograms protein per h (1.2 mM Ca2+), whereas the diacylglycerol lipase was calcium independent hydrolyzing diacylglycerol at a rate of 7.2 pmol/micrograms protein per h. The lipase but not the phospholipase C was inhibited 50% by 1.7 mM para-bromophenacylbromide.     

The alpha 1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C.

Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both of these reactions.     

Intracellular Ca2+ and protein kinase C interact to regulate alpha 1-adrenergic- and bradykinin receptor-stimulated phospholipase A2 activation in Madin-Darby canine kidney cells.

Alpha 1-Adrenergic receptors and bradykinin receptors are two distinct membrane receptors that stimulate phospholipid breakdown and arachidonic acid and arachidonic acid metabolite release. In the current studies, we have examined several mechanisms to assess their possible contribution to arachidonic acid release in the Madin-Darby canine kidney cell line by agonist stimulation of these receptors: 1) activation of phospholipase A2 (PLA2); 2) sequential activation of phospholipase C, diacylglycerol lipase, and monoacylglycerol lipase; and 3) inhibition of the sequential action of fatty acyl-CoA synthetase and lysophosphatide acyltransferase. Experiments were conducted to measure the stimulation of lysophospholipid production by epinephrine and bradykinin, the rate of incorporation of [3H]arachidonic acid into stimulated and unstimulated cells, and the effect on [3H]arachidonic acid release of treating cells with exogenous phospholipase C. The data indicate that stimulation of PLA2 activity is regulated by alpha 1-adrenergic and bradykinin receptors and that this stimulation is mediated, at least in part, by the activation of protein kinase C. We find that the role of diacylglycerol in arachidonic acid release is as an activator of protein kinase C and not as a substrate for a lipase. Moreover, the hormonal agonists do not appear to inhibit fatty acid reacylation. Experiments using the Ca2(+)-sensitive dye fura-2 and the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid suggest that bradykinin activates PLA2 by a transient elevation of intracellular Ca2+. This action appears to be less important for activation of PLA2 by epinephrine. Taken together, these data are consistent with the following conclusions. 1) Hormone-stimulated arachidonic acid release in Madin-Darby canine kidney-D1 cells occurs as a consequence of PLA2 activation. 2) The ability of an agonist both to mobilize Ca2+ and to activate protein kinase C contributes to its efficacy as a stimulator of PLA2-mediated arachidonic acid release.     

Alpha 1-adrenergic receptor-mediated phosphoinositide hydrolysis and prostaglandin E2 formation in Madin-Darby canine kidney cells. Possible parallel activation of phospholipase C and phospholipase A2

alpha 1-Adrenergic receptors mediate two effects on phospholipid metabolism in Madin-Darby canine kidney (MDCK-D1) cells: hydrolysis of phosphoinositides and arachidonic acid release with generation of prostaglandin E2 (PGE2). The similarity in concentration dependence for the agonist (-)-epinephrine in eliciting these two responses implies that they are mediated by a single population of alpha 1-adrenergic receptors. However, we find that the kinetics of the two responses are quite different, PGE2 production occurring more rapidly and transiently than the hydrolysis of phosphoinositides. The antibiotic neomycin selectively decreases alpha 1-receptor-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis without decreasing alpha 1-receptor-mediated arachidonic acid release and PGE2 generation. In addition, receptor-mediated inositol trisphosphate formation is independent of extracellular calcium, whereas release of labeled arachidonic acid is largely calcium-dependent. Moreover, based on studies obtained with labeled arachidonic acid, receptor-mediated generation of arachidonic acid cannot be accounted for by breakdown of phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, or phosphatidic acid. Further studies indicate that epinephrine produces changes in formation or turnover of several classes of membrane phospholipids in MDCK cells. We conclude that alpha 1-adrenergic receptors in MDCK cells appear to regulate phospholipid metabolism by the parallel activation of phospholipase C and phospholipase A2. This parallel activation of phospholipases contrasts with models described in other systems which imply sequential activation of phospholipase C and diacylglycerol lipase or phospholipase A2.     

The role of polyphosphoinositides and their breakdown products in A23187-induced release of arachidonic acid from rabbit polymorphonuclear leucocytes.

