endothelin-1 and 1-oleoyl-2-acetylglycerol

In the present work we used patch pipette techniques to study the properties of a novel Ca(2+)-permeable cation channel activated by the potent coronary vasoconstrictor endothelin-1 (ET-1) in freshly dispersed rabbit coronary artery myocytes. With cell-attached recording bath application of 10 nm ET-1 evoked cation channel currents (I(cat)) with subconductance states of about 18, 34 and 51 and 68 pS, and a reversal potential of 0 mV. ET-1 evoked channel activity when extracellular Ca(2+) was the charge carrier, illustrating significant Ca(2+) permeability. ET-1-induced responses were inhibited by the ET(A) receptor antagonist BQ123 and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also stimulated I(cat), but the protein kinase C (PKC) inhibitor chelerythrine did not inhibit either the OAG- or ET-1-induced I(cat). Inositol 1,4,5-trisphosphate (IP(3)) did not activate I(cat), but greatly potentiated the response to OAG and this effect was blocked by heparin. Bath application of anti-TRPC3 and anti-TRPC7 antibodies to inside-out patches markedly inhibited ET-1-evoked I(cat), but antibodies to TRPC1, C4, C5 and C6 had no effect. Immunocytochemical studies demonstrated preferential TRPC7 expression in the plasmalemma, whereas TRPC3 was distributed throughout the myocyte, and moreover co-localization of TRPC3 and TRPC7 signals was observed at, or close to, the plasma membrane. Flufenamic acid, Gd(3+), La(3+) and extracellular Ca(2+) inhibited I(cat) with IC(50) values of 2.45 microm, 3.8 microm, 7.36 microm and 22 microm, respectively. These results suggest that in rabbit coronary artery myocytes ET-1 evokes a Ca(2+)-permeable non-selective cation channel with properties similar to TRPC3 and TRPC7, and indicates that these proteins may be important components of this conductance.

Topics: Animals; Calcium; Coronary Vessels; Diglycerides; Electric Conductivity; Endothelin-1; Inositol 1,4,5-Trisphosphate; Muscle Cells; Permeability; Rabbits; Signal Transduction; TRPC Cation Channels

Protein kinase C (PKC) has been implicated in the regulation of endothelin-1 production by bovine and porcine endothelial cells. The role of PKC in the synthesis and release of endothelin-1 has not been demonstrated previously in human arterial endothelial cells. The effects of activators and an inhibitor of PKC on endothelin-1 release from cultured human arterial endothelial cells have now been examined. Endothelial monolayers were incubated with the PKC activators phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl 2-acetyl glycerol (OAG) for 30 min to 8 h and the amount of endothelin-1 released into the medium was determined. PMA (0.1 micromol/L) and OAG (0.1 mmol/L) stimulated endothelin-1 release, with maximal increases of 85% and 90%, respectively, apparent at 30 min in aortic endothelial cells, and of 81% and 75%, respectively, apparent at 1 h in pulmonary artery endothelial cells. Endothelin-1 release had returned to control values by 4 h and was approximately one-third to one-half of control values after 8 h of continuous exposure of both cell types to PMA or OAG. Long-term exposure of both types of cells to the PKC inhibitor staurosporine (0.1 micromol/L) reduced both basal endothelin-1 release and the enhancement of release by PMA or OAG. The PKC agonists also stimulated endothelin-1 release by bovine aortic endothelial cells. The maximal effects of PMA and OAG in these cells were apparent at 4 h, at which time endothelin-1 release was increased by 154% and 125%, respectively; after 4 h, endothelin-1 release had decreased, but was still 132% and 120%, respectively, of the control value at 8 h. The PKC activators markedly stimulated prostaglandin I2 (PGI2) release by human pulmonary artery endothelial cells. The enhancement of PGI2 release continued for up to 8 h, at which time 109-fold and threefold increases in PGI2 release were apparent with PMA and OAG, respectively. Therefore, activation of PKC results in a rapid increase in endothelin-1 release by human arterial endothelial cells. The subsequent decrease in endothelin-1 release in the presence of PMA or OAG is presumably attributable to activator-induced downregulation of PKC, which was apparent earlier in human than in nonhuman endothelial cells. The downregulation of endothelin-1 release also occurred much earlier than that of PGI2 release in human pulmonary artery endothelial cells.

Topics: Animals; Aorta; Cattle; Cells, Cultured; Diglycerides; Endothelin-1; Endothelium, Vascular; Enzyme Activation; Epoprostenol; Humans; Protein Kinase C; Pulmonary Artery; Tetradecanoylphorbol Acetate