Release of endothelium‐derived relaxing factor from pig cultured aortic endothelial cells, as assessed by changes in endothelial cell cyclic GMP content, is inhibited by a phorbol ester

Abstract

Cultured aortic endothelial cells of the pig respond to the endothelium‐derived relaxing factor (EDRF) they release with an increase in cyclic GMP content. This response is inhibited by haemoglobin or by l‐NG‐monomethyl‐arginine (l‐NMMA), and has been used to investigate the effects of phorbol esters on EDRF release. Pretreatment with phorbol‐12,13‐dibutyrate (PDB) but not the inactive 4α‐phorbol‐12,13,‐didecanoate (PDD), inhibited increases in cyclic GMP induced by substance P (10−8 m) in a time and concentration‐dependent manner. PDB did not affect basal cyclic GMP levels. PDB (3 × 10−7 m), but not PDD (3 × 10−7 m), also inhibited ATP (10−5 m)‐induced increases in cyclic GMP, but did not affect those induced by bradykinin (10−7 m). Increases in cyclic GMP induced by low (10−7 m) but not high (10−6 m) concentrations of the calcium ionophore A23187 were inhibited by PDB (3 × 10−7 m). This inhibitory effect was due to enhanced destruction of EDRF by superoxide anions rather than inhibition of EDRF release, as the inhibition was abolished in the presence of superoxide dismutase (SOD, 30 u ml−1) and catalase (CAT, 100 u ml−1). SOD and CAT did not affect the inhibitory action of PDB on substance P or ATP‐induced increases in cyclic GMP. Increases in endothelial cell cyclic GMP content induced by sodium nitroprusside (10−5 m) were unaffected by PDB pretreatment. The inhibitory effects of PDB are probably a result of an action of protein kinase C on the steps between receptor occupation and phospholipase C activation.

Elevated D‐glucose induces insulin insensitivity in human umbilical endothelial cells isolated from gestational diabetic pregnancies

“…Diacylglycerol levels and PKC activity are increased in vascular cells exposed to prolonged hyperglycaemia (Inoguchi et al 1994; see also review by King, Kunisaki, Nishio, Inoguchi, Shiba & Xia, 1996), and activation of PKC appears to mediate cytokine-induced increases in ¬_arginine transport in umbilical vein endothelial cells (Pan et al 1995). Activation of PKC cannot fully explain our previous findings that stimulatory actions of insulin on ¬_arginine transport and NO synthesis were attenuated in non-diabetic endothelial cells adapted to elevated ª_glucose (Sobrevia et al 1996), since stimulation of PKC does not regulate basal NO release in porcine endothelial cells (Smith & Lang, 1990) and actually stimulates basal PGIµ synthesis in umbilical vein endothelial cells (Carter, Hallam & Pearson , 1989). Although it is difficult to explain the inhibitory action of insulin on ¬_arginine transport and NO synthesis in fetal endothelial cells isolated from gestational diabetic pregancies, insulin has been shown to downregulate diabetes-induced stimulation of amino acid transport in hepatocytes (Handlogten & Kilberg, 1984), isolated pancreas (Mann & Norman, 1984) and isolated gastric glands (Contreras et al 1997).…”

Modulation of agonist‐induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP

“…However, differential agonist sensitivity to the inhibitory actions of the phorbol esters on EDRF release appears to occur: for example, Smith & Lang (1990) have shown that bradykinin-induced production of EDRF by pig aortic endothelial cells is unaffected, whereas that induced by substance P or ATP is inhibited. This differential sensitivity may be explained by our finding that bradykinininduced calcium mobilisation in bovine aortic endothelial cells is less sensitive to the inhibitory actions of phorbol esters than that induced by other agonists.…”

“…The sustained plateau phase of the increase in [Ca2+]i induced by agonists results from calcium influx (Hallam et al, 1988) through receptor-but not voltage-operated channels (Hallam & Pearson, 1986;Colden-Stanfield et al, 1987;Johns et al, 1987 (Singer & Peach, 1982), the ability of phorbol esters to inhibit EDRF release (Weinheimer et al, 1986; Lewis & Henderson, 1987;Cherry & Gillis, 1988;de Nucci et al, 1988;Smith & Lang, 1990) is likely to result from their ability to inhibit endothelial calcium mobilisation. However, differential agonist sensitivity to the inhibitory actions of the phorbol esters on EDRF release appears to occur: for example, Smith & Lang (1990) have shown that bradykinin-induced production of EDRF by pig aortic endothelial cells is unaffected, whereas that induced by substance P or ATP is inhibited.…”

Fructose Induces the Inflammatory Molecule ICAM-1 in Endothelial Cells

“…Cells were also stimulated with ATP (1 M to 1 mM), which has been shown to increase endothelial cGMP level or other vasodilation factors that could be theoretically associated with NO production. [32][33][34] Intracellular ATP concentration was thought to be approximately 1.0 to 30 M. 35,36 ELISA for MCP-1…”