Dominique Demolle

Rapid phosphorylation of a 27 kDa protein induced by tumor necrosis factor

Tumor necrosis factor (TNF) has been shown to induce the phosphorylation of a 27 kDa protein in a time‐ and concentration‐dependent manner in HeLa D98/AH2, ME 180 and bovine aortic endothelial cells. This phosphorylation could be reproduced by the calcium ionophore, A23187. However, this phosphorylation was not observed in L929 cells, for which TNF is highly cytotoxic, suggesting that it might play a role in actions of TNF other than the induction of cell death.

Stimulation of prostacyclin release from aortic smooth muscle cells by purine and pyrimidine nucleotides.

ATP and ATP gamma S(10-100 microM) stimulated the release of prostacyclin (PGI2) from bovine aortic smooth muscle cells. This effect was reproduced by UTP, ITP and partially by GTP. ADP and ADP beta S, the P2X-selective agonist alpha, beta-methylene ATP (APCPP), AMP and adenosine were all inactive. This effect of ATP gamma S was not inhibited by Reactive Blue 2, an antagonist of P2Y receptors. The stimulation of PGI2 production in aortic smooth muscle cells by these nucleotides thus seems to involve receptors distinct from both P2X and P2Y subtypes, which are responsible for smooth muscle contraction and PGI2 release from endothelial cells, respectively.

Cholera and pertussis toxins amplify prostacyclin synthesis in aortic smooth muscle cells

Pretreatment of bovine aortic smooth muscle cells in culture with pertussis toxin (PT) or cholera toxin (CT) potentiated the synthesis of prostacyclin (PGI2) induced by 5‐hydroxytryptamine (5‐HT) and phorbol‐12‐myristate, 13‐acetate (PMA). The production of PGI2 by explants from the bovine aortic media was also synergistically stimulated by 5‐HT and CT, whereas PT was inactive. These data are consistent with the hypothesis that guanosine 5′‐triphosphate binding proteins are directly involved in the control of phospholipases which release free arachidonic acid for prostaglandin synthesis.

Dipyridamole and vascular prostacyclin production

The action of dipyridamole on the vascular production of prostacyclin (PGI2) has been investigated. Dipyridamole (1-100 microM) did not induce a significant stimulation of PGI2 release in any of the following experimental models: rings of rabbit aorta, cultured endothelial cells from bovine aorta or human umbilical vein, cultured explants of bovine aortic smooth muscle. The activity of known stimuli of PGI2 release (ADP, suloctidil, serotonin) and the capacity of dipyridamole to inhibit adenosine uptake into endothelial cells were carefully checked. Pretreatment of the rabbit aorta with dipyridamole (10-100 microM) prolonged the transient stimulation of PGI2 release induced by mechanical deendothelialization: this effect was probably due to a partial protection of the cyclooxygenase against oxidative self-inactivation. Our largely negative results are consistent with the current theory that the antiplatelet action of dipyridamole is mediated by adenosine and not by PGI2.

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.

P2-purinergic receptors in vascular endothelial cells: from concept to reality.

ATP exerts at least 2 actions on arterial endothelial cells: it stimulates the release of endothelium-derived relaxing factor, a still unidentified vasodilator, and of prostacyclin, a potent inhibitor of platelet aggregation. A study of agonist specificity indicates that these responses are mediated by P2-purinergic receptors. We have now demonstrated that in these cells, the P2-receptors are coupled to a phospholipase C hydrolysing phosphatidylinositol-bisphosphate and that this coupling involves a pertussis toxin-sensitive GTP-binding regulatory protein.

