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

Contribution à l'étude des actions de l'ATP et de la sérotonine sur les cellules vasculaires

Doctorat en sciences médicalesinfo:eu-repo/semantics/nonPublishe

Contribution à l'étude des actions de l'ATP et de la sérotonine sur les cellules vasculaires

Doctorat en sciences médicalesinfo:eu-repo/semantics/nonPublishe

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.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

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.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Metabolism of arachidonic acid in rabbit iris and retina.

Both the iris and the retina of the rabbit released prostaglandins (PG) E2, F2 alpha, 6-keto-F1 alpha and thromboxane (Tx) B2, when incubated in vitro. PGE2 was the major cyclooxygenase product formed by each tissue. The kinetics of PGE2 release by the iris and the retina were similar: high initial output followed by a decline to a steady-state value. The production of PGE2 was inhibited by indomethacin and stimulated by ionophore A23187. The iris and the retina converted exogenous arachidonic acid into 12- and 15-hydroxy-eicosatetraenoic acid (HETE): inhibition by eicosatetraynoic acid (ETYA) indicated the involvement of lipoxygenase enzymes. This lipoxygenase activity was important relatively to cyclooxygenase in the retina, but was only a minor pathway in the iris. Leukotriene (LT) B4 was released by the iris and the retina in amounts smaller than PGE2, but compatible with a biological activity: ionophore A23187 stimulated LTB4 production in both tissues. Our data support the hypothesis that PGE2 and LTB4 could play a role in the initiation of ocular inflammation.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Pattern of protein phosphorylation in aortic endothelial cells. Modulation by adenine nucleotides and bradykinin.

In bovine aortic endothelial cells, ATP (10-100 microM) and bradykinin (0.1-1.0 microM) enhanced the phosphorylation of two major protein substrates with apparent molecular masses of 95 and 28 kDa. The action of ATP involved P2y purinoceptors. The kinetics were distinct for the two phosphopeptides. The phosphorylation of the 95-kDa protein was rapid (within 30 s) but transient (maintained for only 2 min). This time course agrees with that observed for the increase of the cytosolic Ca2+ level induced by ATP in these cells. Ionophore A23187 (greater than or equal to 100 nM) induced this phosphorylation for a longer period (5-10 min), whereas phorbol 12-myristate 13-acetate (PMA) was completely inactive. The enhancement of the 28-kDa protein phosphorylation was detectable after a 5-min lag and was maintained for at least 20 min. PMA (50 nM) stimulated weakly the phosphorylation of the 28-kDa protein, whereas A23187 (100-300 nM) was even more effective than ATP and bradykinin. The 95-kDa phosphoprotein seems to be related to a 100-kDa substrate of calmodulin-dependent protein kinase III recently identified as elongation factor-2. The 28-kDa protein, which was resolved as three variants in bidimensional gel electrophoresis, appears very similar to a slightly heavier phosphoprotein from thrombin-stimulated human platelets. In addition, bidimensional electrophoresis allowed the detection of at least 10 substrates (from 18 to 46 kDa) whose phosphorylation was enhanced equally well by ATP, bradykinin, and A23187 and only partially by PMA. In conclusion, protein phosphorylation induced by ATP and bradykinin in aortic endothelial cells seems to be catalyzed mostly by Ca2+-dependent kinases, distinct from protein kinase C.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Prostacyclin-stimulating drugs: new prospects.

SKF 525-A (proadifen), a well-known inhibitor of drug metabolism and cytochrome P-450 activity, stimulated the release of prostacyclin (PGI2) from the rabbit aorta in vitro. The PGI2-stimulating activity of SKF 525-A was characterized by specific structural requirements: activity was abolished by the deletion of the terminal propyl chain and increased by its elongation into an isobutyl chain; chlorination of the phenyl rings increased the potency. SKF 525-A increased the production of PGI2 by cultured endothelial cells from bovine aorta and human umbilical vein, but had no effect on cultured smooth muscle from the bovine aortic media. In human platelets, SKF 525-A inhibited prostaglandin and thromboxane production induced by A23187, thrombin and ADP. Simultaneous stimulation of endothelial PGI2 and inhibition of platelet TxA2 represents an original pharmacological profile: SKF 525-A might thus constitute the prototype of a new class of antiplatelet drugs.Comparative StudyIn VitroJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

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.In VitroJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe