Inhibition of prostaglandin synthesis in human endothelial cells treated with metabolic inhibitors

Endothelial cell injury is often associated with increased synthesis of prostaglandin (PG)I₂. We observed, however, that endothelial cells treated with metabolic inhibitors which reduce cellular ATP content develop an injury pattern characterized by reduced PGI₂ synthesis. This study examined the relationship between cell injury, arachidonic acid metabolism and ATP content in human umbilical vein endothelial cells treated with 2-deoxyglucose (2DG), a glycolytic inhibitor, and oligomycin (OG), a respiratory chain inhibitor. Either inhibitor alone significantly reduced cellular ATP concentrations, but only OG reduced basal PG synthesis. The combination of 2DG and OG, however, was more effective than either agent alone in reducing cellular ATP content (≥ 50% of control) and inhibiting basal and agonist-stimulated PGI₂ synthesis. This reduced PGI₂ synthesis preceded ⁵¹ chromium release, lactic dehydrogenase release and was not associated with a net release of arachidonic acid from cell membranes. Histamine, A23187 and bradykinin stimulated PGI₂ synthesis in untreated but not in 2DG and OG treated cells. Exogenous arachidonic acid increased PGI₂ synthesis to a similar extent in both 2DG and OG treated and untreated cells. Therefore, reduced PG synthesis in 2DG and OG treated endothelial cells is not due to inhibition of cyclooxygenase. Furthermore, reduced PG synthesis in these cells occurs prior to cell injury and is not strictly associated with cellular ATP depletion.     

Prostacyclin does not play any cytoprotective role in endothelial cell injury induced by 15-hydroperoxy-eicosatetraenoic acid, an arachidonate lipoxygenase product

Among various arachidonic acid metabolites examined, only 15(S)-hydroxperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), a lipoxygenase product, caused a time- and dose-dependent injury to bovine endothelial cells in culture. There also occurred a significant inhibition of endothelial prostacyclin (PGI2) production due to 15-HPETE. But there were obvious dissociations in time course and dose dependence between 15-HPETE-induced cellular injury and 15-HPETE-induced inhibition of PGI2 synthesis. In addition, the cytotoxicity of 15-HPETE was not aggravated even when the endothelial monolayers were pretreated with several inhibitors of PGI2 synthesis. Also, some stable analogues of PGI2 had no protective effect on the injury. These results suggest that the reduced production of PGI2 caused by 15-HPETE is not directly associated with the onset of cellular injury, and that PGI2 does not play any cytoprotective role in endothelial cell injury induced by at least such lipid peroxides as 15-HPETE.     

Docosatetraenoic acid in endothelial cells: formation, retroconversion to arachidonic acid, and effect on prostacyclin production

Cultured bovine aortic endothelial cells convert arachidonic acid to docosatetraenoic acid and also take up docosatetraenoic acid from the extracellular fluid. After a 24-h incubation with biosynthetically prepared [3H]docosatetraenoic acid, about 20% of the cellular fatty acid radioactivity was converted to arachidonic acid. Furthermore, in pulse-chase experiments, the decrease in phospholipid docosatetraenoic acid content was accompanied by an increase in arachidonic acid, providing additional evidence for retroconversion. These findings suggest that one possible function of docosatetraenoic acid in endothelial cells is to serve as a source of arachidonic acid. The endothelial cells can release docosatetraenoic acid when they are stimulated with ionophore A23187, but they do not form appreciable amounts of eicosanoids from docosatetraenoic acid. Enrichment of the endothelial cells with docosatetraenoic acid reduced their capacity to produce prostacyclin (PGI2) in response to ionophore A23187. This may be related to the fact that docosatetraenoic acid enrichment caused a 40% reduction in the arachidonic acid content of the inositol phosphoglycerides. In addition, less prostacyclin was formed when the enriched cells were incubated with arachidonic acid, suggesting that docosatetraenoic acid also may act as an inhibitor of prostaglandin synthesis in endothelial cells.     

