title: Endothelin-induced atrial natriuretic peptide release from cultured neonatal cardiac myocytes: the role of extracellular calcium and protein kinase-C.
abstract: Regulation of atrial natriuretic peptide (ANP) secretion from neonatal rat myocytes cultured on microcarriers was studied using endothelin-1 (ET-1) as a secretagogue. Myocytes were cultured for 3 days on microcarriers, packed in a chromatography column, and perifused with Krebs-Henseleit bicarbonate buffer. ANP secretion was measured by RIA, and the cytosolic free calcium concentration ([Ca2+]f) was measured continuously during secretion by the fluorescent calcium indicator fura-2. In perifused atrial and ventricular cells, basal values for [Ca2+]f were 146 and 167 nM, and immunoreactive ANP (IR-ANP) secretion rates were 61 and 65 pg/min.mg protein, respectively. ET-1 at concentrations of 1, 10, and 100 nM caused a concentration-dependent increases in [Ca2+]f and IR-ANP secretion in atrial myocytes. The maximal increases in [Ca2+]f and IR-ANP secretion were 30% and 100%, respectively. Diltiazem (1 microM), an inhibitor of voltage-sensitive Ca2+ channels, inhibited [Ca2+]f increments, but had no effect on ET-induced IR-ANP secretion. Staurosporine (10 nM), a protein kinase-C inhibitor, augmented [Ca2+]f changes, but inhibited the sustained phase of ET-induced IR-ANP secretion (P less than 0.05). Diltiazem abolished the stimulatory effect of staurosporine on [Ca2+]f and its inhibitory effect on IR-ANP secretion. ET-1 caused increases in [Ca2+]f and IR-ANP secretion in ventricular myocytes similar to those in atrial myocytes. Peptides corresponding in size to pro-ANP and ANP-(1-28) were detected in the original cell culture medium and perifusion effluent, and ET-1 did not change their concentration ratio in the eluate. Lactate dehydrogenase was not detected in the effluents before or during ET infusion, showing that the increase in IR-ANP secretion was not due to cell damage. This study shows that ET stimulates atrial and ventricular ANP secretion. The results also suggest that sustained ET-induced atrial ANP secretion is dependent on protein kinase-C, but does not require the influx of extracellular calcium.
authors.affiliation: Department of Medical Biochemistry, University of Oulu, Finland.
authors.forename: P A
authors.initials: PA
authors.lastname: Uusimaa
authors.forename: I E
authors.initials: IE
authors.lastname: Hassinen
authors.forename: O
authors.initials: O
authors.lastname: Vuolteenaho
authors.forename: H
authors.initials: H
authors.lastname: Ruskoaho
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D009320:Atrial Natriuretic Factor / Q000096:biosynthesis / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004110:Diltiazem / Q000494:pharmacology; D016232:Endothelins / Q000494:pharmacology*; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D007213:Indomethacin / Q000494:pharmacology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D010477:Perfusion; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D019311:Staurosporine
chemical_list: D000470:Alkaloids; D016232:Endothelins; D009320:Atrial Natriuretic Factor; D007770:L-Lactate Dehydrogenase; D011493:Protein Kinase C; D004110:Diltiazem; D019311:Staurosporine; D002118:Calcium; D007213:Indomethacin