α1-Adrenoceptor subtypes on rat afferent arterioles assessed by radioligand binding and RT-PCR

Abstract

We utilized [3H]prazosin saturation and competition radioligand binding studies to characterize the expression of α1-adrenoceptors in preglomerular vessels. mRNA for adrenoceptor subtypes was assayed using RT-PCR. The vessels were isolated using an iron oxide-sieving method. [3H]prazosin bound to a single class of binding sites ( K d0.087 ± 0.012 nM, Bmax 326 ± 56 fmol/mg protein). Phentolamine displaced [3H]prazosin (0.2 nM) with a p K i of 8.37 ± 0.09. Competition with the selective α1A-adrenoceptor antagonist 5-methylurapidil fit a two-site model (p K i9.38 ± 0.21 and 7.04 ± 0.15); 59 ± 3% of the sites were high-affinity, and 41 ± 3% were low-affinity binding sites. Competition with the α1D-adrenoceptor antagonist 8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) fit a one-site model with low affinity (p K i 6.83 ± 0.03). The relative contents of α1A-, α1B-, and α1D-adrenoceptor mRNAs were 64 ± 5, 25 ± 5, and 11 ± 1%, respectively. Thus there was a very good correlation between mRNA and receptor binding for the subtypes. These data indicate a predominance of the α1A-adrenoceptor subtype in rat renal resistance vessels, with smaller densities of α1B- and α1D-adrenoceptors.