Enhanced formation of PGI2, a potent hypotensive substance, by aortic rings and homogenates of the spontaneously hypertensive rat.

Intact rings and homogenates of aorta from spontaneously hypertensive rats (SHR) contain enhanced capacity over normal rats (NR) to convert arachidonic acid into PGI2. The PGI2 synthetic system in SHR is stimulated to a greater extent than NR by norepinephrine. Indomethacin blocks this stimulation. PGE2 and PGF2alpha were detected in much smaller amounts in homogenates (undetected in rings) but their formation was not enhanced by the hypertensive tissue. The identity of PGI2 was based on 1) direct pharmacological assay on the rat blood pressure. In this system identical vasodepressor responses to PGI2 are observed after intracarotid and intrajugular administration 2) indirectly as 6-keto PGF1alpha isolated after incubation of aortic homogenates with tritiated arachidonic acid and 3) indirectly by GC-MS assay of PGE2, PGF2alpha and 6-keto PGF1alpha formed during incubation of aortic homogenates with excess unlabeled arachidonic acid. These results provide additional support to our recent hypothesis that PGI2, of aortic origin, might actively participate in the regulation of systemic blood pressure. Its enhanced formation by intact hypertensive vascular tissue reflects an increase in the number of enzyme molecules immediately available to the substrate. This could probably be an adaptive response to the elevated levels of catecholamines in the circulation.     

Metabolism of prostacyclin. III. Urinary metabolite profile of 6-keto PGF1 alpha in rat.

The in vivo metabolism of 6-keto PGF1 alpha was investigated in rats. Following continuous intravenous infusion for 14 days the urinary metabolites were isolated and identified. A substantial amount of unchanged 6-keto PGF1 alpha was recovered in the urine. The metabolic pattern very closely resembles that of PGI2 in rats. Metabolites were found which represented 15-dehydrogenation, beta-oxidation, omega and omega-1-hydroxylation and oxidation. Previous work showed that 6-keto PGF1 alpha is very poorly oxidized by 15-PGDH. We administered 15-[H3]-PGI2 and 15-[H3]-6-keto PGF1 alpha to rats and measured urinary tritiated water as an index for in vivo 15-PGDH activity. The results showed that PGI2 and 6-keto PGF1 alpha were both oxidized to the 15-keto product, although the rate of oxidation of PGI2 was greater than that of 6-keto PGF1 alpha. We concluded that the administered PGI2 was oxidized by 15-PGDH before hydrolysis to 6-keto PGF1 alpha. A portion of the dose is probably hydrolzyed before 15-dehydrogenation.     

Effect of alpha-adrenergic stimulation on prostacyclin and renin release in the rat kidney

The relationship between renin secretion and PGI2 production, in response to intrarenal infusion of norepinephrine, was examined in the isolated perfused rat kidney. Infusion of norepinephrine in a dose which caused substantial vasoconstriction (100 ng/min), markedly increased urinary excretion of 6-keto PGF1 alpha, the stable derivative of PGI2, without significantly altering renin secretion measured in the effluent perfusate. No change in urinary 6-keto PGF1 alpha occurred when vasoconstriction was prevented by infusing the alpha-adrenoceptor blocking drug phenoxybenzamine (2 x 10(3) ng/min) alongside norepinephrine (100 ng/min). However, under these conditions there was marked stimulation of renin secretion which, as has been demonstrated previously, is mediated by a beta-adrenoceptor. There were significant increases in urine flow rates during both vasoconstrictor and non-vasoconstrictor infusions. These findings clearly indicate that in the rat kidney prostacyclin production and renin release in response to norepinephrine are dissociated.     

Sex differences in gallbladder prostaglandin synthesis mediated by acute inflammation

The influences of sex and acute inflammation on prostaglandin biosynthesis in rabbit gallbladder were examined by radiochromatography. Male rabbit gallbladder microsomes converted small amounts of labelled arachidonate to total prostaglandin synthesis with PGE2, 6-keto PGF1 alpha (stable metabolite of PGI2) and PGF2 alpha as the major products synthesized. Microsomes from the male rabbit gallbladder inflamed by bile duct ligation for 3 days increased total prostaglandin synthesis five-fold with 6-keto PGF1 alpha being the major prostaglandin produced. Female rabbit gallbladder microsomes converted three times more arachidonate to total prostaglandin synthesis than did microsomes from the male rabbit. Bile duct ligation did not alter total prostaglandin biosynthesis in the female rabbit gallbladder, but significantly decreased synthesis of PGE2, thromboxane B2 and PGF2 alpha and increased synthesis of 6-keto PGF1 alpha. These data suggest that although bile duct ligation had different effects on male and female gallbladder total prostaglandin synthesis, 6-keto PGF1 alpha is the major product induced by this stimulus for acute inflammation.     