Cultured endothelial cells increase their capacity to synthesize prostacyclin following the formation of a contact inhibited cell monolayer

The synthesis of the prostaglandins (PG), prostacyclin (PGI2), PGE2, and thromboxane A2 (TXA2), has been investigated in actively growing and contact-inhibited bovine aortic endothelial cell cultures. Cells were stimulated to synthesize prostaglandins by exposure to exogenous arachidonic acid or to the endoperoxide PGH2 and by the liberation of endogenous arachidonic acid from cellular lipids with melittin or ionophore A23187. Increased capacity of the cells to synthesize PGI2 and PGE2 was observed as a function of time in culture, regardless of the type of stimulation. TXA2 production increased with time only upon stimulation of the cells with ionophore A23187. This increased PG synthetic capacity was independent of cell density since it was mainly observed in confluent, nondividing endothelial cell cultures. The fact that increased PGI2 production in confluent cells was also observed with PGH2, a direct stimulator of PGI2 synthetase, implies that this process is independent of the arachidonate concentration within the cells or in the culture medium. This increased capacity is likely to reflect an increased activity of the PG synthetase system associated with the formation of a contact inhibited endothelial cell monolayer. A similar time-dependent increase in the PGI2 production capacity was also observed during growth of cultured bovine corneal endothelial cells.     

The influence of gamma radiation on arachidonic acid release and prostacyclin synthesis

Cultured pulmonary artery endothelial cells produce PGI2 as their primary prostaglandin. Conditions which inhibit cell division have been shown to accelerate the synthesis of this compound. Exposure of endothelial cells to gamma radiation results in an irreversible cessation of growth and enhanced production of PGI2. The level of PGI2 measured after radiation exposure exceeds that observed in cultures rendered quiescent by serum reduction. This indicates a role for gamma radiation in the elevation of PGI2 levels which is distinct from its effect on cell division. Results presented indicate that exposure to gamma radiation does not, in and of itself, elevate PG levels but capacitates cells for enhanced production when presented with appropriate stimuli. Increased PGI2 synthesis appears to be a result of an observed increase in arachidonic acid release and an activation of cyclooxygenase.     

Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells

Human umbilical vein endothelial cells incorporate eicosapentaenoic acid (EPA) when this fatty acid is present in the culture medium. From 30 to 70% of the uptake remains as EPA, and much of the remainder is elongated to docosapentaenoic acid. All of the cellular glycerophospholipids become enriched with EPA and docosapentaenoic acid, with the largest increase in EPA occurring in the choline glycerophospholipids. When this fraction is enriched with EPA, it exhibits a large decrease in arachidonic acid content. Cultures exposed to tracer amounts of [1-14C]linolenic acid in 5% fetal bovine serum convert as much as 17% of the radioactivity to EPA. The conversion is reduced, however, in the presence of either 20% fetal bovine serum or 50 microM linolenic acid. Like arachidonic acid, some newly incorporated EPA was released from the endothelial cells when the cultures were exposed to thrombin. However, as compared with arachidonic acid, only very small amounts of EPA were converted to prostaglandins. Cultures enriched with EPA exhibited a 50 to 90% reduction in capacity to release prostacyclin (PGI2) when subsequently stimulated with thrombin, calcium ionophore A23187, or arachidonic acid. The degree of inhibition was dependent on the time of exposure to EPA and the EPA concentration, and it was not prevented by adding a reversible cyclooxygenase inhibitor, ibuprofen, during EPA supplementation. EPA appears to decrease the capacity of the endothelial cells to produce PGI2 in two ways: by reducing the arachidonic acid content of the cell phospholipid precursor pools and by acting as an inhibitor of prostaglandin production. These findings suggest that regimens designed to reduce platelet aggregation and thrombosis by EPA enrichment may also reduce the capacity of the endothelium to produce PGI2.     

Activity of prostaglandin biosynthetic pathways in rat pancreatic islets

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.     

