Antiproliferative effect of nitric oxide on rat glomerular mesangial cells via inhibition of mitogen-activated protein kinase.

The effect of nitric oxide (NO) donors and lipopolysaccharide (LPS) on the proliferation of rat glomerular mesangial cells was characterized. Exogenous application of a NO donor inhibited serum-induced proliferation in a time- and dose-dependent manner. S-Nitrosoglutathione (GSNO) also increased cGMP generation and arachidonic acid release, but it did not cause any measurable increase in the cytosolic Ca2+ concentration. Chelation of cytosolic Ca2+ or inhibition of mitogen-activated protein kinase (MAPK) kinase had an inhibitory effect on proliferation, but neither enhanced the antiproliferative effect of GSNO. In contrast, inhibition of guanylate cyclase or phospholipase A2 had no effect on proliferation, but partially reversed GSNO-induced antiproliferation by approximately 98 and 65%, respectively. GSNO did not cause cell death. Incubation of cells with LPS induced endogenous NO generation and had an antiproliferative effect. LPS-induced antiproliferation was reversed completely by inhibition of nitric oxide synthase and partially by inhibition of guanylate cyclase or phospholipase A2. GSNO or LPS inhibited serum-induced MAPK activation, and both effects were partially reversed by inhibition of guanylate cyclase or phospholipase A2. Inclusion of 8-bromo-cGMP or arachidonic acid in the growth medium resulted in a similar antiproliferative effect. In conclusion, in rat glomerular mesangial cells, MAPK inhibition and an antiproliferative effect could be induced by either an increase in the cellular concentration of NO or exposure of the cells to LPS. Part of the effect of NO was attributable to the increased cellular cGMP generation and arachidonic acid release.     

Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid

Addition of thrombin to human platelets results in production of lysophosphatidic acid. Such synthesis of lysophosphatidic acid can be inhibited by mepacrine, an inhibitor of the phospholipase A2 which attacks phosphatidic acid to give lysophosphatidic acid. In the present study, mepacrine was used at a concentration of 2.5-20 microM, sufficient to block aggregation and lysophosphatidic acid formation induced by 0.1 U/ml thrombin. Mepacrine, at this concentration, also blocked thrombin-induced phosphorylation of platelet myosin light chain and a 47 kDa protein, thrombin-induced secretion and thrombin-induced release of arachidonic acid from platelet phospholipids. However, mepacrine also partly inhibited the formation of phosphatidic acid in response to thrombin, consistent with some simultaneous inhibition of phospholipase C. Lysophosphatidic acid (2.5-22 microM) overcame the mepacrine block in thrombin-stimulated aggregation, protein phosphorylation and secretion without stimulating the release of arachidonic acid from platelet phospholipids or the formation of lysophosphatidic acid, and only slightly increasing phosphatidic acid formation. The results suggest that lysophosphatidic acid primarily acts distal to mepacrine inhibition of phospholipase A2 and phospholipase C and are consistent with the possibility that lysophosphatidic acid might be a mediator of part of the effects of low-dose thrombin on human platelets.     

The activation by Ca2+ of platelet phospholipase A2. Effects of dibutyryl cyclic adenosine monophosphate and 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate.

Thrombin-induced release of arachidonic acid from human platelet phosphatidylcholine is found to be more than 90% impaired by incubation of platelets with 1 mM dibutyryl cyclic adenosine monophosphate (Bt2 cyclic AMP) or with 0.6 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist. Incorporation of arachidonic acid into platelet phospholipids is not enhanced by Bt2 cyclic AMP. The addition of external Ca2+ to thrombin-treated platelets incubated with Bt2 cyclic AMP or TMB-8 does not counteract the observed inhibition. However, when divalent cation ionophore A23187 is employed as an activating agent, much less inhibition is produced by Bt2 cyclic AMP or TMB-8. The inhibition which does result can be overcome by added Ca2+. Inhibition of arachidonic acid liberation by Bt2 cyclic AMP, but not by TMB-8, can be overcome by high concentrations of A23187. When Mg2+ is substituted for Ca2+, ionophore-induced release of arachidonic acid from phosphatidylcholine of inhibitor-free controls is depressed and inhibition by Bt2 cyclic AMP is slightly enhanced. The phospholipase A2 activity of platelet lysates is increased by the presence of added Ca2+, however, the addition of either A23187 or Bt2 cyclic AMP is without effect on this activity. We suggest that Bt2 cyclic AMP may promote a compartmentalization of Ca2+, thereby inhibiting phospholipase A activity. The compartmentalization may be overcome by ionophore. By contrast, TMB-8 may immobilize platelet Ca2+ stores in situ or restrict access of Ca2+ to phospholipase A in a manner not susceptible to reversal by high concentrations of ionophore.     

