Characteristics of Endothelin Receptors and Intracellular Signalling in Porcine Inner Medullary Collecting Duct Cells

Abstract

In porcine kidneys we investigated the characteristics of endothelin (ET) receptors that are present in papillary tissue but not in glomeruli. Therefore, porcine inner medullary collecting duct (IMCD) cells were separated by Percoll density gradient centrifugation after enzymatic and hypotonic treatment of minced papillary tissue. Studies were performed in fresh cell suspensions and in cells in primary culture. Changes in cytosolic free Ca2+ concentration [Ca2+]i were measured by the use of fura-2. Optimum binding of ET-1 was obtained by incubation for 120 min at 37 degrees C, pH 7.0 when maximal protein content was 40 micrograms. Analysis with the LIGAND program showed an average number of binding sites (Bmax) of 26.0 +/- 30.5 fmol/mg protein and dissociation constant (Kd) of 90.5 +/- 28.6 pmol/L for ET-1 and Bmax of 246.9 fmol/mg protein and Kd of 162.5 pmol/L for ET-3. ET-1, 10(-9) to 10(-6) mol/L, dose dependently raised [Ca2+]i four to tenfold, respectively, from a mean basal level of 41 nmol/L. This rise was significantly attenuated by TMB-8 and by verapamil. Preincubation with Ni2+ almost completely prevented the increment in [Ca2+]i. ET-1 slightly suppressed basal and significantly attenuated arginine vasopressin (AVP)-induced cyclic adenosine monophosphate (cAMP) synthesis. Thus, porcine IMCD cells possess a single class of super high affinity ETB receptors (ETB1). ET-1 raises [Ca2+]i through release from intracellular stores, activation of L-type calcium channels and, probably to a larger extent, through stimulation of other channels, eg, T-type calcium channels or unselective cation channels.(ABSTRACT TRUNCATED AT 250 WORDS)