Prostaglandin synthesis by the cochlea

The exogenous and endogenous syntheses of prostaglandins (PG's) by the cochlea of adult mongolian gerbils were studied in vitro. After incubation of the whole membraneous cochlea with [3H]-arachidonic acid (AA), syntheses of PGF2 alpha, 6-keto PGF1 alpha, PGE2, thromboxane (TX) P2 and PGD2 were evidenced in this order. The synthesis of radioactive PG's was almost completely inhibited by incubation with 10(-5) M indomethacin. No significant amounts of those PG's were detected by radioimmunoassay (RIA) in the cochlea obtained from animals killed by microwave irradiation at 5.0 kw for 0.8 sec. However, when the homogenate of the whole membraneous cochlea obtained from animals without microwave irradiation was incubated at 37 degrees C for 0-15 min, PGD2, PGE2, PGF2 alpha and 6-keto PGF1 alpha were found to be formed from endogenous AA in the cochlea by RIA. PG's were formed already at 0 time to considerable level (PGD2, PGF2 alpha and 6-keto PGF1 alpha, 90-120 pg/cochlea; PGE2, 370 pg/cochlea), reached to the maximum level (PGD2, PGF2 alpha and 6-keto PGF1 alpha, 170-200 pg/cochlea; PGE2, 500 pg/cochlea) at a 5-min incubation, and then gradually decreased. On the other hand, the amount of TXB2 was lower than the detection limit by RIA (less than 50 pg/cochlea) even after the incubation. The cochlea was dissected into three parts: organ of Corti + modiolus (OC + M), lateral wall (LW), and cochlear nerve (CN), and then PG's formed by these tissues were determined after a 5-min incubation of the homogenates. In the CN and OC + M, PGE2 was the major PG (100 and 160 pg/tissue, respectively), and the amounts of PGD2, PGF2 alpha and 6-keto PGF1 alpha were about 1/3 of those of PGE2. In the LW, the amounts of PGD2, PGE2, PGF2 alpha and 6-keto PGF1 alpha were about the same level (70-100 pg/LW).     

Specific binding of a stable prostacyclin analogue (Iloprost) to rat oxyntic mucosa

It has been reported that prostacyclin (PGI2) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI2 is inactivated under physiological conditions it has not been possible to demonstrate specific PGI2 binding to the rat stomach. Therefore a stable PGI2 analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE2 nor 6 keto PGF1 alpha displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 X 10(-11) M and 7.1 X 10(-8) M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI2 was less potent than unlabelled Iloprost in displacing 3H-Iloprost from its binding site. The addition of PGE2 to the incubation medium resulted in an increase in 3H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI2-like agents but not for either PGE2 or 6 keto PGF1 alpha.     

Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin

We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB2, 6-keto PGF1 alpha, 2,3-dinor-TXB2, 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TXB2 using acetylcholinesterase from Electrophorus Electricus coupled to TXB2, 6-keto PGF1 alpha and 11-dehydro-TXB2. Urinary PGI2 and TXA2 breakdown products and their metabolites were extracted from 3-40 ml of urine corresponding to 100 mumoles creatinine. Measurements were performed after Sep-Pak extraction and thin layer chromatography separation in a system that allows separation between dinor- and parent derivatives. Because of the relatively high cross reactivity (10-15%) of the anti-TXB2 serum with 2,3-dinor TXB2 and the anti-6-keto PGF1 alpha serum with 2,3-dinor-6-keto PGF1 alpha, measurements were done using 3 antisera (anti-TXB2 and anti-6-keto PGF1 alpha diluted 1/50,000, anti 11-dehydro-TXB2 diluted 1/200,000). The reproducibility of the technique was assessed by measuring the same urine stored frozen in aliquots together with each series of samples (Coefficient of variation 6-12% (n = 20), depending on the compound). In addition, the use of a different solvent system for the thin layer chromatography did not affect the results although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtained by other methodologies. In addition, comparisons made between EIA and gas chromatography/mass spectrometry analysis showed good correlation between the urinary metabolites as determined by each technique (r = 0.98).     