Ca2(+)-dependent synthesis of prostaglandin I2 and mobilization of arachidonic acid from phospholipids in cultured endothelial cells permeabilized with saponin

The feasibility of using saponin as a permeabilization agent to study the effect of free Ca2+ concentration ([Ca2+]f) on prostaglandin I2 (PGI2) synthesis and mobilization of arachidonic acid from membrane phospholipids was investigated in cultured bovine pulmonary artery endothelial cells (BPAEC). Treatment of BPAEC with 20 micrograms/ml saponin caused selective permeabilization of the plasma membrane as determined by measurements of the release of lactate dehydrogenase and beta-hexosaminidase. In cells prelabeled with [3H]arachidonic acid for 22 h, permeabilization with 20 micrograms/ml saponin induced PGI2 synthesis and release of [3H]arachidonic acid from membrane phospholipids. These effects were dependent upon [Ca2+]f in the range 72 nM to 5 microM. Release of [3H]arachidonic acid from phospholipid classes was determined in suspensions of BPAEC prelabeled with [3H]arachidonic acid and permeabilized with 20 micrograms/ml saponin. At [Ca2+]f optimal for PGI2 synthesis, 16.2% of the total incorporated [3H]arachidonic acid was released from phosphatidylinositol (3.4%), phosphatidylethanolamine (3.5%) and phosphatidylcholine (9.3%). The time course and dependence upon [Ca2+]f of [3H]arachidonic acid release from phospholipids correlated with PGI2 synthesis. The amount of PGI2 synthesized in permeabilized BPAEC was similar to that in cell cultures treated with the calcium ionophore A23187. In comparison, however, PGI2 synthesis induced by A23187 was associated with less release of [3H]arachidonic acid from membrane phospholipids, e.g., 2.3% versus 16.2%. The greater loss of [3H]arachidonic acid from phospholipids in saponin-permeabilized BPAEC was most likely due to the loss of cell integrity and/or nonspecific effects of the detergent on phospholipases. Despite these limitations, the Ca2+ dependence observed for PGI2 synthesis and [3H]arachidonic acid mobilization suggest that saponin-permeabilization may provide a useful system for studies of the intracellular events triggered by the rise in intracellular Ca2+ which culminate in PGI2 synthesis.     

Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells.

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca2+]i) and the intracellular level of inositol 1,4,5-trisphosphate (IP3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP3 levels, [Ca2+]i and PGI2 release. An increase in [Ca2+]; and PGI2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca2+]i or PGI2 release. The inability of ATP to cause an increase in [Ca2+]i or PGI2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca2+]i and PGI2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca2+]i and PGI2 release, further evidence that PGI2 release is dependent upon an increase in [Ca2+]i. The increase in [Ca2+]i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A2, prevented the stimulation of PGI2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI2 synthesis, prior to the release of PGI2 from BPAE cells. These findings suggest that the increase in PGI2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca2+]i and the subsequent activation of a phosphatidylcholine-specific phospholipase A2. ATP analogs modified in the adenine base or phosphate moiety caused PGI2 release with a rank order of agonist potency of adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) greater than 2-methylthioATP (2-MeSATP) greater than ATP, whereas alpha, beta methyleneATP and beta, gamma methyleneATP had no effect on PGI2 release.     

Physiological concentrations of ADP stimulate the release of prostacyclin from bovine aortic endothelial cells

ADP (0.2-200 microM) stimulated the synthesis of prostacyclin (PGI2), as reflected by the release of 6-keto-prostaglandin F1 alpha (6-K-PGF1 alpha), in endothelial cells cultured from bovine aorta. This effect of ADP was mimicked by ATP, whereas AMP and adenosine were completely inactive. The release of 6-K-PGF1 alpha triggered by ADP was rapid and onset (within 5 min), transient (10 min) and followed by a period of refractoriness to a new ADP challenge. Growing and confluent cells were equally responsive to ADP. ADP stimulated the release of free arachidonic acid from the endothelial cells. ADP could thus exert two opposite actions on platelet aggregation in vivo: a direct stimulation and an inhibition mediated by PGI2. This last action might contribute to limit thrombus formation to areas of endothelial cell damage.     

