The beta(1)- and beta(2)-adrenoceptor subtypes in cultured rat inner medullary collecting duct cells

We investigated beta-adrenoceptor subtype(s) expressed in cultured rat inner medullary collecting duct (IMCD) cells. In radioligand binding assay, [I-125]iodocyanopindolol bound to IMCD cell membranes, representing a single class of binding sites (dissociation constant = 96.1 pill, maximum binding capacity = 18.2 fmol/mg protein, n = 8). In competition studies, ICI-89406 (beta(1)-antagonist) and ICI-118551 (beta(2)-antagonist) bound with high affinity, fitting a two-site model. Isoproterenol increased intracellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation (half-maximal effective concentration = 200 nM). Propranolol completely inhibited isoproterenol-induced cAMP accumulation [half-maximal inhibitory concentration (IC50) = 270 nM]. ICI-89406 and ICI-118551 inhibited cAMP accumulation by 50% (IC50 = 1.5 mu M and 1.7 mu M, respectively). The combined addition of ICI-89406 and ICI-118551 resulted in a curve indistinguishable from that of propranolol. The beta(1)- and beta(2)-adrenoceptor mRNAs have been demonstrated using reverse transcription-polymerase chain reaction. In initial and terminal IMCD cells, propranolol (3 mu M) inhibited isoproterenol-stimulated cAMP accumulation by 80%, whereas ICI-89406 (3 mu M) and ICI-118551 (3 mu M) resulted in only partial inhibition (50%). We conclude that both beta(1)- and beta(2)-adrenoceptors are expressed in initial and terminal IMCD cells in primary culture.