Role of protein kinase C in the control of vascular prostacyclin: Study of phorbol esters effect in bovine aortic endothelium and smooth muscle

In bovine aortic endothelial cells, phorbol 12-myristate, 13-acetate induced a smaller stimulation of prostacyclin release than ionophore A23187: the combination of both agents was highly synergistic. The responses of the bovine aortic smooth muscle were very different in the 2 preparations studied. In media explants cultured for short periods, neither phorbol 12-myristate, 13-acetate, nor A23187, alone or in combination, were able to increase prostacyclin release, whereas serotonin was an effective stimulus. In cultured smooth muscle cells, outgrown from the explants, phorbol 12-myristate, 13-acetate increased prostacyclin release to the same levels as A23187 or serotonin. It is concluded that increased cytosolic Ca⁺⁺ level and protein kinase C activity induce a synergistic stimulation of endothelial prostacyclin. On the other hand, the phenotypic modulation of the arterial smooth muscle, from a contractile to a synthetic state, seems to be associated with a profound change in the control of prostacyclin.     

Pertussis toxin and H-7 distinguish mechanisms involved in eicosanoid release from lipopolysaccharide-primed macrophages. Eicosanoid release from lipopolysaccharide-primed macrophages

Release of eicosanoids is an important response of macrophages to inflammation and bacterial infection. At low concentrations, bacterial lipopolysaccharide (1-2 micrograms/ml) fails to stimulate eicosanoid release in resident peritoneal macrophages but primes the macrophages for a greatly enhanced release of eicosanoids on stimulation with the calcium ionophore A23187 (0.1 microM) or with phorbol 12-myristate 13-acetate (50 nM), an activator of protein kinase C. Incubation of macrophages with Bordetella pertussis toxin, prior to priming with lipopolysaccharide, inhibited the release of both cyclooxygenase and lipoxygenase products upon A23187 stimulation. Pertussis toxin treatment of macrophages had no effect on eicosanoid release when the stimulus was phorbol 12-myristate 13-acetate. The presence of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an effective inhibitor of protein kinase C, during lipopolysaccharide priming and subsequent stimulation significantly inhibited eicosanoid release when phorbol 12-myristate 13-acetate was the stimulus, but did not affect eicosanoid release stimulated by A23187. Based on these results, at least two mechanisms, distinguished by apparent differences in sensitivity to pertussis-toxin-sensitive, guanine-nucleotide-binding proteins and protein kinase C, are involved in eicosanoid secretion by lipopolysaccharide-activated macrophages in response to A23187 and phorbol 12-myristate 13-acetate.     

Prostacyclin production by the bovine aortic smooth muscle

It is well known that cultured aortic smooth muscle cells, the phenotype of which has modulated from contractile to synthetic, are able to release prostacyclin (PGI2). We have studied the release of PGI2 from cultured explants of bovine aortic media, which represent an homogeneous population of smooth muscle cells with a contractile phenotype. These explants released spontaneously huge amounts of PGI2, which was the major eicosanoid produced. PGI2 release was stimulated by serum and by serotonin. This experimental model seems useful to evaluate the contribution of smooth muscle to the biosynthesis of PGI2 by the arterial wall.     

Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation

The biochemical events that lead to thrombin-stimulated release of von Willebrand factor and prostacyclin synthesis in cultured endothelial cells are examined. Treatment of human umbilical vein endothelial cells with thrombin results in an instantaneous increase in phospholipid methylation which can be blocked by 3-deazaadenosine, a methyltransferase inhibitor. 3-Deazaadenosine also blocks the thrombin-induced Ca2+ influx into endothelial cells and the release of von Willebrand factor, indicating that these processes are coupled. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) and the Ca2+ ionophore A23187 both bypass the phospholipid methylation and directly stimulate Ca2+ influx and von Willebrand factor release. In contrast to the stimulus-induced von Willebrand factor release, the thrombin-induced prostacyclin synthesis cannot be blocked by 3-deazaadenosine. Similarly, incubation of endothelial cells with EDTA has no influence on the thrombin-induced prostacyclin synthesis, and PMA has no stimulatory effect on prostacyclin synthesis. These observations indicate that thrombin induces different metabolic responses in endothelial cells: phospholipid methylation followed by a Ca2+ influx, which subsequently leads to release of von Willebrand factor, and liberation of arachidonic acid from phospholipids for prostacyclin formation, which is independent of phospholipid methylation and Ca2+ influx.     