IL-1 and related cytokines enhance thrombin-stimulated PGI2 production in cultured endothelial cells without affecting thrombin-stimulated von Willebrand factor secretion or platelet-activating factor biosynthesis

We examined the effects of various cytokines on alpha-thrombin-stimulated prostaglandin (PG) I2 production, von Willebrand factor (vWF) secretion, and platelet-activating factor (PAF) synthesis in cultured human umbilical vein endothelial cells (HUVEC). A 24-h pretreatment with IL-1 beta doubled the low level of constitutive PGI2 production. In contrast, alpha-thrombin increased PGI2 production fivefold in untreated HUVEC. The most striking increase in PGI2 production was observed in IL-1 beta-treated HUVEC that were subsequently stimulated with thrombin. PGI2 production was two to three times greater than in untreated, thrombin-stimulated HUVEC and nearly eightfold greater than in IL-1 beta-treated but unstimulated HUVEC. Enhanced thrombin-stimulated PGI2 production was also observed in HUVEC pretreated with the related cytokines IL-1 alpha, TNF, or lymphotoxin. This cytokine effect was selective for PGI2 production because none of these cytokines altered either constitutive or thrombin-stimulated vWF secretion or PAF biosynthesis. IL-1 beta enhancement of thrombin-stimulated PGI2 production was concentration and time dependent and required protein synthesis. IL-1 beta pretreatment also enhanced PGI2 production in response to another agonist, histamine, and to exogenously added substrates, arachidonic acid or PGH2. Our results indicate that activation by IL-1 and related cytokines selectively primes endothelial cells for enhanced PGI2 production, but not vWF secretion or PAF synthesis, in response to thrombin and histamine. The evidence suggests that this effect is mediated through specific induction of biosynthetic enzymes for PGI2.     

17 alpha-ethinylestradiol decreases production and release of prostacyclin in cultured human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) were incubated with the estrogen 17 alpha-Ethinylestradiol (30 ng/ml) in order to examine its regulating influence on synthesis and release of prostacyclin (PGI2). ATP (1 mg/ml) was used to stimulate PGI2-production through the purine receptors. We demonstrated that this level of estrogen decreases PGI2-synthesis by 11% and PGI2-release by 32% within 300 sec. Longer incubation times (48 hrs) resulted in the same inhibitory effect. Intracellular ATP content and methyl-3H-thymidine uptake demonstrated that the decrease of prostacyclin-concentration is not caused by reduced viability of the cells but by a direct inhibitory effect on prostacyclin synthesis and release.     

Cytoprotective effect of reduced glutathione in hydrogen peroxide-induced endothelial cell injury

The kinetic effects of hydrogen peroxide (H2O2) on cultured endothelial cells isolated from bovine carotid artery were studied. The cytoprotective effects of glutathione (GSH) on H2O2-induced cell injury were also investigated. H2O2-induced a dose- and time-dependent cell injury in cultured endothelial cells. H2O2-induced cell injury was blocked by simultaneous treatment by catalase, but not by superoxide dismutase. H2O2 also induced endogenous PGI2 biosynthesis, and the maximum PGI2 production was reached after 1 h treatment. Stimulation of PGI2 production was parallel with arachidonate release from H2O2-treated cells. However the prostaglandin biosynthesis enzyme activity in cells was inhibited by H2O2 treatment. When the cells were treated with GSH, the intracellular GSH reached a plateau after 3 h treatment. Both H2O2-induced cell injury and PGI2 production were significantly inhibited by the 3 h pretreatment with GSH. The cytoprotective effect of GSH was completely inhibited by buthionine sulfoximine which is a specific inhibitor of gamma-glutamylcysteine synthetase. The results indicate that the cytoprotective effect of GSH on H2O2-induced cell injury in cultured bovine carotid artery endothelial cells depends on the increase in intracellular GSH content.     