Stimulation of rabbit polymorphonuclear leucocytes with A23187 causes phospholipase C mediated breakdown of polyphosphoinositides, as evidenced by accumulation of [3H]inositol-labelled inositol bisphosphate and inositol trisphosphate. At the same time the polyphosphoinositides and the products of their breakdown, diacylglycerol and phosphatidic acid, label rapidly with radioactive arachidonic acid. Enhancement of polyphosphoinositide labelling is not as great as enhancement of diacylglycerol or phosphatidic acid labelling, suggesting additional early activation of a second independent synthetic pathway to the last named lipids. Experiments using double (3H/14C) labelling, to distinguish pools with different rates of turnover, suggest the major pool of arachidonic acid used for synthesis of lipoxygenase metabolites turns over more slowly than arachidonic acid in diacylglycerol, but at about the same rate as arachidonic acid esterified in phosphatidylcholine or phosphatidylinositol. Further, when cells are prelabelled with [14C]arachidonic acid, then stimulated for 5 min, it is only from phosphatidylcholine, and to a lesser extent phosphatidylinositol, that radiolabel is lost. Release of arachidonic acid is probably via phospholipase A2, since it is blocked by the phospholipase A2 inhibitor manoalide. The absence of accumulated lysophosphatides can be explained by reacylation and, in the case of lysophosphatidylinositol, deacylation. The importance of phospholipase A2 in phosphatidylinositol breakdown contrasts with the major role of phospholipase C in polyphosphoinositide hydrolysis. Measurements of absolute free fatty acid levels, as well as studies showing a correlation between production of radiolabelled hydroxyeicosatetraenoic acids and release of radiolabel from the phospholipid pool, both suggest that hydrolysis of arachidonic acid esterified into phospholipids is the limiting factor regulating formation of lipoxygenase metabolites. By contrast with A23187, fMet-Leu-Phe (a widely used polymorphonuclear leucocyte activator) is a poor stimulant for arachidonic acid release unless a 'second signal' (e.g. cytochalasin B, or a product of A23187-stimulated cells) is also present. In the presence of cytochalasin B, fMet-Leu-Phe, like A23187, stimulates release of radiolabelled arachidonic acid principally from phosphatidylcholine.     

The inhibition of arachidonic acid metabolism in human platelets by RHC 80267, a diacylglycerol lipase inhibitor

The diacylglycerol lipase inhibitor, RHC 80267, 1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane, was tested for its ability to block the release of arachidonic acid from human platelets. At a concentration (10 microM) reported to completely inhibit diacylglycerol lipase in fractions of broken platelets, RHC 80267 had no effect on diacylglycerol lipase activity or the release of arachidonic acid from washed human platelets stimulated with collagen. At a high concentration (250 microM), the compound inhibited the formation of arachidonyl-monoacylglycerol by 70% and the release of arachidonate by 60%. However, at this concentration RHC 80267 was found to inhibit cyclooxygenase activity, phospholipase C activity and the hydrolysis of phosphatidylcholine (PC) (presumably by inhibiting phospholipase A2). The phospholipase C inhibition was attributed to the inhibition of prostaglandin H2 formation, as it was alleviated by the addition of the endoperoxide analog, U-46619. PC hydrolysis was only partially restored with U-46619, suggesting that RHC 80267 directly alters phospholipase A2 activity. The inhibition of arachidonate release observed was accounted for by the inhibition of PC hydrolysis. We conclude that RHC 80267, because of its lack of specificity at concentrations needed to inhibit diacylglycerol lipase, is an unsuitable inhibitor for studying the release of arachidonic acid in intact human platelets.     

Involvement of phospholipase D activation in endothelin-1-induced release of arachidonic acid in osteoblast-like cells

In a previous study, we have shown that endothelin-1 (ET-1) activates phospholipase D independently from protein kinase C in osteoblast-like MC3T3-E1 cells. It is well recognized that phosphatidylycholine hydrolysis by phospholipase D generates phosphatidic acid, which can be further degraded by phosphatidic acid phosphohydrolase to diacylglycerol. In the present study, we investigated the role of phospholipase D activation in ET-1-induced arachidonic acid release and prostaglandin E₂ (PGE₂) synthesis in osteoblast-like MC3T3-E1 cells. ET-1 stimulated arachidonic acid release dose-dependently in the range between 0.1 nM and 0.1 μM. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the ET-1-induced arachidonic acid release in a dose-dependent manner as well as the ET-1-induced diacylglycerol formation. 1,6-bis-(cyclohexyloxyminocarbonylamino)-hexane (RHC-80267), an inhibitor of diacylglycerol lipase, significantly suppressed the ET-1-induced arachidonic acid release. The pretreatment with propranolol and RHC-80267 also inhibited the ET-1-induced PGE₂ synthesis. These results strongly suggest that phosphatidylcholine hydrolysis by phospholipase D is involved in the arachidonic acid release induced by ET-1 in osteoblast-like cells. J. Cell. Biochem. 64:376–381. © 1997 Wiley-Liss, Inc.     