Prostacyclin production by the bovine aortic smooth muscle

It is well known that cultured aortic smooth muscle cells, the phenotype of which has modulated from contractile to synthetic, are able to release prostacyclin (PGI₂). We have studied the release of PGI₂ from cultured explants of bovine aortic media, which represent an homogeneous population of smooth muscle cells with a contractile phenotype. These explants released spontaneously huge amounts of PGI₂, which was the major eicosanoid produced. PGI₂ release was stimulated by serum and by serotonin. This experimental model seems useful to evaluate the contribution of smooth muscle to the biosynthesis of PGI₂ by the arterial wall.     

Endothelin stimulates diacylglycerol accumulation and activates protein kinase C in cultured vascular smooth muscle cells

Endothelin, a novel peptide isolated from the conditioned medium of endothelial cells, causes a slow, sustained contraction of vascular smooth muscle, but its mechanism of action remains unclear. To determine whether the diacylglycerol/protein kinase C signalling pathway is stimulated by endothelin, we exposed cultured rat aortic smooth muscle cells to endothelin and measured diacylglycerol accumulation and protein kinase C-dependent protein phosphorylation. Endothelin stimulated a dose-dependent, biphasic increase in diacylglycerol, which was sustained for at least 20 min. This peptide also induced a prolonged phosphorylation of an acidic protein with a molecular weight of 76,000, which was detectable by 30 s and sustained for at least 20 min. This phosphorylation could be mimicked by phorbol 12-myristate 13-acetate, but not by ionomycin, and was markedly reduced when protein kinase C was down-regulated by a 24-h pretreatment with phorbol 12,13-dibutyrate. These results suggest that endothelin causes a robust stimulation of the diacylglycerol/protein kinase C pathway in cultured vascular smooth muscle cells, and that this mechanism may contribute importantly to the physiologic events stimulated by endothelin in intact blood vessels, including slow, tonic contraction and Ca2+ influx.     

Phorbol12-Myristate13-Acetate Relieves the Inhibitory Effect of A23187 on Erythroid Differentiation of K562 Cells Induced by Dimethylsulfoxide

The differentiating effect of DMSO on K562 cells was studied against the background of pretreatment of the cells by the modulators of activities of protein kinase C and Ca signaling, phorbol 12-myristate 13-acetate, and ionophore A23187. The 2-hour pretreatment of K562 cells with A23187 (1 M), rather than phorbol 12-myristate 13-acetate (0.1 M), led to inhibition of the differentiating effect of DMSO (0.1%) during the subsequent four days of incubation. When the cells were pretreated jointly with A23187 and phorbol 12-myristate 13-acetate, the DMSO-induced erythroid differentiation was restored. Analysis of the DNA-degrading effect of the reagents used on K562 cells by fluorescent dyes under the same conditions suggests that this activity is induced in the presence of DMSO in the cells pretreated with a combination of phorbol 12-myristate 13-acetate and A23187 or without pretreatment.     

Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells

The subcellular colocalization of prostacyclin synthase (PGIS) with prostaglandin H synthase (PGHS) has not been delineated. To test the hypothesis that its colocalization with PGHS is crucial for prostacyclin synthesis, we determined subcellular locations of PGIS, PGHS-1, and PGHS-2 in bovine aortic endothelial cells by immunofluorescent confocal microscopy. PGIS and PGHS-1 were colocalized to nuclear envelope (NE) and endoplasmic reticulum (ER) in resting and adenovirus-infected bovine aortic endothelial cells. PGIS and PGHS-2 were also colocalized to ER in serum-treated or adenovirus-cyclooxygenase-2-infected cells. By contrast, PGIS was not colocalized with PGHS-2 in cells induced with phorbol 12-myristate 13-acetate where PGHS-2 was visualized primarily in vesicle-like structures. The lack of colocalization was accompanied by failed prostacyclin production. Resting ECV304 cells did not produce prostacyclin and had no detectable PGHS-1 and PGIS proteins. Confocal analysis showed abnormal colocalization of PGIS and PGHS-1 to a filamentous structure. Interestingly, the abundant PGIS and PGHS-1 expressed in adenovirus-infected ECV304 cells were colocalized to NE and ER, which synthesized a large quantity of prostacyclin. These findings underscore the importance of colocalization of PGHS and PGIS to ER and NE in prostacyclin synthesis.     