The effects of prostacyclin (PGI2) on tissues which detect prostaglandins (PG'S).

The effects of prostacyclin (PGI2) and its stable metabolite 6-oxo-PGF1alpha on various bioassay tissues are compared with those of PGE2 and PGF2alpha, using the cascade superfusion method. On vascular smooth muscle, PGI2 caused relaxation of all tissues tested except the rabbit aorta. PGE2 relaxed rabbit coeliac and mesenteric artery but contracted bovine coronary artery and had no effect on rabbit aorta. 6-oxo-PGF1alpha was ineffective at the concentrations tested. On gastro-intestinal smooth muscle, PGI2 contracted strips of rat and hamster stomach and the chick rectum. It was less potent than PGE2 or PGF2alpha. None of these substances contracted the cat terminal ileum. 6-oxo-PGF1alpha was inactive on these tissues at the doses tested. PGI2 was less active than PGE2 or PGF2alpha in contracting guinea-pig trachea and rat uterus; 6-oxo-PGF1alpha was active only on the rat uterus. Thus, PGI2 can be distinguished from the other stable prostaglandins using the cascade method of superfusion.     

Prostacyclin induces a surprising long-lasting motility in quiescent uterine strips (indomethacin-treated) isolated from ovariectomized rats

Dose-response curves for several prostaglandins (PGI2; PGD2; PGF2 and PGE2); BaCl2 or prostaglandin metabolites (15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha; 6-keto-PGF1 alpha and 6-keto PGE1 in quiescent (indomethacin-treated) uterine strips from ovariectomized rats, were constructed. All PGs tested as well as BaCl2, triggered at different concentrations, evident phasic contractions. Within the range of concentrations tested the portion of the curves for the metabolites of PGF2 alpha was shifted to the right of that for PGF2 alpha itself; the curve for 6-keto-PGF1 alpha was displaced to the right of the curve for PGI2 and that for 6-keto-PGE1 to the left. It was also demonstrated that the uterine motility elicited by 10(-5) M PGF2 alpha and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI2;6-keto-PGF1 alpha and BaCl2, but not the contractions following 6-keto-PGE1, which disappeared much earlier. The contractile tension after PGF2 alpha; 15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha and PGI2, increased as time progressed whilst that evoked by 6-keto-PGF1 alpha or BaCl2 fluctuated during the same period around more constant levels. The surprising sustained and gradually increasing contractile activity after a single dose of an unstable prostaglandin such as PGI2, on the isolated rat uterus rendered quiescent by indomethacin, is discussed in terms of an effect associated to its transformation into more stable metabolites (6-keto-PGF1 alpha, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.     

Altered levels of tissue and urinary prostacyclin in rats subjected to pancreas transplantation

Significant increases of TXB2 and PGE2 are reported to occur in pancreas transplantation. These increases are prevented with scavengers of oxygen-free radicals. In this communication, we report on changes of prostacyclin metabolites such as tissue 6-keto prostaglandin F1 alpha and urinary 2,3-dinor 6-keto prostaglandin F1 alpha in rats subjected to pancreas transplantation after different periods of organ cold preservation ischemia as well as the effect of superoxide dismutase (SOD) on these changes. For this purpose, male Lewis rats were classified as follows: Group I, Control; Group II, syngenic pancreas transplantation after 15 min of organ preservation in Collins solution at 4 degrees C; Group III, same as II but with 12 hours of organ preservation; Group IV, same as III, but with SOD pretreatment. Results have shown significant posttransplantation increases of both tissue 6-keto PGF1 alpha and urinary 2, 3 dinor 6-keto PGF1 alpha, the latter being a useful marker to evaluate systemic prostacyclin (PGI2) production by rat pancreas. This effect was prevented when the organ had been exposed to SOD during the period of cold preservation ischemia. These results confirm the implication of oxygen-free radicals (OFR) in the ischemia-reperfusion process associated to rat pancreas transplantation leading to enhanced arachidonic acid metabolism.     

