CHARACTERISTICS OF ENDOTHELIN RECEPTORS AND INTRACELLULAR SIGNALING IN PORCINE INNER MEDULLARY COLLECTING DUCT CELLS

In porcine kidneys we investigated the characteristics of endothelin ( ET) receptors that are present in papillary tissue but not in glomerul i. Therefore, porcine inner medullary collecting duct (IMCD) cells wer e separated by Percoll density gradient centrifugation after enzymatic and hypotonic treatment of minced papillary tissue. Studies were perf ormed in fresh cell suspensions and in cells in primary culture. Chang es in cytosolic free Ca2+ concentration [Ca2+]i were measured by the u se of fura-2. Optimum binding of ET-1 was obtained by incubation for 1 20 min at 37-degrees-C, pH 7.0 when maximal protein content was 40 mug . Analysis with the LIGAND program showed an average number of binding sites (B(max)) of 26.0 +/- 30.5 fmol/mg protein and dissociation cons tant (K(d)) of 90.5 +/- 28.6 pmol/L for ET-1 and B(max) of 246.9 fmol/ mg protein and K(d) of 162.5 pmol/L for ET-3. ET-1, 10(-9) to 10(-6) m ol/L, dose dependently raised [Ca2+]i four to tenfold, respectively, f rom a mean basal level of 41 nmol/L. This rise was significantly atten uated by TMB-8 and by verapamil. Preincubation with Ni2+ almost comple tely prevented the increment in [Ca2+]. ET-1 slightly suppressed basal and significantly attenuated arginine vasopressin (AVP)-induced cycli c adenosine monophosphate (cAMP) synthesis. Thus, porcine IMCD cells p ossess a single class of super high affinity ET(B) receptors (ET(B1)). ET-1 raises [Ca2+]i through release from intracellular stores, activa tion of L-type calcium channels and, probably to a larger extent, thro ugh stimulation of other channels, eg, T-type calcium channels or unse lective cation channels. ET-1 suppresses AVP-induced cAMP synthesis su ggesting an important role in the renal regulation of body fluid balan ce.