Prostacyclin-stimulating drugs: new prospects

SKF 525-A (proadifen), a well-known inhibitor of drug metabolism and cytochrome P-450 activity, stimulated the release of prostacyclin (PGI2) from the rabbit aorta in vitro. The PGI2-stimulating activity of SKF 525-A was characterized by specific structural requirements: activity was abolished by the deletion of the terminal propyl chain and increased by its elongation into an isobutyl chain; chlorination of the phenyl rings increased the potency. SKF 525-A increased the production of PGI2 by cultured endothelial cells from bovine aorta and human umbilical vein, but had no effect on cultured smooth muscle from the bovine aortic media. In human platelets, SKF 525-A inhibited prostaglandin and thromboxane production induced by A23187, thrombin and ADP. Simultaneous stimulation of endothelial PGI2 and inhibition of platelet TxA2 represents an original pharmacological profile: SKF 525-A might thus constitute the prototype of a new class of antiplatelet drugs.     

Synergistic Stimulation of Interleukin 6 Release and Gene Expression by Phorbol Esters and Interleukin 1β in Rat Cortical Astrocytes: Role of Protein Kinase C Activation and Blockade

Abstract: The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12-myristate 13-acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.     

Endothelial cells in culture produce a vasoconstrictor substance

We report that cultured vascular endothelial cells release into the culture medium a vasoconstrictor peptide, a substance we call an endothelium-derived constricting factor (EDCF). Conditioned medium from cultured bovine aortic and pulmonary artery endothelial cells caused sustained, dose-dependent isometric constriction of vascular rings isolated from bovine coronary and pulmonary arteries and rat and guinea pig pulmonary arteries and aortas. The medium also caused vasoconstriction when infused into isolated, perfused rabbit hearts and rat kidneys. Conditioned medium from bovine aortic intimal explants also contained constrictor activity, whereas medium from denuded intimal explants, cultured microvascular endothelial cells, vascular smooth muscle cells, or lung fibroblasts did not. Constrictor activity increased progressively in the culture medium over 2-12 h of incubation. Thrombin stimulated the release of constrictor activity; hypoxia, anoxia and meclofenamate had no effect and the calcium ionophore A23187 inhibited EDCF release. The EDCF caused a characteristic slow-onset and sustained constriction of the vascular rings that relaxed slowly over 60-90 min following removal. The constriction was not affected by inhibitors of arachidonic acid metabolism or by antagonists of serotonergic, histaminergic, alpha-adrenergic, opioid, leukotriene, angiotensin II, or substance P receptors; constriction was reversed partly by verapamil and acetylcholine and completely by nitroprusside and isoproterenol. EDCF was heat stable, not extractable into organic solvents, and completely destroyed by trypsin and neutral protease. Cycloheximide blocked the production of EDCF. These properties and the results of polyacrylamide gel filtration experiments suggested that EDCF was a peptide with a molecular weight of 3,000 daltons. These findings show that endothelial cells in culture produce a vasoconstrictor substance and support the idea that endothelial cell products play a role in mediating vascular tone.     

Effect of Age on Brain Cortical Protein Kinase C and Its Mediation of 5-Hydroxytryptamine Release

The effects of age on the activity and translocation of protein kinase C (PKC) and on the facilitation of 5-hydroxytryptamine (5-HT, serotonin) release induced by PKC activation with the phorbol ester phorbol 12-myristate 13-acetate were investigated. The activities of cortical PKC and its translocation in response to K+ depolarization and phorbol ester stimulation were reduced during aging in Fischer-344 rats. Parietal cortical brain slices from 6-, 12-, and 24-month-old animals were preloaded with [3H]5-HT and release was evoked by 65 mM K+ or the calcium ionophore A23187. 5-HT release induced by either K+ or A23187 was found to be reduced in 12- and 24-month-old as compared to 6-month-old animals. This decrease was not reversed by high extracellular Ca2+. Activation of PKC resulted in a facilitated transmitter release in tissue from 6- and 12-month-old animals but reduced [3H]5-HT release in slices from 24-month-old animals. These responses were prevented by the putative PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), but not by increasing extracellular or intracellular Ca2+. The results demonstrate an age-related change (1) in brain PKC activity and translocation and (2) in a physiological response to PKC stimulation. These results may have implications for other PKC-mediated functions that are altered during senescence.     

