Characterization of prazosin-sensitive alpha 2 B-adrenoceptors expressed by cultured rat IMCD cells

Abstract

alpha 2-Adrenoceptor subtype expression was investigated in cultured rat inner medullary collecting duct (IMCD) cells using radioligand binding studies, Northern blot analysis, and adenosine 3',5'-cyclic monophosphate (cAMP) assays. [3H]rauwolscine bound to a single class of alpha 2-adrenoceptors with high affinity [Kd = 1.7 +/- 0.3 nM, maximum binding (Bmax) = 45.2 +/- 10.8 fmol/mg protein]. alpha 2-Adrenoceptor ligands inhibited [3H]rauwolscine binding with a rank order of potency characteristic of interaction with the alpha 2B-adrenoceptor [inhibitory constant (Ki) values (in nM) rauwolscine (1.95) greater than ARC-239 (8.52) greater than prazosin (237) greater than oxymetazoline (30,000)]. Northern blot analysis was performed using poly(A)+ RNA isolated from 90% confluent rat IMCD cells and probes derived from alpha 2-adrenoceptor DNA sequences from the rat nonglycosylated alpha 2B-adrenoceptor and the human alpha 2A-adrenoceptor. The alpha 2B probe hybridized to a 4.2-kb band under high stringency conditions, but the alpha 2A-adrenoceptor probe did not hybridize to this band. In functional studies, the full alpha 2-adrenoceptor agonists epinephrine and UK-14,304 potently inhibited vasopressin-stimulated cAMP accumulation by 50 to 70% [half-maximal response (EC50) (in nM) epinephrine = 11.2, UK-14,304 = 6.4]. Guanabenz and clonidine were partial agonists, inhibiting cAMP accumulation by 30 to 40% and were less potent than the full agonists [EC50 (in nM) 56.0 guanabenz and 94.5 clonidine]. Epinephrine-induced inhibition of cAMP accumulation was blocked by rauwolscine, prazosin, and ARC-239 but not by the alpha 1-adrenoceptor antagonist corynanthine. We conclude that rat IMCD cells in primary culture express functional alpha 2-adrenoceptors of the alpha 2B-subtype.