Relationship between arachidonic acid release and Ca2(+)-dependent exocytosis in digitonin-permeabilized bovine adrenal chromaffin cells.

The relationship between Ca2(+)-dependent arachidonic acid release and exocytosis from digitonin-permeabilized bovine adrenal chromaffin cells was investigated. The phospholipase A2 inhibitors mepacrine, nordihydroguaiaretic acid and indomethacin had no effect on either arachidonic acid release or secretion. The phospholipase A2 activator melittin had no effect on secretion. The specific diacylglycerol lipase inhibitor RG80267 had no effect on secretion, but decreased basal arachidonic acid release to such an extent that the level of arachidonic acid in treated cells in response to 10 microM-Ca2+ was equivalent to that of control cells in the absence of Ca2+. Staurosporine, a protein kinase C inhibitor, was found to abolish Ca2(+)-dependent arachidonic acid release completely, but had only a slight inhibitory effect on Ca2(+)-dependent secretion. It is concluded that arachidonic acid is not essential for Ca2(+)-dependent exocytosis in adrenal chromaffin cells.     

Dependence of secretory responses to gonadotropin-releasing hormone on diacylglycerol metabolism. Studies with a diacylglycerol lipase inhibitor, RHC 80267

The role of diacylglycerol (DG) as a source of arachidonic acid during gonadotropin-releasing hormone (GnRH) stimulation of gonadotropin secretion was analyzed in primary cultures of rat anterior pituitary cells. An inhibitor of DG lipase (RHC 80267, RHC) caused dose-dependent blockade of GnRH-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. The DG lipase inhibitor did not alter gonadotropin responses to arachidonic acid, and addition of arachidonic acid reversed its inhibition of GnRH-stimulated LH and FSH release. In [3H]arachidonic acid-prelabeled cells, incubation with RHC increased the accumulation of [3H]DG. These results suggest that DG lipase participates in GnRH action and that arachidonic acid mobilization from DG is involved in the mechanism of gonadotropin release. Gonadotropin responses to tetradecanoyl phorbol acetate and dioctanoyl glycerol were not altered by RHC, and the addition of these activators of protein kinase C (Ca2+- and phospholipid-dependent enzyme) did not prevent the inhibition of GnRH-induced gonadotropin release by RHC. Activation of phospholipase A2 by melittin increased LH and FSH secretion, whereas blockade of this enzyme by quinacrine reduced GnRH-stimulated hormone release. However, RHC did not diminish the gonadotropin response to melittin. The inhibitory actions of RHC and quinacrine were additive and were reversed by concomitant treatment with arachidonic acid. Ionomycin also increased LH and FSH release, and the gonadotropin responses to the ionophore were unaltered by RHC but were reduced by quinacrine. Incubation of cells in Ca2+-depleted (+/- [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) medium reduced but did not abolish the LH and FSH releasing activity of GnRH. Treatment with RHC also reduced the gonadotropin responses to GnRH under Ca2+-depleted conditions. These observations indicate that RHC inhibition of GnRH action is not due to nonspecific actions on Ca2+ entry, protein kinase C activation and actions, nor phospholipase A2 enzyme activity. The results of this study provide further evidence for an extracellular Ca2+-independent mechanism of GnRH action, and suggest that GnRH causes mobilization of arachidonic acid by two distinct lipases, namely, phospholipase A2 and DG lipase, during stimulation of gonadotropin secretion.     

Arachidonic acid release from diacylglycerol in human neutrophils. Translocation of diacylglycerol-deacylating enzyme activities from an intracellular pool to plasma membrane upon cell activation