Serotonin-induced deoxyribonucleic acid synthesis in vascular smooth muscle cells involves a novel, pertussis toxin-sensitive pathway

Serotonin-induced DNA synthesis in bovine aortic smooth muscle cells was totally abolished by pretreatment of cultures with 5 ng/ml pertussis toxin. The half maximally effective concentration of toxin was approximately 10 pg/ml. Pertussis toxin did not affect platelet-derived growth factor (PDGF)-stimulated DNA synthesis and actually enhanced the mitogenic effect of the phorbol ester, phorbol 12-myristate 13-acetate. Pertussis toxin did not inhibit serotonin-stimulated inositol phosphate accumulation or increases in intracellular calcium or cAMP concentrations under conditions sufficient to completely inhibit serotonin-induced (3H)thymidine incorporation. These results demonstrate that a novel, pertussis-sensitive pathway is required for serotonin-, but not platelet-derived growth factor-induced DNA synthesis in vascular smooth muscle cells. The pertussis-sensitive step does not involve cAMP, phosphoinositide hydrolysis, mobilization of intracellular calcium, or phorbol ester-sensitive protein kinase C activity.     

Sequential incorporation of 3H- and 14C-arachidonic acid during 24 h in rabbit colonic explants cultured in vitro.

Arachidonic acid (AA) might be released from a number of different intracellular pools. This aspect was studied by sequential incorporation of 3H- and 14C-AA during 24 h in rabbit colonic biopsies in culture. Mucosal explants were subjected to 3H-AA (0-6 h) and 14C-AA (6-24 h) followed by a 2-h washout. Lipid extraction of biopsies revealed a distinct pattern where 3H, in relative terms, dominated in phosphatidylinositol (PI) and phosphatidylethanolamine (PE) and 14C was more abundant in neutral lipids, whereas the remaining lipids had equal proportions of labellings. When stimulating double labelled biopsies with 1 microM phorbol 12-myristate 13-acetate (PMA), 10 micrograms/ml A23187 and a combination thereof, the 14C-labelling was in general released in larger amounts, i.e. a maximum of 7.5% with the combined drugs vs 5% for 3H. However, the relative release of 3H increased upon stimulation especially for PMA + A23187. The release of labelling was directly coupled to a stimulation dependent production of PGE2 in the order PMA less than A23187 less than PMA + A23187, maximum release = 8.2 +/- 2.4 ng/h and 3 biopsies. This study shows the existence of different AA-pools, where PI and PE (3H-labelling) appears to have a slow turnover and representing a terminus in a redistribution chain and where neutral lipids (14C-labelling) have a more rapid turnover. Upon stimulation more AA appears to be recruited from pools with slow turnover.     

Neural differentiation of amphibian gastrula ectoderm exposed to phorbol ester

Summary Ectoderm from early gastrula stages of amphibians was isolated and treated with phorbol 12-myristate 13-acetate. The ectoderm formed neural tissue and in a few cases also mesenchyme and melanophores. The control explants formed atypical epidermis. In explants treated with phorbol 12-myristate 13-acetate the mitotic rate was increased.     

Inhibition of protein kinase C by H-7 potentiates the release of oleic, linoleic and arachidonic acids in A23187-stimulated human neutrophils

This present report describes the effect of H-7, a protein kinase C inhibitor, on the release of oleic, linoleic and arachidonic acids in A23187-stimulated neutrophils. Surprisingly, the inhibitor potentiated the release of all three unsaturated fatty acids in neutrophils stimulated with A23187 alone. In contrast, released oleic acid, linoleic acid and arachidonic acid in phorbol 12-myristate 13-acetate-primed neutrophils were attenuated by 35, 47 and 33%, respectively, in the presence of H-7 (300 microM). Phorbol 12-myristate 13-acetate (PMA) had no effect on A23187-stimulated release of saturated fatty acids. Both PMA and H-7 when used alone had no effect on the release of saturated or unsaturated fatty acids. We, therefore, conclude that H-7 may have effects other than inhibiting PMA-primed responses including superoxide generation, degranulation and arachidonic acid release in human neutrophils.     

Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms.