Longitudinal and cross-sectional studies of the relationship between 6-keto PGF1 alpha and high density lipoproteins

Two studies were performed to examine the relationship between Prostacyclin (PGI2) and high density lipoprotein cholesterol (HDL-C). A longitudinal study examined the stable metabolite of PGI2, 6-keto PGF1 alpha, along with HDL-C, and total cholesterol (TC) before (Week 0), during (Week 4), and after (Week 9) an eight week aerobic conditioning program. 6-keto PGF1 alpha was measured by radioimmunoassay using 125I, and HDL-C and TC were spectrophotometrically analysed. Maximal oxygen uptake and resting heart rate data obtained at Week 0 confirmed that the training group (E) was not different from the sedentary group (C). Results obtained at Week 9 indicated that maximal oxygen uptake was higher (p less than 0.05) and resting heart rate lower (p less than 0.05) in the E group, while these variables remained unchanged in group C. Levels of 6-keto PGF1 alpha, HDL-C, and TC did not differ between groups E and C when Week 0 was compared with Week 9. However, within the E group, significantly lower concentrations of 6-keto PGF1 alpha (p less than 0.05) and HDL-C (p less than 0.05) were found at Week 4 compared with Week 0. A cross-sectional study involving 52 subjects examined the relationship between 6-keto PGF1 alpha and several cardiovascular disease risk factors which included HDL-C. The only significant correlation (r = 0.50 p less than 0.05) observed was between 6-keto PGF1 alpha and HDL-C.     

Effect of prostaglandins against alloxan-induced diabetes mellitus

Previously, we observed that alloxan-induced in vitro cytotoxicity and apoptosis in an insulin secreting rat insulinoma, RIN, cells was prevented by prior exposure to prostaglandin (PG) E(1), PGE(2), PGI(2), PGF(1)(alpha), and PGF(3)(alpha) (P<0.05 compared to alloxan), whereas thromboxane B(2) (TXB(2)) and 6-keto-PGF(1)(alpha) were ineffective. In an extension of these studies, we now report that prior intraperitoneal administration of PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) prevented alloxan-induced diabetes mellitus in male Wistar rats, whereas PGI(2), TXB(2), and 6-keto PGF(1)(alpha) were not that effective. PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) not only attenuated chemical-induced diabetes mellitus but also restored the antioxidant status to normal range in red blood cells and pancreas. These results suggest that PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) can abrogate chemically induced diabetes mellitus in experimental animals and attenuate the oxidant stress that occurs in diabetes mellitus.     

Appearance of prostaglandins, thromboxane B2, and hepoxilin A3 in the circulation of the normal and diabetic (BB) rat after arachidonic acid administration--correlation with plasma insulin

The temporal in vivo expression of the eicosanoids (products of the cyclooxygenase pathway and one product of the 12-lipoxygenase pathway, hepoxilin A3) was investigated after bolus intravenous injection of arachidonic acid in the normal rat and in the genetic rat model of type I insulin-dependent diabetes, the diabetic BB rat. The temporal relationship between the expression of these products and plasma insulin concentrations was also investigated to determine whether any correlation existed between the rise in plasma insulin levels and any of the newly formed eicosanoids. Measurements of the eicosanoids present in whole blood were carried out using the deuterium isotope dilution technique involving separation of pentafluorobenzyl esters, O-methyl oximes, and trimethylsilyl ether derivatives by high-resolution gas chromatography and specific detection by negative ion chemical ionisation mass spectrometry in the selected ion mode. Injection of arachidonic acid resulted in large and statistically significant increases in the blood concentrations of all products within 1 min, with thromboxane B2 (the stable product of thromboxane A2) and trioxilin A3 (the stable product of hepoxilin A3) being the highest (4.5-12 ng/mL). The mean concentrations of thromboxane B2 and trioxilin A3 in blood appeared greater in the diabetic BB rat than in the normal rat, while the opposite was found for 6-keto PGF1 alpha (the stable product of prostacyclin). The apparent greater ratio of thromboxane B2 to 6-keto PGF1 alpha in the diabetic BB rat than in the normal rat supports a prothrombotic nature of platelets associated with diabetes.     