Evidence for a two-step process in prostaglandin secretion. Intracellular accumulation of prostacyclin precedes its release from human endothelial cells in culture

Cultured endothelial cells (EC) from human umbilical vein were incubated with [U-14C]arachidonic acid (AA) followed by a challenge with thrombin (2 units/ml) or calcium ionophore A23187 (5 microM) for 0.5-10 min at 37 degrees C. In both cases, AA was rapidly liberated from phospholipids and converted into prostaglandin I2 (PGI2), as determined by the radioactivity of the stable derivative 6-keto-PGF1 alpha. Maximal liberation of AA and synthesis of PGI2 were achieved within 2 min, but the two compounds first accumulated in EC prior to their release into supernatants. This finding, which was never reported before, raises the question of the mechanism of AA and PG release through the cell membranes and offers a convenient model to investigate this still obscure process.     

Endothelial cell prostacyclin production induced by activated neutrophils

A bovine aortic endothelial cell (EC) line released prostacyclin (greater than 1 pmol/10(+5) EC cells) when incubated with fMet-Leu-Phe (FMLP)-stimulated rat and human neutrophils (PMNs). This prostaglandin (PG) I2 was shown to come from the ECs and not from the PMNs by radioactive, high-performance liquid chromatography, and immunochemical criteria. Both FMLP-stimulated rat peritoneal and human peripheral PMNs as well as their stimulated cell-free supernatants and unstimulated sonicates could elicit the release of PGI2 from ECs. Since phorbol myristate acetate stimulated PMN adherence but elicited little PGI2 release from ECs, the PGI2 stimulation in ECs is unrelated to PMN adhesion. The addition of catalase and superoxide dismutase to FMLP-stimulated PMNs enhanced rather than reduced PGI2 formation, indicating that activated oxygen products of the PMN are not responsible for the induction of PGI2. Incubation of ECs with leukotriene (LT) B4, LTC4, or LTD4 did not trigger PGI2 release nor did aspirin pretreatment of the PMNs reduce the PGI2 induction. These data suggest that arachidonic acid metabolites of the PMNs were not responsible for the PGI2 induction. Available data indicates that the PMN factor that stimulates PGI2 from ECs is either released concomitantly with the azurophilic granules or is closely related to this event.     

On the role of arachidonic acid metabolites in mast-cell mediated mitogenesis in the rat

We investigated whether the mitogenic response induced by local mast-cell secretion in the rat mesentery was affected by suppression of phospholipase A2, lipoxygenase, or cyclooxygenase in arachidonic acid metabolism. Enzyme inhibitor was given in a single intravenous dose 5 min before intraperitoneal injection of the mast-cell secretagogue 48/80. Mepacrine, a phospholipase A2 inhibitor, suppressed the generation of both leukotrienes (SRS) and prostaglandins (PG), whereas the lipoxygenase inhibitor BW 755C reduced the generation of SRS, and the cyclooxygenase inhibitor indomethacin significantly suppressed the generation of PG. None of the enzyme inhibitors affected the basal mesenteric histamine content or histamine release in the mesentery after exposure to 48/80, and none of them affected mast-cell-mediated mitogenesis in the mesentery as judged by specific DNA activity and mitosis counting. The stimulation of DNA synthesis and mitosis initiated by secreting mast cells is apparently not mediated or modulated by synthesis of leukotrienes, prostaglandins, or other known arachidonic acid metabolites.     