Nitric oxide inhibits cell growth in cultured human thyrocytes

Effect of NO induced by interleukin-1 (IL-1) or IL-1/interferon-_γ (IL-1/IFN-_γ) was investigated on cell growth using primary cultures of human thyrocytes. Cytokine-induced NO production was associated not only with an increase in cyclic GMP (cGMP) formation but also with an inhibition of cell growth determined by bromo-deoxyuridine (Br-dU) incorporation into DNA. When NO synthesis was blocked by N^G-monomethyl-L-arginine (L-MMA), cGMP formation was prevented in parallel with NO production and inversely a restoration of cell growth was evident. S-nitroso-N-acetyl-penicillamine, a NO donor, but not a cell permeable cGMP analog, 8-bromo-cGMP, inhibited cell growth in a dose-dependent manner. The present findings strongly indicate that endogenous NO produced by the cytokine treatment as well as exogenous NO, has a cGMP-independent inhibitory action on human thyrocyte growth.     

Ethanol interferes with collagen-induced platelet activation by inhibition of arachidonic acid mobilization

The effect of ethanol on signal generation in collagen-stimulated human platelets was evaluated. Incubation of washed human platelets with physiologically relevant concentrations of ethanol (25-150 mM) resulted in a dose-dependent inhibition of aggregation and secretion in response to collagen (0.5-10 micrograms/ml), but did not inhibit shape change. In platelets labeled with [3H]arachidonic acid, ethanol significantly inhibited the release of arachidonic acid from phospholipids, in both the presence and the absence of indomethacin. Thromboxane B2 formation was also inhibited in proportion to the reduction in free arachidonic acid. There was a close correlation between the extent of inhibition of arachidonic acid release and secretion. The inhibition of platelet aggregation and secretion by ethanol was partially overcome by the addition of exogenous arachidonic acid. In the presence of indomethacin, ethanol had no effect on the activation of phospholipase C by collagen as determined by the formation of inositol phosphates and phosphatidic acid. Moreover, ethanol had no effect on the mobilization of intracellular calcium by collagen and only minimally inhibited the early phases of the phosphorylation of myosin light chain (20 kDa) and a 47-kDa protein, a known substrate for protein kinase C. Arachidonic acid formation was also inhibited by ethanol in response to ionomycin under conditions where phospholipase C activation was prevented. The results suggest that the functional effects of ethanol on collagen-stimulated platelets are due, at least in part, to an inhibition of phospholipase A2.     

Cyclic AMP and Cyclic GMP suppress TNFα-induced hepatocyte apoptosis by inhibiting FADD up-regulation via a protein kinase A-dependent pathway

Cyclic AMP (cAMP) and cyclic GMP (cGMP) suppress apoptosis in many cell types, including hepatocytes. We have previously shown that membrane-permeable cAMP and cGMP analogs attenuate tumor necrosis factor α plus actinomycin D (TNFα/ActD)-induced apoptosis in hepatocytes at a step upstream of caspase activation and cytochrome c release. Recently we have also shown that FADD levels increase 10 folds in response to TNFα/ActD. Therefore we hypothesized that cAMP and cGMP would inhibit FADD upregulation. We show here that cyclic nucleotide analogs dibutyryl cAMP (db-cAMP) and 8-bromo-cGMP (Br-cGMP) inhibit cell death and the cleavages of multiple caspases including caspase-10, -9, -8, -3, and -2, as well as suppress FADD protein up-regulation in TNFα/ActD-induced apoptosis. The inhibitory effects of cAMP were seen at lower concentrations than cGMP. Both cAMP and cGMP prevented FADD overexpression and cell death in hepatocytes transfected with the FADD gene. A protein kinase A (PKA) inhibitor, KT 5720, reversed the inhibition of FADD protein levels induced by cAMP or cGMP. In conclusion, our findings indicate that cAMP and cGMP prevent TNFα/ActD-induced apoptosis in hepatocytes and that this occurs in association with a near complete inhibition of the upregulation of FADD via a PKA-dependent mechanism. Supported by the National Institutes of Health Grant GM-44100 (to T.R.B).     