We have studied the capacity of human neutrophils to release arachidonic acid from diacylglycerol, employing 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol and 1-[1-14C]stearoyl-2-arachidonoyl-sn-glycerol as exogenous substrates. We have found that arachidonic acid is removed from diacylglycerol by the sequential action of two enzymes. First, the sn-1 position is split by 1-diacylglycerol lipase activity, and then, arachidonic acid is released from the resulting 2-monoacylglycerol by a 2-monoacylglycerol lipase. The specific activity of the 2-monoacylglycerol lipase, using 2-[1-14C]arachidonoyl-sn-glycerol as exogenous substrate, was at least 9-fold higher than that of 1-diacylglycerol lipase, indicating that the action of the 1-diacylglycerol lipase is the rate-limiting step in arachidonic acid release from diacylglycerol. Postnuclear supernatants from A23187-treated cells showed a 2.5-fold increase in both lipase activities. The arachidonic acid-releasing diacylglycerol lipase system showed an optimum pH of 4.5 and was not inhibited by EGTA or stimulated by Ca2+, Mg2+, Mn2+, Zn2+, or Co2+. However, arachidonic acid release was inhibited by Hg2+, suggesting the involvement of sulfhydryl groups in catalytic activity. The subcellular distribution of both 1-diacylglycerol lipase and 2-monoacylglycerol lipase activities was examined in resting and A23187-treated human neutrophils by fractionation of postnuclear supernatants on continuous sucrose gradients. Both lipases were localized mainly in the membrane of gelatinase-containing granules, which were resolved from cytosol, plasma membrane, phosphasomes, and specific and azurophilic granules. When neutrophils were stimulated by the calcium ionophore A23187, a drastic shift of the 1-diacylglycerol lipase and 2-monoacylglycerol lipase toward the plasma membrane was detected. This shift was due to fusion of gelatinase-containing granules with the plasma membrane upon neutrophil stimulation. As a result of the membrane fusion process, the capacity to release arachidonic acid from diacylglycerol was increased. This translocation from the membrane of gelatinase-containing granules to the plasma membrane may play an important role in regulating the diacylglycerol level in stimulated human neutrophils.     

A major role for phospholipase A2 in antigen-induced arachidonic acid release in rat mast cells.

Cross-linking of IgE receptors by antigen stimulation leads to histamine release and arachidonic acid release in rat peritoneal mast cells. Investigators have reported a diverse distribution of [3H]arachidonate that is dependent on labelling conditions. Mast cells from rat peritoneal cavity were labelled with [3H]arachidonic acid for different periods of time at either 30 or 37 degrees C. Optimum labelling was found to be after 4 h incubation with [3H]arachidonate at 30 degrees C, as judged by cell viability (Trypan Blue uptake), responsiveness (histamine release) and distribution of radioactivity. Alterations in 3H-radioactivity distribution in mast cells labelled to equilibrium were examined on stimulation with antigen (2,4-dinitrophenyl-conjugated Ascaris suum extract). The results indicated that [3H]arachidonic acid was lost mainly from phosphatidylcholine and, to a lesser extent, from phosphatidylinositol. A transient appearance of radiolabelled phosphatidic acid and diacylglycerol indicated phosphatidylinositol hydrolysis by phospholipase C. Pretreatment with a phospholipase A2 inhibitor, mepacrine, substantially prevented the antigen-induced liberation of [3H]arachidonic acid from phosphatidylcholine. It can be thus concluded that, in the release of arachidonic acid by antigen-stimulated mast cells, the phospholipase A2 pathway, in which phosphatidylcholine is hydrolysed, serves as the major one, the phospholipase C/diacylglycerol lipase pathway playing only a minor role.     

Chemotactic peptide stimulation of arachidonic acid release in HL60 cells, an interaction between G protein and phospholipase C mediated signal transduction

The mechanism of phospholipase A2 activation by chemotactic peptide was investigated in human promyelocytic HL60 cells. N-Formyl-methionyl-leucyl-phenylalanine (fMetLeuPhe) and the non-hydrolyzable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induced arachidonic acid release in permeabilized and metabolically inhibited HL60 cells, a preparation in which calcium was buffered and inositol phospholipid hydrolysis was inhibited. Inositol phosphate generation and arachidonic acid were shown to be temporally dissociated. These results suggest that receptor-dependent phospholipase C activity is not required for fMetLeuPhe to induce arachidonic acid release. However, fMetLeuPhe effects were highly calcium-dependent and inhibition of phospholipase C reduced fMetLeuPhe stimulation of arachidonic acid release even in the permeabilized cell preparation. We conclude that although phospholipase A2 activation is linked to the fMetLeuPhe receptor independent of phospholipase C, actions of phospholipase C to mobilize calcium and release diacylglycerol may be important to phospholipase A2 activation in the intact cell.     