Eicosapentaenoic acid metabolism in brain microvessel endothelium: effect on prostaglandin formation

Mouse brain microvessel endothelial cells convert eicosapentaenoic acid (EPA) to prostaglandin (PG) E3, PGI3, and several hydroxy fatty acid derivatives. Similar types of products are formed by these microvessel endothelial cells from arachidonic acid. The formation of PGI2 and PGE2 is reduced, however, when the brain microvessel endothelial cultures are incubated initially with EPA. Exposure to linolenic or docosahexaenoic acid also decreased the capacity of these microvessel endothelial cells to form PGI2 and PGE2, but the reductions were smaller than those produced by EPA. Like the endothelial cultures, intact mouse brain microvessels convert EPA into eicosanoids, and incubation with EPA reduces the subsequent capacity of the microvessels to produce PGI2 and PGE2. Brain microvessel endothelial cells took up less EPA than arachidonic acid, primarily due to lesser incorporation into the inositol, ethanolamine, and serine glycerophospholipids. By contrast, considerably more EPA than arachidonic acid was incorporated into triglycerides. These findings suggest that the microvessel endothelium may be a site of conversion of EPA to eicosanoids in the brain and that EPA availability can influence the amount of dienoic prostaglandins released by the brain microvasculature. Furthermore, the substantial incorporation of EPA into triglyceride suggests that this neutral lipid may play an important role in the processing and metabolism of EPA in brain microvessels.     

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.     

The effects of ionizing radiation on the pulmonary endothelial cell uptake of alpha-aminoisobutyric acid and synthesis of prostacyclin

The effects of gamma irradiation (150-3000 rad) on prostacyclin synthesis (PGI2) and Na+-dependent amino acid uptake (alpha-aminoisobutyric acid, AIB) were assessed in vitro in bovine pulmonary artery endothelial cells grown in plastic culture dishes. A dose-dependent increase in both PGI2 synthesis and AIB was found 24 h after irradiation at exposure levels greater than 600 rad. The increase in PGI2 synthesis [297% of sham-irradiated values at 3000 rad, P less than 0.01] was due to an increase in release of arachidonic acid from plasma membrane stores as well as stimulation of cyclooxygenase and/or prostacyclin synthetase enzymes. The increase in AIB uptake (75% increase at 3000 rad compared to sham-exposure values) correlated with the increased synthesis of PGI2 (r = 0.94). There was also a dose-dependent increase in the number of cells that became detached from the culture dishes during the 24-h period after irradiation. The changes in PGI2 synthesis and AIB uptake induced by gamma irradiation differed if the endothelial cells were grown on cover slips, indicating that the endothelial response to irradiation may be dependent on the interaction between the endothelial cell and its extracellular basement membrane matrix.     

Eicosanoid production by the coronary microvascular endothelium

Cultured rabbit coronary microvessel endothelial (RCME) cells have been used as an in vitro model to study the regulation of microvascular endothelial cell prostaglandin (PG) production by hormones, vasoactive drugs, and inflammatory mediators in an environment that can be tightly controlled and that is unaffected by interactions with other cell types, physical stimulation, or alterations in oxygenation. The most potent stimuli for RCME cell PG secretion were substances associated with inflammation, including histamine, interleukin 1, leukotriene D4, fMet-Leu-Phe, interferon-gamma, and exogenous phospholipases. Inhibition of calcium availability by lower [Ca2+]o or by treatment with calcium channel blockers reduced A23187-stimulated PG release but increased PG synthesis from exogenous arachidonic acid (AA). These observations suggest that Ca2+ may regulate several steps in the pathway leading to PG synthesis and release. Elevated intracellular [Ca2+] may, on the one hand, promote PG production by stimulating phospholipase A2 leading to AA release and, on the other hand, limit the magnitude of the response by increasing the rate of AA reacylation. Glucocorticoids reduce PG production by RCME cells via an action that requires new protein and mRNA synthesis and appears to involve the production of an endothelial cell-derived phospholipase inhibitory protein, or "endocortin." Thus, microvascular endothelial cells can both contribute to (by the release of PGs and possibly platelet-activating factor-acether) and limit (by the production of endocortins) the degree of a local inflammatory response in the heart.