Further studies on the mechanism of action of human plasma in stimulating prostacyclin production by rat smooth muscle cells.

Human normal plasma stimulates prostacyclin (PGI2) production by vascular cells. This plasma activity may be greatly modified in different pathological conditions. The purpose of this study was to characterize some aspects of the mechanism of action of plasma in modulating PGI2 release. Cultured rat aortic smooth muscle cells were used. Citrated plasma from healthy donors stimulated PGI2 production in a concentration-dependent way. Plasma-derived serum containing increasing concentrations of platelets had the same PGI2 stimulating activity as citrated plasma. Plasma stimulation of PGI2 production was accompanied by release of endogenously incorporated arachidonic acid (AA) from the cell membrane. Similarly to AA, plasma induced PGI2 synthesis only once, a second or third challenge producing a reduced response from the cells. Cells stimulated twice with plasma responded to AA like unstimulated cells while cells stimulated twice with AA were poorly responsive to subsequent stimulation with plasma. When the cells were repeatedly stimulated with AA in the presence of plasma no refractoriness was apparent. This study suggests that plasma increases PGI2 synthesis by the release of endogenous substrate from the cell membrane and by protecting the cells from self-inactivation during AA conversion to prostaglandins.

Maternal smoking and prostacyclin production by cultured endothelial cells from umbilical arteries.

A study was made of the production of prostacyclin (PGI2) by cultured endothelial cells from the umbilical veins of mothers who smoked and of matched mothers who did not smoke. Cells were obtained from 58 umbilical cords from 22 apparently healthy women, 11 mild smokers (less than 15 cigarettes per day), and 25 heavy smokers (more than 15 cigarettes per day). Production of PGI2 by the cells was calculated by measuring the amount of 6-keto-prostaglandin F1 alpha released in the culture medium after the addition of arachidonic acid, by means of a specific radioimmunoassay. Endotheliai cells from mild and heavy smokers were significantly less able to grow and to reach confluency than cells from nonsmokers. Smoking also resulted in a marked reduction in the capacity of cultured cells to produce PGI2. This was particularly apparent for the cells from heavy smokers. Smoking during pregnancy appears to induce some modifications in the enzymes of the prostacyclin pathway which persist in endothelial cells undergoing replication in primary culture even in the absence of the pathogenic factor.

PIP: A study was made of the production of prostacyclin (PGI2) by cultured endothelial cells from the umbilical veins of mothers who smoked and of matched mothers who did not. Cells were obtained from 58 umbilical cords from 22 apparently healthy women, 11 mild smokers (15 cigarettes/day), and 25 heavy smokers (15 cigarettes/day). Production of PGI2 by the cells was calculated by measuring the amount of 6-keto-prostaglandin F1alpha released in the culture medium after the addition of arachidonic acid, by means of a specific radioimmunoassay. Endothelial cells from mild and heavy smokers were significantly less able to grow and reach confluency than cells from nonsmokers. Smoking also resulted in a marked reduction in the capacity of cultured cells to produce PFI2. This was particularly apparent for the cells from heavy smokers. Smoking during pregnancy appears to induce some modifications in the enzymes of the prostacyclin pathway which persist in endothelial cells undergoing replication in primary culture even in the absence of the pathogenic factor.

Specific binding of human fibrinogen to cultured human fibroblasts. Evidence for the involvement of the E domain.

Highly purified human fibrinogen was labelled with radioactive iodine and its interaction with cultured human embryo lung fibroblasts (MRC5) was examined. The cell monolayer was incubated at 37 degrees C with 125I-fibrinogen in phosphate/saline buffer containing 1 mM Ca2+ and 1 mM Mg2+. A direct interaction between 125I-fibrinogen and MRC5 was observed. The binding was time-dependent, reached saturation at 10 min and was regulated by the density of the cell monolayer. Non-labelled fibrinogen inhibited the interaction but unrelated proteins, including fibronectin, ovalbumin or myoglobin, did not. Monospecific Fab fragments, directed to fibrinogen, inhibited binding by 55.3% at a 50/1 molar ratio while non-immune Fab produced a 1.5% inhibition at similar concentration. Autoradiography of the display of fibroblast-bound 125I on a 7.5% polyacrylamide gel showed that the extract exhibited electrophoretic bands characteristic of the constitutive B beta and gamma chains of the fibrinogen molecule. An apparent affinity constant, Ka = 6.7 +/- 0.2 X 10(6) M-1, was estimated from binding isotherms. After a 30-min incubation time the interaction between 125I-fibrinogen and the cells was completely reversible and displaceable by a large molar excess of non-labelled fibrinogen. When compared to fibroblasts (MRC5 or W138), cultures of human embryo epithelial cells (EUE) failed to interact with 125I-fibrinogen, providing evidence for the specificity of binding for fibroblast monolayers. Plasmin-degradation fragments D and E, of 100000 and 50000 relative molecular mass respectively, were tested for their capacity to inhibit fibrinogen binding. At a 1/400 125I-fibrinogen/fragment molar ratio, fragment E inhibited binding by 30% while fragment D produced a 3% inhibition only. Altogether, the results demonstrate that human fibroblasts possess specific binding sites for fibrinogen, which exhibit the characteristics of a receptor system regulated by the culture state of the cells. In addition the structural features, which are necessary for the interaction of fibrinogen with the cells, are probably located in the E domain of the molecule.

Prostacyclin production by human endothelial and bovine smooth muscle cells in culture. Effect of repeated stimulation with arachidonic acid, thrombin and ionophore A23187.

Prostacyclin (prostaglandin I2) is the major product of arachidonic acid metabolism in vascular cells. Its physiological role may be linked to the ability of the cells to respond continuously with prostaglandin I2 production to a variety of stimuli. We report that human endothelial cells or bovine smooth muscle cells in culture respond with prostaglandin I2 synthesis to a first but not to a second stimulation with arachidonic acid. The development of this refractoriness was independent of the arachidonic acid concentration used (6.6-25 microM) and lasted for about 6 h. The same time was required for the cells to recover completely after inhibition of cyclooxygenase activity by aspirin. Neither cis-polyunsaturated fatty acids (linoleic or oleic acids) nor stearic acid (a long-chain saturated fatty acid) prevented the generation of prostaglandin I2 by arachidonic acid. Similarly to arachidonic acid, thrombin and ionophore A23187 could elicit vascular prostaglandin I2 synthesis only once. Pretreatment of the cells with arachidonic acid rendered the cells unresponsive to any other stimulus. These results indicate that the mechanism of the refractoriness induced by arachidonic acid was different from that induced by the other stimuli. It is proposed that vascular cells cannot be stimulated continuously to produce prostaglandin I2, but this process is regulated by different feedback mechanisms.