Adrenergic stimulation of vascular prostacyclin: role of α1-receptors in smooth muscle cells

Epinephrine and norepinephrine (1-10 microM) stimulated the release of prostacyclin (PGI2) from the rabbit aorta in vitro. The stimulation was maintained for at least 2 h in the continuous presence of epinephrine. Phenylephrine mimicked this effect, whereas the selective alpha 2-agonist UK-14,304 was completely ineffective. The action of epinephrine was abolished by prazosin (1 microM) and was maintained in the presence of yohimbine. Epinephrine or phenylephrine neither increased the basal release of PGI2 from bovine aortic endothelial cells nor potentiated the stimulatory action of adenine nucleotides, which is mediated by P2-purine receptors. The response to epinephrine was lost in freshly deendothelialized strips of rabbit aorta, possibly because of cyclooxygenase self-inactivation. The response recovered however following overnight incubation of these strips in a cell culture medium. The response to epinephrine was mimicked by neither phorbol 12-myristate,13-acetate nor ionophore A23187. It was not inhibited by pretreatment with pertussis toxin. It is concluded that adrenergic agents stimulate the vascular production of PGI2, by activating alpha 1-receptors located on smooth muscle cells.

Stimulation of aortic smooth muscle prostacyclin by serotonin: role of distinct receptors in contractile and synthetic states.

It has been shown previously that serotonin stimulates the production of prostacyclin by bovine aortic smooth muscle cells in culture, via 5-HT2 receptors (Coughlin SR, Moskowitz MA, Antoniades HN, Levine L. Proc Natl Acad Sci USA 1981;78:7134-7138). These cells express a synthetic phenotype, whereas the majority of the smooth muscle cells in the media from adult arteries are in a contractile state. We have now compared 5-HT stimulated prostacyclin production in bovine aortic media explants, a preparation of contractile smooth muscle, with cultured smooth muscle cells derived from the explants. In the 1-10 microM range, serotonin stimulates the release of prostacyclin from the explants of bovine aortic media, cultured for a short period. In the presence of cocaine (30 microM), 1 microM was sufficient to produce a maximal effect. The stimulatory action of serotonin was sustained with time and did not induce a lasting desensitization. The effect of serotonin on the explants was inhibited only partially (+/- 30%) by ketanserin, a selective and potent 5-HT2 antagonist. It was mimicked by 5-carboxamido-tryptamine, a 5-HT1 agonist, but was only weakly inhibited by methiothepin, a 5-HT1 antagonist. As expected, in cultured smooth muscle cells, 5-carboxamido-tryptamine was only a weak agonist in stimulating prostacyclin production. In conclusion, it appears that the serotonin effect on prostacyclin production is mediated by different receptors in media explants from bovine aortic media and cultured cells obtained by outgrowth from these explants: a 5-HT2 receptor in the smooth muscle cells in culture and a receptor presenting some similarities with 5-HT1 receptors in the explants.

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.

P2‐Purinergic Receptors on Vascular Endothelial Cells

ATP and ADP, via P,, receptors, stimulate the release of nitric oxide’ and of prostacyclin *.’ from aortic endothelial cells and increase their rate of proliferation? These actions might have a physiological importance in the interaction between platelets and the vascular endothelium. The release of prostacyclin will limit the extent of platelet aggregation following a lesion of the endothelium, while the mitogenic effect will accelerate the repair of that lesion. Bovine aortic endothelial cells (BAECs) provided a useful model to elucidate the transduction mechanisms associated with PZy receptors. The occupancy of these receptors induces the hydrolysis of several phospholipids by various phospholipases, generating different second messengers. These responses have different time courses and are triggered by distinct mechanisms (FIG. 1). ATP and ADP induce a rapid and transient accumulation of inositol trisphosphate ( lP3), reflecting the hydrolysis of phosphatidylinositol bisphosphate (PIP,) by phospholipase C (FIG. lA).’ The rise of cytoplasmic Ca2+ that results6 is responsible for a burst of prostacyclin release, probably via the activation of a Ca2+-dependent phospholipase A,.’ The coupling of the P,, receptors and the phospholipase C seems to involve a GTP-binding protein (FIG. lC).* ATP and ADP also induce a sustained increase of the choline level inside endothelial cells (FIG. lB), an increase likely to result from the activation of phospholipase D, which hydrolyzes phosphatidylcholine into phosphatidic acid and choline? Phosphatidic acid might play a role in the mitogenic effect of ATP.” Depletion of protein kinase C by a prolonged exposure to phorbol 12-myristate 13-acetate abolished the ATP-stimulated release of choline metabolites (FIG. 1D). This indicates that the activation of protein kinase C by diacylglycerol released from PIP, plays a crucial role in the stimulation of phospholipase D.’