Prostacyclin-independence in beta-adrenoceptor mediated renin release from dog renal cortical slices

There is some controversy regarding whether stimulation of renin release by the beta-adrenergic system is dependent on prostaglandin (PG) production. We have examined this problem in renal cortical slices of the dog and have obtained the following results: (1) Isoproterenol (4 X 10(-6) M) stimulated renin release, but had no effect on the formation of 6-keto PGF1 alpha, a stable metabolite of PGI2; (2) Indomethacin (2 X 10(-5) M) had no effect on isoproterenol stimulated renin release, but inhibited 6-keto PGF1 alpha formation; (3) Dibutyryl cyclic AMP (10(-3) M) stimulated both renin release, and 6-keto PGF1 alpha release. Indomethacin (2 X 10(-5) M) did not inhibit dibutyryl cyclic AMP-stimulated renin release, but did inhibit the production of 6 keto PGF1 alpha. These results indicate that the beta-adrenoceptor mediated renin release does not depend on the formation of PGI2, but renin release is dependent on cyclic AMP formation.     

High glucose levels modulate eicosanoid production in uterine and placental tissue from non-insulin-dependent diabetic rats during late pregnancy

Severe uterine and placental disturbances have been described in diabetes pathology. The relative severity of these changes appears to correlate with high glucose levels in the plasma and incubating environment. In order to characterize changes in eicosanoid production we compared uterine and placental arachidonic acid conversion from control and non-insulin-dependent diabetes mellitus (NIDDM) rats on day 21 of pregnancy, into different prostanoids, namely PGE2, PGF22alpha, TXB2 (indicating the production of TXA2) and 6-keto-PGF1 (indicating the generation of PGI2). PGE2, PGF2alpha and TXB2 production was higher and 6-keto-PGF1alpha was similar in diabetic compared to control uteri. PLA2 activity was found diminished in the NIDDM uteri in comparison to control. A role for PLA2 diminution as a protective mechanism to avoid prostaglandin overproduction in uterine tissue from NIDDM rats is discussed. Placental tissues showed an increment in TXB2 generation and a decrease in 6-keto PGF1alpha level in diabetic rats when compared to control animals. Moreover, when control uterine tissue was incubated in the presence of elevated glucose concentrations (22 mM), similar generation of 6-keto PGF1alpha and elevated production of PGE2, PGF2alpha and TXB2 were found when compared to those incubated with glucose 11 mM. Placental TXB2 production was higher and 6-keto PGF1alpha was lower when control tissues were incubated in the presence of high glucose concentrations. However, high glucose was unable to modify uterine or placental prostanoid production in diabetic rats. We conclude that elevated glucose levels induced an abnormal prostanoid profile in control uteri and placenta, similar to those observed in non-insulin-dependent diabetic tissues.     

Epidermal growth factor (EGF) and tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate PG synthesis and thymidine incorporation in cultured porcine thyroid cells

This is the first report to show that epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate the production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in the thyroid gland. In cultured porcine thyroid cells, EGF and TPA stimulate PGE2 and 6-keto PGF1 alpha production; the maximum PG levels were obtained after 3-4 h incubation with EGF or TPA; the addition of as little as 10(-11) M EGF or 5 X 10(-11) M TPA resulted in increases in PGE2 and 6-keto PGF1 alpha, and the maximum levels were obtained with 10(-8)-10(-7) M EGF or TPA. This report also shows that EGF and TPA stimulate [3H] thymidine incorporation.     

Arachidonic acid metabolism in isolated rat aorta. Dependence of prostacyclin biosynthesis on extracellular potassium concentration

Slices of rat aorta were incubated in Krebs-Ringer bicarbonate buffer for measurements of immunoreactive 6-ketoprostaglandin F1 alpha, thromboxane (TX) B2, prostaglandin (PG)E2, and PGF2 alpha, and in Tris buffer (pH 9.3) for determination of prostacyclin (PGI2)-like activity. No significant generation of TXB2, PGE2, or PGF2 alpha by rat aortic tissue could be detected. The time-dependent release of 6-keto-PGF1 alpha Krebs-Ringer bicarbonate buffer closely correlated with PGI2 generation in alkaline Tris buffer. During a 30-min incubation period, 6-keto-PGF1 alpha, release was 79.8 +/- 3.3 pmol/mg at a buffer potassium concentration of 3.9 mmol/liter and significantly increased by 23% to 98.3 +/- 8.5 pmol/mg (P less than 0.025) in the absence of potassium in the incubation medium. A smaller decrease in buffer potassium concentration to 2.1 mmol/liter and an increase to 8.8 mmol/liter did not significantly alter aortic 6-keto-PGF1 alpha release. Changes in the incubation buffer sodium concentration from 144 mmol/liter to either 138 or 150 mmol/liter at a constant potassium concentration of 3.9 mmol/liter did not alter the recovery of 6-keto-PGF1 alpha. Our results support the concept that PGI2 is the predominant product of arachidonic acid metabolism in rat aorta. They further show that PGI2 can be recovered quantitatively as 6-keto-PGF1 alpha under the present in vitro conditions. In addition, this in vitro study points to the potassium ion as a modulator of vascular PGI2 synthesis with a stimulation at low potassium concentrations.     