Role of cyclic GMP in atrial natriuretic factor stimulation of Na+,K+,Cl- cotransport in vascular smooth muscle cells

Atrial natriuretic factor (ANF) has been shown to bind to specific receptors on vascular smooth muscle cells (VSMC) and to cause an increase in intracellular cyclic GMP (cGMP) content. We have recently demonstrated that a prominent Na+,K+,Cl- cotransport system is present in VSMC and that a permeable cGMP analog (8-bromo-cGMP) stimulates activity of the cotransporter. We have also shown that the ANF peptide, rat atriopeptin III, stimulates Na+,K+,Cl- cotransport and elevates intracellular cGMP levels in VSMC. In the present study, we tested the hypothesis that ANF stimulation of Na+,K+,Cl- cotransport occurs via an increase in cGMP levels. When the quinolinedione, 6-anilo-5,8-quinolinedione (LY83583) (10 microM), was used to block formation of cGMP in VSMC from primary cultures of rat thoracic aorta, it was found that both basal and rat atriopeptin III (100 nM)-stimulated Na+,K+,Cl- cotransport were significantly inhibited. The effect of LY83583 was dose-dependent and the half-maximal inhibitory concentration was 0.5 microM. LY83583 also inhibited cotransport in the presence of a maximal concentration of 8-bromo-cGMP. However, this inhibition was not seen in cells also treated with 2-O-propoxyphenyl-8-azapurin-6-one (M&B 22,948), an inhibitor of cGMP phosphodiesterase. M&B 22,948 alone also increased levels of cotransport. Since inhibition of cGMP formation blocks ANF-stimulated Na+,K+,Cl- cotransport and inhibition of cGMP breakdown enhances Na+, K+, Cl- cotransport, we conclude that ANF stimulation of Na+,K+,Cl- cotransport in VSMC is mediated via increase in intracellular cGMP levels.     

Effect of diarachidonin on prostaglandin E2 synthesis in rabbit kidney medulla slices.

The effect of diarachidonin on the synthesis of prostaglandin E2 in rabbit kidney medulla slices was examined. The addition of diarachidonin stimulated prostaglandin E2 production in a dose-dependent manner. At three concentrations (10, 50 and 100 microM), increases in prostaglandin E2 formation induced by exogenous diarachidonin were 2-fold greater than those induced by exogenous arachidonic acid. Diacylglycerol or phosphatidic acid from egg lecithin had little or no effect on prostaglandin E2 production. Moreover, EGTA failed to inhibit diarachidonin-stimulated prostaglandin E2 formation, indicating that the stimulatory effect of diarachidonin is not mediated through the activation of endogenous phospholipase A2 (including phosphatidic acid-specific phospholipase A2). These results are discussed in the light of our former hypothesis that arachidonic acid release from kidney medulla phospholipids might occur through the sequential action of a phospholipase C coupled to diacylglycerol and monoacylglycerol lipases [Fujimoto, Akamatsu, Hattori & Fujita (1984) Biochem. J. 218, 69-74].     

Role of phospholipase D in the cAMP signal transduction pathway activated during fibroblast contraction of collagen matrices

Fibroblast contraction of stressed collagen matrices results in activation of a cAMP signal transduction pathway. This pathway involves influx of extracellular Ca2+ ions and increased production of arachidonic acid. We report that within 5 min after initiating contraction, a burst of phosphatidic acid release was detected. Phospholipase D was implicated in production of phosphatidic acid based on observation of a transphosphatidylation reaction in the presence of ethanol that resulted in formation of phosphatidylethanol at the expense of phosphatidic acid. Activation of phospholipase D required extracellular Ca2+ ions and was regulated by protein kinase C. Ethanol treatment of cells also inhibited by 60-70% contraction-dependent release of arachidonic acid and cAMP but had no effect on increased cAMP synthesis after addition of exogenous arachidonic acid or on phospholipase A2 activity measured in cell extracts. Moreover, other treatments that inhibited the burst of phosphatidic acid release after contraction--chelating extracellular Ca2+ or down-regulating protein kinase C--also blocked contraction activated cyclic AMP signaling. These results were consistent with the idea that phosphatidic acid production occurred upstream of arachidonic acid in the contraction- activated cAMP signaling pathway.     

