PGI2 Synthase Research Articles

Prostanoid synthesis by aortic rings in human blood: selective increase of prostacyclin mediated by a serum factor.

Synthesis of vascular epoprostenol (PGI2) and platelet thromboxane (TX) A2 is influenced by the coagulation cascade in incompletely understood ways. To elucidate this, prostanoids were determined in human blood anticoagulated by different drugs and incubated with and without rat aortic rings. Control incubations were performed in Hanks balanced salt solution. PGI2 and TXA2 synthesis were assessed by radioimmunoassay of their stable hydrolysis products 6-oxo-prostaglandin (PG) F1 alpha and TXB2. Fresh aortic rings incubated in Hanks solution with a thrombin inhibitor (TCK) synthesized similar quantities of 6-oxo-PGF1 alpha in the presence or absence of sodium citrate. In contrast, the intracellular calcium antagonist TMB-8 inhibited 6-oxo-PGF1 alpha synthesis. In contrast to the finding in Hanks solution, sodium citrate inhibited 6-oxo-PGF1 alpha synthesis by fresh aortic rings incubated in blood anticoagulated with TCK. However, TXB2 synthesis was not affected by citrate. Blood incubated alone at 37 degrees C in plain glass tubes generated a small amount of immunoreactive 6-oxo-PGF1 alpha. A thromboxane synthase inhibitor, OKY1581, increased immunoreactive 6-oxo-PGF1 alpha. However, blood anticoagulated with TCK and incubated similarly, generated no detectable 6-oxo-PGF1 alpha either in the presence or absence of OKY1581, showing that 6-oxo-PGF1 alpha synthesis in the previous experiments was dependent on the vascular rings. OKY1581 had little or no effect on 6-oxo-PGF1 alpha synthesis in incubations of fresh aortic rings with blood anticoagulated with TCK, despite inhibition of TXB2 synthesis. However, OKY1581 increased 6-oxo-PGF1 alpha synthesis by rings pretreated with acetylsalicylic acid (ASA) when incubated in blood, presumably by diversion of platelet endoperoxide to vascular PGI2 synthase. Sodium citrate did not influence the increase in 6-oxo-PGF1 alpha synthesis by ASA pretreated aortic rings caused by OKY1581 in whole blood. This implies that the PGI2 stimulating activity of whole blood in the absence of citrate exerts its effect proximal to PGI2 synthase. It is concluded that a low molecular weight serum factor formed during activation of the intrinsic coagulation pathway in blood, modulates PGI2/TXA2 balance by an action on vascular cyclo-oxygenase, possibly by an effect on intracellular calcium.

Increase in concentrations of prostaglandin endoperoxide synthase and prostacyclin synthase in human myometrium in late pregnancy

Concentrations of prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH synthase) and prostacyclin synthase (PGI synthase) were quantified with specific radioimmunometric assays inhuman myometrium during the last trimester of pregnancy (n=23) and in non-pregnant controls (n=8). Pregnant myometrium contained 3 times more PGH synthase per mg microsomal protein than non-pregnant myometrium (p < 0.01) but there was no increase with increasing gestational age in the third trimester nor with the onset of labor. In pregnancy, as compared to the non-pregnant state, there was no significant change in the PGI synthase content of myometrial microsomes, but significantly more PGI synthase was recovered in other subcellular fractions (p < 0.01). This suggests that pregnancy affects preferential changes in the subcellular distribution of PGI synthase in myometrial cells. Relative to its PGI synthase content pregnant myometrium contained twice as much PGH synthase as non-pregnant myometrium (p < 0.01). This may offer further evidence that PGH synthase rather than PGI synthase itself is the rate limiting factor in myometrial PGI 2 production. On the other hand, the much larger increase in PGH synthase than in PGI synthase in pregnant as compared to non-pregnant myometrium, may serve to promote preferential synthesis of prostaglandins that are potent myometrial stimulants and of critical importance in human parturition.

Concentrations of prostaglandin endoperoxide synthase and prostaglandin I2 synthase in the endothelium and smooth muscle of bovine aorta.