Vasopressin induces V1 receptors to activate phosphatidylinositol- and phosphatidylcholine-specific phospholipase C and stimulates the release of arachidonic acid by at least two pathways in the smooth muscle cell line, A-10

The rat thoracic aortic smooth muscle cell line, A-10, expresses vasopressin receptors of the V1 subtype. Vasopressin treatment of these cells stimulated the release of arachidonic acid and the formation of diacylglycerol and phosphocholine. These responses to vasopressin were inhibited by the V1-specific antagonist SK&F 100273, indicating that these were receptor-mediated phenomena. The mechanisms by which V1 receptors mediate arachidonic acid release appeared to be unaffected by cycloheximide or actinomycin D, suggesting that the release is independent of protein and RNA synthesis. The V1 receptors also appeared to be coupled to a phospholipase C which can hydrolyze phosphatidylcholine, a possible source of the released arachidonic acid. Phosphocholine and diacylglycerol were also generated. The release of arachidonic acid, phosphocholine, or diacylglycerol was not affected by prior treatment of the cells with pertussis toxin (islet-activating protein). Thus, the release of these second messengers is not mediated by the guanine nucleotide-binding protein Gi or other pertussis toxin-sensitive substrates. We conclude that V1 receptors induce the release of arachidonic acid and the formation of diacylglycerol and phosphocholine via the activation of both a phosphatidylinositol- and phosphatidylcholine-specific phospholipase C.     

The inhibition of arachidonic acid mobilization in human platelets by R59 022, a diacylglycerol kinase inhibitor

R59 022 (6-[2-[4-[(4-fluorophenyl)phenylmethylene]-1- piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one) has been suggested as an inhibitor of diacylglycerol kinase in erythrocyte membranes and intact platelets. In the present study, we have investigated the effects of this drug on arachidonic acid mobilization occurring in response to thrombin in intact human platelets. Our results indicate that release of arachidonic acid from membrane phospholipids such as phosphatidylcholine and phosphatidylinositol was severely impaired by R59 022 and the extent of inhibition amounted to 77% and 84%, respectively, as compared to controls. This resulted in a dramatic decrease in the accumulation of free arachidonic acid (labeled/unlabeled) and the percent inhibition of free arachidonic acid accumulation amounted to 80-90% as compared to controls. Furthermore, the drug caused a significant accumulation of thrombin-induced diacylglycerol (labeled) without affecting the formation of labeled phosphatidic acid (PA). We found no significant changes in the radioactivity of either phosphatidylethanolamine or phosphatidylserine following stimulation with thrombin in the presence or absence of R59 022. We conclude that the observed inhibition of thrombin-induced arachidonic acid mobilization by R59 022 may be due to its effects on the activities of diacylglycerol lipase/phospholipase A2. In addition, the failure of further stimulation of thrombin-induced PA by R59 022 may indicate that PA-specific phospholipase A2 is either not involved in the release of arachidonic acid or is not a major source for arachidonic acid release in thrombin-stimulated human platelets. These findings may prove to be important when this drug is used as a selective inhibitor of diacylglycerol kinase.     

Inhibition of adenylylcyclase activity in mouse cerebellum membranes upon hydrolysis of triacylglycerols by triacylglycerol lipase, but not phospholipids by phospholipase A(2)

We previously showed that arachidonic acid and related unsaturated free fatty acids (U-FFAs) inhibit the activity of adenylylcyclase in brain membranes of mice. The level of U-FFAs elevates when the hydrolysis of triacylglycerols (TAGs) and phospholipids is promoted. In this study, we examined whether activation of triacylglycerol lipase (TAG lipase) and phospholipase A(2) (PLA(2)) results in the inhibition of adenylylcyclase activity in cerebellum membranes of mice. Incubation of Intralipos with TAG lipase in the presence of membranes mainly released oleic acid and linoleic acid and caused > or =95% inhibition of adenylylcyclase activity. In contrast, PLA(2), though releasing substantial amounts of U-FFAs, increased the enzymatic activity. To account for this difference, we examined how by-products formed in U-FFA release by TAG lipase and PLA(2) operated on the arachidonic acid-induced inhibition. Lysophosphatidylcholne and some other lysophospholipids, produced by PLA(2), enhanced the adenylylcyclase activity and attenuated the inhibitory effect of arachidonic acid. On the other hand, no such effects were found with by-products of TAG lipase-mediated lipolysis. Rather, monoacylglycerols having U-FFAs, possibly formed by TAG lipase, potentiated the arachidonic acid-induced inhibition of adenylylcyclase. Bovine serum albumin, added into the mixture for the pretreatment of membranes with TAG lipase, prevented the inhibition of adenylylcyclase. These results indicate that by-products formed in U-FFA release have a crucial role for the U-FFA's action on adenylylcyclase and that U-FFAs released from TAG are an inhibitor of adenylylcyclase. It may be that albumin in plasma, and thus FFA-binding proteins within cells, are of importance in protecting adenylylcyclase upon U-FFA release.     