Platelet activation by tetraprenol via stimulation of phospholipase A2 action

Only tetraprenol (n = 4), among the (n)-polyprenols studied, induced activation of rabbit platelets. Tetraprenol-induced responses, including platelet aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release, were greatly inhibited by a thromboxane A2 (TXA2) receptor antagonist and a cyclooxygenase inhibitor, indicating an essential role for endogenously produced TXA2. The TXA2-mimetic agonist U46619 induced platelet aggregation, Ca2+ mobilization and phospholipase C action but did not induce arachidonic acid release. These results suggest that arachidonic acid is not released via phospholipase C but by phospholipase A2, and this is also supported by the finding that phospholipase C action was inhibited by depletion of extracellular Ca2+, while arachidonic acid release was not. Full arachidonic acid release was found to be induced by the synergistic action of U46619 and tetraprenol. Therefore, the initial, most essential response induced by tetraprenol is a small arachidonic acid release by phospholipase A2, which results in initial TXA2 formation. Further action of phospholipase C as well as Ca2+ mobilization and aggregation were induced by the initially formed TXA2 while further activation of phospholipase A2 required the synergistic action of tetraprenol and TXA2.     

Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.     

Vitamin E inhibits platelet phospholipase A2

One of the most important functions of phospholipase A2 is the release of arachidonic acid from membrane phospholipids for the synthesis of biologically active eicosanoids. We have demonstrated in our laboratory that vitamin E inhibits platelet phospholipase A2 in a dose-dependent manner. Rats fed a 100 ppm or a 1000 ppm vitamin E diet exhibit diminished phospholipase A2 activity compared to those fed a vitamin E-free diet. Addition of vitamin E to a sonicated platelet suspension resulted in further suppression of the phospholipase A2 activity in all groups of rats. In order to gain insight into the mechanism of vitamin E inhibition of platelet phospholipase A2, we partially purified this enzyme by gel filtration chromatography. Enzyme activity was localized in the soluble supernatant fraction of a high-speed spin. This partially purified rat platelet phospholipase A2 had an absolute requirement for Ca2+ and was inhibited by various forms of tocopherol. Tocol inhibited the enzyme to a greater extent than either D- or DL-alpha-tocopherol, while there was little or no effect from DL-alpha-tocopherol acetate. These results emphasize the importance of the hydroxyl moiety on the chromanol of the vitamin E molecule for its inhibitory action, compared to that of the methyl groups which are absent in tocol. This inhibitory action of vitamin E on platelet phospholipase A2 suggests a crucial function for vitamin E in regulating arachidonate release from the membrane phospholipids and its subsequent metabolism.     

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.     

Cyclic AMP and platelet prostaglandin synthesis.

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3'5'-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of 14C-arachidonic acid by washed platelets. Prostaglandin E1 (PGE1), PGE1 with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B2. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE1 with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of 14C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

Calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells

We provided evidence that calcium-calmodulin plays a major role in bradykinin-induced arachidonic acid release by bovine aortic endothelial cells. In cells labeled for 16 hr with ³H-arachidonic acid, ionomycin and Ca²⁺-mobilizing hormones such as bradykinin, thrombin and platelet activating factor induced arachidonic acid release. However, arachidonic acid release was not induced by agents known to increase cyclic AMP (forskolin, isoproterenol) or cyclic GMP (sodium nitroprusside). Bradykinin induced the release of arachidonic acid in a dose-dependent manner (EC₅₀ = 1.6 ± 0.7 nM). This increase was rapid, reaching a maximal value of fourfold above basal level in 15 min. In a Ca²⁺-free medium, bradykinin was still able to release arachidonic acid but with a lower efficiency. Quinacrine (300 μM), a blocker of PLA₂, completely inhibited bradykinin-induced arachidonic acid release. The B₂ bradykinin receptor antagonist HOE-140 completely inhibited bradykinin-induced arachidonic acid release. The B₁-selective agonist DesArg⁹-bradykinin was inactive and the B₁-selective antagonist [Leu⁸]DesArg⁹-bradykinin had no significant effect on bradykinin-induced arachidonic acid release. The phospholipase C inhibitor U-73122 (100 μM) decreased bradykinin-induced arachidonic acid release. The calmodulin inhibitor W-7 (50 μM) drastically reduced the bradykinin- and ionomycin-induced arachidonic acid release. Also, forskolin decreased bradykinin-induced arachidonic acid release. These results suggest that the activation of PLA₂ by bradykinin in BAEC is a direct consequence of phospholipase C activation. Ca²⁺-calmodulin appears to be the prominent activator of PLA₂ in this system. © 1996 Wiley-Liss, Inc.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Activation of phospholipase A2 and beta-thromboglobulin release in human platelets: comparative effects of thrombin and fluoroaluminate stimulation.