Stimulation of ovine myometrial activity by 6-keto prostaglandin E1

6-keto prostaglandin E1 (6KE) is a metabolite of PGI2, which we have shown previously inhibits spontaneous myometrial activity. In the present study we examined the effects of 6KE on uterine electrical and mechanical activity in non-pregnant ovariectomized sheep. 6KE stimulated uterine activity in a dose-dependent fashion. The effect was enhanced by pre-treatment with estradiol (E2). It was not influenced by pre-treatment with meclofenamic acid and was not associated with significant changes in the concentrations of 13,14 dihydro 15-keto PGF2 alpha in vena cava plasma. After E2 treatment, 6KE had 0.2-0.3 of the stimulatory activity of PGF2 alpha. In the absence of E2, the uterine response to both 6KE and PGF2 alpha was decreased. In animals in which spontaneous myometrial activity was inhibited by PGI2, the uterus remained responsive to 6KE. We conclude that in the ovariectomized non-pregnant sheep 6KE stimulates uterine activity, and that the effect is independent of endogenous PG production.     

Thrombin-induced prostacyclin biosynthesis in human endothelium: role of guanine nucleotide regulatory proteins in stimulus/coupling responses

The regulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelium (HUVEC) was investigated. HUVEC monolayer generation of PGI2 was monitored by RIA of 6-keto PGF1 alpha and dose-dependent increases observed with human alpha- and gamma-thrombins, histamine, or arachidonate. Alpha thrombin (10 nM) produced levels of 6-keto PGF1 alpha approximating responses with 1 microM gamma-thrombin, 5 microM arachidonate, or 10 microM histamine. Diisopropyl phosphorofluoridate-inactivated alpha-thrombin did not stimulate PGI2 release, demonstrating that catalytic activity was required for thrombin-stimulated PGI2 release. Sodium fluoride (NaF), at concentrations known to activate guanine nucleotide regulatory proteins (G proteins), directly stimulated HUVEC PGI2 synthesis in a dose-dependent and time-dependent manner (20 mM NaF, 4.4 +/- 0.5-fold increase at 10 min, 11.9 +/- 1.5-fold increase at 30 min). Neither alpha-thrombin nor NaF-stimulated PGI2 release was dependent upon the availability of extracellular Ca++). The hypothesis that G proteins are involved in agonist-stimulated PGI2 synthesis was further supported by studies using digitonin-permeabilized HUVEC monolayers challenged with another G protein activator, guanosine 5'-0-3-thiotrisphosphate (GTP gamma S), which effected significant dose-dependent increases in PGI2 synthesis compared with control levels of 6-keto PGF1 alpha. In contrast, the G-protein inhibitor GDP beta S, (guanosine 5'-0-2-thiodiphosphate), attenuated alpha-thrombin-mediated prostaglandin generation. Treatment of HUVEC monolayers with pertussis toxin (1 microgram/ml) did not inhibit the PGI2 synthesis stimulated by either alpha-thrombin, NaF, or histamine but catalyzed the ADP ribosylation of a 40 kDa membrane protein which cross-reacted with antisera against a synthetic peptide corresponding to an amino acid sequence common to the alpha-subunit of other G-proteins. Preincubation of HUVEC microsomal membranes with alpha-thrombin diminished pertussis toxin-catalyzed ADP ribosylation in a time-dependent manner. These data suggest that thrombin stimulation of PGI2 synthesis by HUVEC monolayers requires the catalytically functional enzyme and further suggests that the thrombin-occupied receptor is coupled to phospholipase activities by a pertussis toxin-insensitive guanine nucleotide regulatory protein in human endothelial cell membranes.