Using cultured human umbilical vein endothelial cells, in which phosphatidylcholine (PC) is equally pulse-labelled by various eicosanoid precursor fatty acids (EPFAs), we have studied the remodelling of EPFAs among the phospholipid classes and subclasses with and without activation, and the relationship of this remodelling process to the selective release of arachidonic acid (AA) by phospholipase A2-mediated cell stimulation. When endothelial cells are pulse-incubated with radiolabelled EPFA for 15 min, greater than 80% of cell-associated radioactivity is present in phospholipids, among which greater than 60% is found in 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl PC). After removing unincorporated radioactivity, reincubation of the pulse-labelled cells for up to 6 h results in progressive decrease in EPFA-labelled diacyl PC, increase in AA- or eicosapentaenoic acid (EPA)-labelled 1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmalogen PE) and increase only in AA-labelled 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl PC). This redistribution of radiolabelled phospholipids is not altered by the presence of excess non-radiolabelled EPFAs. When aspirin-treated EPFA-labelled endothelial cells are stimulated with ionophore A23187, a very selective release of AA is noted in comparison with eicosatrienoate (ETA) or EPA, accompanied by an equivalent decrease in AA-labelled diacyl PC and specific increase in AA-labelled plasmalogen PE and alkyl PC. These selective changes in AA radioactivity induced by A23187 are enhanced 2-fold by pretreating the AA-labelled cells with phorbol 12-myristate 13-acetate, which by itself induces no changes. The changes in radioactivity induced by A23187 without and with phorbol ester among the released AA, the diacyl PC and the plasmalogen PE are significantly correlated with each other. These results indicate that human endothelial cells incorporate EPFAs (AA, ETA, EPA) equally into diacyl PC but selectively release AA esterified into diacyl PC with specific remodelling into plasmalogen PE and alkyl PC.     

Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells

ATP and ADP, in concentrations ranging from 1-100 microM, increased the release of [3H]choline and [3H]phosphorylcholine (P-choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP gamma S and ADP beta S) and adenosine 5'-(beta, gamma imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5'-(alpha, beta methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 microM), the tumor promoter phorbol 12-myristate 13-acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 microM). Down-regulation of protein kinase C, following a 24-hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P-choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).     

Inhibitory and stimulatory effects of phorbol ester on vasopressin-induced cellular responses in cultured rat aortic smooth muscle cells

In rat aortic smooth muscle cells, vasopressin (AVP) induces prostacyclin (PGI2) production, probably as the consequence of phospholipase C activation. Our study analyzes the effects of phorbol 12-myristate 13-acetate (PMA)-induced protein kinase C (PKC) activation on AVP-induced inositol 1,4,5-trisphosphate formation, cytosolic free Ca2+ concentration [( Ca2+]c), and PGI2 production. PMA rapidly decreased PKC activity in the cytosol of smooth muscle cells, while increasing it transiently in the membranes with a maximum around 20 min. Prior exposure of the cells to PMA resulted in a transient inhibition of both AVP-induced inositol 1,4,5-trisphosphate formation and [Ca2+]c rise. This was inversely correlated with membraneous PKC activity and partially reversed by the PKC inhibitor staurosporine. In contrast, pretreating the cells with PMA markedly potentiated A23187 or AVP-induced PGI2 production. Under those conditions, AVP-induced PGI2 production did not correlate either with PMA-induced membranous PKC activity or with AVP-induced PLC activation. However, this potentiating effect of PMA was reversed by staurosporine and was not mimicked by the 4 alpha-phorbol, an inactive analogue of PMA. Thus, the possibility is raised that, while inhibiting AVP-induced PLC activation, PMA-induced PKC activation increases the Ca2+ sensitivity of the cellular signaling system leading to PGI2 production.     

Release of eicosanoids from cultured rat aortic endothelial cells; studies with arachidonic acid and calcium ionophore A23187

The release of prostanoids from the three different vascular cell types derived from rat aortic explants has been studied in vitro. Under resting conditions and when incubated with exogenous arachidonic acid (AA, 10 microM), the endothelial cells (EC) produced the highest concentration of prostacyclin (PGI2 PGE2 PGF2 alpha TxA2). In contrast, PGE2 was the major prostanoid produced by the smooth muscle cells and fibroblasts. Pretreatment of EC with aspirin (10 microM) or indomethacin (10 microM) effectively inhibited the production of prostanoids by these cells. Incubation with the calcium ionophore A23187 (10 microM) did not stimulate production of PGI2 or leukotriene B4 (LTB4) by EC. However, treatment of EC with a combination of A23187 and AA led to production of amounts of both PGI2 and LTB4 which were greater than the summed values for the different drug treatments. These findings indicate that the concentration of substrate, AA, is a limiting factor in prostanoid formation by these cultured vascular cells but that rat EC are relatively poor in the enzymes required for leukotriene formation.