Several reports have suggested that the activity of platelet phospholipase A2 is modulated by GTP-binding protein(s) whose nature and properties need to be defined. Fluoroaluminate is known to activate G-proteins and this leads to a number of cellular responses including the activation of phospholipases. This paper demonstrates that human platelets, prelabelled with [3H]arachidonic acid, produce free arachidonic acid when stimulated with fluoroaluminate and this effect is time- and dose-dependent. The production of arachidonic acid is not inhibited by neomycin, a PI-cycle inhibitor, but is completely abolished by mepacrine, an inhibitor of both phospholipase A2 and C. At low concentration of fluoroaluminate (10 mM NaF) phospholipase A2 but not phospholipase C is activated. In addition, fluoroaluminate treatment releases beta-thromboglobulin (beta-TG) and this effect is not inhibited by acetylsalicylic acid. Under identical conditions both neomycin and mepacrine suppress the release of arachidonic acid and beta-TG induced by thrombin. Sodium nitroprusside, which increases cGMP levels in platelets, inhibits arachidonic acid liberation and beta-TG release in thrombin-stimulated platelets but has no effect in fluoroaluminate-treated platelets; cGMP was reported to suppress phospholipase C activation. These results are consistent with the hypothesis that, in thrombin-stimulated platelets, the liberation of arachidonic acid and beta-TG are strictly dependent on the activation of phospholipase C. We have also provided evidence for the existence of a phospholipase A2 activated by a G-protein which is independent from the degradation of phosphoinositides and, contrary to phospholipase C, it is not down regulated by cGMP.     

Phosphatidic and lysophosphatidic acid production in phospholipase C-and thrombin-treated platelets. Possible involvement of a platelet lipase.

Incubation of 32P-labelled platelets with Clostridium welchii phospholipase C greatly stimulates 32P-incorporation into phosphatidic and lysophosphatidic acids. A net synthesis is demonstrated for both phospholipids, which exhibit identical specific radioactivities. Phosphatidic acid production roughly parallels the phospholipase C-induced aggregation, whereas lysophosphatidic acid appears secondarily during cell lysis. The same qualitative variations are observed during thrombin-induced aggregation. At the physiological pH used throughout the incubations, platelets display no phospholipase A activity towards phosphatidic acid, whereas diglycerides are deacylated by platelet lysates. On the basis of these findings, a mechanism for phosphatidic and lysophosphatidic acid production is proposed, involving a phosphorylation of the di- and monoglycerides formed upon phospholipase C and lipase action. The possible role of such a pathway in regulating arachidonic acid release from phospholipids during platelet activation is discussed.     

Shape change induced in human platelets by platelet-activating factor. Correlation with the formation of phosphatidic acid and phosphorylation of a 40,000-dalton protein

Washed human platelets that have been separated from plasma in the presence of prostacyclin are activated by the addition of platelet activating factor (PAF). Activation (shape change, serotonin release, and aggregation) correlates closely with the formation of phosphatidic acid and the phosphorylation of a 40,000-dalton protein. Platelet shape change, formation of phosphatidic acid, and protein phosphorylation precede aggregation and are induced at lower concentrations of PAF than those required to induce release of serotonin and platelet aggregation. Platelet shape change, formation of phosphatidic acid, and protein phosphorylation induced by PAF are not affected by trifluoperazine or indomethacin. This indicates that these responses are independent of the liberation of arachidonic acid from platelet phospholipids and the metabolism of arachidonic acid via cyclooxygenase and lipoxygenase. These responses are, however, inhibited by prostacyclin. Platelet shape change is the first measurable physiologic response to platelet agonists and may be associated with the stimulation of phospholipase C, inducing formation of 1,2-diacylglycerol and its phosphorylated product, phosphatidic acid. Transient formation of 1,2-diacylglycerol may also induce the specific activation of the protein kinase C that phosphorylates a 40,000-dalton protein.     

Cyclic AMP and platelet prostaglandin synthesis

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of ¹⁴C-arachidonic acid by washed platelets. Prostaglandin E₁ (PGE₁), PGE₁ with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B₂. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE₁ with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of ¹⁴C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.