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%.     

Phospholipase A2 in macrophage plasma membrane releases arachidonic acid from phosphatidylinositol

A high level of arachidonic acid release from [2-14C]arachidonylphosphatidylinositol (PI) was observed at neutral pH (6.0-7.0) in the presence of purified plasma membranes of guinea pig peritoneal macrophages. This activity was at least 10-fold higher than that with arachidonylphosphatidylcholine (PC) or phosphatidylethanolamine (PE) as substrate. The accumulation of [14C]diacylglycerol and [14C]phosphatidic acid was not detected at any time, and arachidonic acid release from [14C]arachidonyldiacylglycerol was not detectable either. The data suggest that arachidonic acid release from PI may not occur via the phospholipase C pathway. In this paper, we demonstrate the possibility that arachidonic acid release from PI at neutral pH in the macrophage plasma membrane is dependent on the action of phospholipase A2 (EC 3.1.1.4) -like activity. The maximum arachidonic acid release was dependent upon both pH and substrate. Particularly, the activity of arachidonic acid release from PI at neutral pH was very high compared with that from PC or PE. We suggest that phosphatidylinositol phospholipase A2 (EC 3.1.1.52) may play an important role in providing arachidonic acid for subsequent metabolic activity in the macrophages.     

Increase of [Ca(2+)]i and release of arachidonic acid via activation of M2 receptor coupled to Gi and rho proteins in oesophageal muscle

We have previously shown that acetylcholine-induced contraction of oesophageal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachidonic acid interact synergistically to activate protein kinase C. We have therefore investigated the relationship between cytosolic Ca(2+) and activation of phospholipase A(2) in response to acetylcholine-induced stimulation, by measuring the intracellular free Ca(2+) ([Ca(2+)]i), muscle tension, and [3H] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca(2+)]i and arachidonic acid release in a dose-dependent manner. In Ca(2+)-free medium, acetylcholine did not produce contraction, [Ca(2+)]i increase, and arachidonic acid release. In contrast, after depletion of Ca(2+) stores by thapsigargin (3 microM), acetylcholine caused a normal contraction, [Ca(2+)]i increase and arachidonic acid release. The increase in [Ca(2+)]i and arachidonic acid release were attenuated by the M2 receptor antagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hexahydro siladifenidol. Increase in [Ca(2+)]i and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findings indicate that contraction and arachidonic acid release are mediated through muscarinic M2 coupled to Gi or rho protein activation and Ca(2+) influx. Acetylcholine-induced contraction and the associated increase in [Ca(2+)]i and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid and a phospholipase D inhibitor pCMB. They increased by the action of the inhibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal circular muscle acetylcholine-induced [Ca(2+)]i increase and arachidonic acid release are mediated through activation of M2 receptor coupled to Gi or rho protein, resulting in the activation of phospholipase A(2) and phospholipase D to activate protein kinase C.     

Diacylglycerol lipase pathway is a minor source of released arachidonic acid in thrombin-stimulated human platelets

It has been postulated that the diacylglycerol lipase pathway is a predominant source of the free arachidonic acid which is released from phospholipids upon the exposure of human platelets to thrombin. The amount of released arachidonic acid and other fatty acids in thrombin-stimulated platelets was determined in the presence of BW755C, the cyclooxygenase/lipoxygenase inhibitor, and in relation to phosphatidylinositol degradation and phosphatidic acid formation. A stearic acid:arachidonic acid molar ratio approaching unity would be expected in the free fatty acid fraction if the latter pathway were a major source of released arachidonic acid. Our results indicate that the diacylglycerol lipase pathway contributes a maximum of 3-4 nmol of arachidonic acid/2 X 10(9) platelets or 12-15% of the total arachidonic acid released (25.8 nmol/2 X 10(9) platelets) upon exposure to thrombin (2 units/ml) for 4 min. Trifluoperazine inhibited most of the thrombin-dependent free arachidonic acid release but only 15% of the absolute loss of arachidonic acid from phosphatidylinositol. Therefore, we conclude that the diacylglycerol lipase pathway represents only a minor source of the free arachidonic acid that is released upon thrombin stimulation of human platelets.