MECHANICAL AND BIOCHEMICAL RESPONSES TO ENDOTHELIN-1 AND ENDOTHELIN-3IN BOVINE BRONCHIAL SMOOTH-MUSCLE

1 In this study, mechanical responses to endothelin-1 and endothelin-3 were examined in bovine bronchial smooth muscle. In addition, the inv olvement of phosphatidylinositol 4,5-bisphosphate hydrolysis (PIP2) in the responses to these peptides was assessed by measurement of inosit ol (1,4,5) trisphosphate (I(1,4,5)P3) production using a specific mass assay. 2 ET-1 evoked contractions of bovine bronchi which were concen tration-dependent and initiated at between 10(-9) M and 10(-8) M. ET-1 -evoked responses were unaffected by slight elevation of tone with pot assium chloride (3 x 10(-2) M), methacholine (10(-6) M) or U46619 (10( -7) M). 3 Contractions to ET-1 were not altered by pre-incubation with atropine (10(-5) M), indomethacin (10(-5) M), nifedipine (10(-5) M), phosphoramidon (3.67 x 10(-5) M) or by removal of the epithelium. 4 ET -3 evoked small contractions which were not concentration-dependent. I n the presence of phosphoramidon (3.67 x 10(-5) M) however, concentrat ion-dependent contractions were obtained to ET-3 which were unaffected by atropine (10(-5) M) or by removal of the epithelium, but were sign ificantly attenuated by indomethacin (10(-5) M). Nifedipine (10(-5) M) virtually abolished this response. 5 Both ET-1 and ET-3 (in the prese nce of phosphoramidon)-evoked contractions were significantly enhanced by the presence of the phorbol ester phorbol 12,13-dibutyrate (10(-8) M). Neither ET-1-, nor ET-3-mediated responses were antagonized by th e protein kinase C (PKC) inhibitor, Ro 31-8220 (3 x 10(-9)-3 x 10(-8) M). 6 ET-1 (3 x 10(-7) M) evoked a biphasic rise in levels of I(1,4,5) P3 which was unaltered by preincubation with atropine, whilst ET-3 (10 (-10)-3 x 10(-7) M) failed to alter levels of I(1,4,5)P3 at any time p oint examined, even in the presence of phosphoramidon (3.67 x 10-5 M). 7 These results suggest that, in bovine bronchial smooth muscle, ET-1 does not evoke contraction via cyclo-oxygenase metabolites, does not evoke release of the neurotransmitter substance acetylcholine, or requ ire calcium influx via dihydropyridine-sensitive channels. ET-1 evokes I(1,4,5)P3 production, but stimulation of protein kinase C may not be critical for the associated contraction. In contrast, ET-3-evoked con tractions are partly mediated by cyclo-oxygenase metabolites. ET-3 doe s not stimulate PIP2 hydrolysis, nor activate PKC, but may, either dir ectly or as a requirement of intermediates released in response to ET- 3, rely upon extracellular calcium.