Platelets adhere to the subendothelial layer of newly deendothelialized arteries. Attachment can be reduced with exogenous prostacyclin (PGI2). Thus, the subendothelium may be unable to produce sufficient PGI2 to prevent platelet adherence and subsequent platelet-platelet interaction. Consistent with this explanation are data from an earlier report (1977. Moncada S., A. G. Herman, E. A. Higgs, and J. R. Vane. Thromb. Res. 11:323-344) indicating that the smooth muscle layer of aorta has only 10-15% of the capacity of endothelial cells to synthesize PGI2. We have measured the concentrations of PGI2 synthase and prostaglandin endoperoxide (PGH) synthase in bovine aorta and obtained results quite different from those described in this earlier report. Tandem immunoradiometric assays for PGI2 synthase and PGH synthase antigens were used to quantitate these proteins in detergent-solubilized homogenates of endothelial cells and smooth muscle tissue prepared from 10 different bovine aorta. The concentrations of PGI2 synthase in endothelial cells and smooth muscle were found to be the same. However, the concentration of PGH synthase in endothelial cells averaged greater than 20 times that of smooth muscle. Results similar to those determined by immunoradiometric assay were also obtained when PGH synthase and PGI2 synthase catalytic activities were measured in preparations of endothelial and smooth muscle cells. Furthermore, when bovine aorta and renal arteries were subjected to immunocytofluorescence staining using monoclonal antibodies to PGI2 synthase, fluorescence staining of equivalent intensity was detected in both the endothelial cells and the smooth muscle. Moreover, the intensity of fluorescence was similar throughout cross-sections of vascular smooth muscle, indicating that there is no gradient in PGI2 synthase concentrations between the endothelium and adventitia. Our results indicate that the propensity of platelets to adhere to the subendothelium of deendothelialized arteries and form aggregates cannot be attributed simply to an inability of the denuded vasculature to produce PGI2 from PGH2, but may be a consequence of the low PGH synthase activity of smooth muscle. Consistent with this concept are the results of Eldor et al. (1981. J. Clin. Invest. 67:735-741) who reported that increases in PGH synthase activity are associated with formation of a nonthrombogenic neointima.

Bimodal distribution of the prostaglandin I2 synthase antigen in smooth muscle cells.

Vascular and nonvascular smooth muscles in 12 different organs from the gastrointestinal, respiratory, and urogenital tracts of the rabbit, cow, dog, sheep, pig, and rat were examined for prostaglandin I2 (PGI2) synthase immunoreactivity by indirect immunocytofluorescence using monoclonal antibodies against the enzyme. Each of 35 different smooth muscle layers tested stained for the PGI2 synthase antigen except the circular smooth muscle of the rabbit large intestine. Interestingly, PGI2 synthase-positive fluorescent staining of smooth muscle was always observed to be associated with both the nuclear and plasma membranes. Our results indicate that most smooth muscle, both vascular and nonvascular, has the capacity to synthesize PGI2. Moreover, the fact that the PGI2 synthase antigen is associated with at least two different organelles in each cell suggests that there are two independent PGI2-synthesizing systems in smooth muscle; one on the nuclear membrane, and one on the plasma membrane. PGI2 formed at these different sites may subserve different functions within the same cell.

35] Monoclonal antibodies against PGI2 synthase: An immunoradiometric assay for quantitating the enzyme

This chapter describes the preparation of two hybridoma lines producing monoclonal antibodies against two different antigenic sites on the PGI 2 synthase enzyme molecule. The hybridoma lines are derived from mice immunized with a partially purified preparation of PGI 2 synthase from bovine aorta. Using these antibodies an immunoradiometric assay is developed with which to quantitate PGI 2 synthase protein concentrations. For assay a complex of IgG l bound to S. aureus is prepared by adding 100μg of purified IgG 1 in 0.1 M sodium citrate, pH 7.0, to 1 ml of a 10% suspension of rabbit anti-mouse IgG-S. aureus cell complex. After 5 min the S. aureus cells are pelleted by centrifugation and resuspended in 1 ml of 0.1 M Tris-chloride, pH 8.0, containing 0.5% Triton X-100. This IgG1 S. aureus complex is stable for at least 2 weeks when stored at 4 °. Further, a standard curve for quantitating PGI2 synthase protein is generated. The method should be useful for measuring changes in PGI 2 synthase protein concentrations in tissues during physiological stresses such as aging and the development of atherosclerosis. The antibodies should also be of value in the immunocytochemical localization of PGI 2 synthase at both the cellular and subcellular levels.