PHARMACOLOGICAL AND IMMUNOCYTOCHEMICAL CHARACTERIZATION OF SUBTYPES OF ALPHA-1-ADRENOCEPTORS IN DOG AORTA

In this study, the effects of nine alpha-1 adrenoceptor antagonists [p razosin, WE 4101 (WB), chloroethylclonidine (CEC), 5-methylurapidil (5 -MU), BMY 7378 (BMY), MDL 73005EF (MDL73), MDL 72832 (MDL72), RS 17053 (RS) and SK&F 105854 (SKF)] were studied on contractile responses to phenylephrine (PE) of the endothelium-denuded dog aorta in vitro. All antagonists, except CEC, 5-MU and RS, produced concentration-dependent competitive inhibition of contractile responses of the aorta to PE. T he rightward shift of the concentration-response curves of PE yielded constant pK(B), values with increasing antagonist concentrations in mo st cases allowing a single pooled value to be determined: for prazosin , a pK(B), of 8.99 +/- 0.11 (n = 20, K-B, of 1.03 nM); for WE, a pK(B) , of 8.75 +/- 0.08 (n = 23, K-B, of 1.76 nM); for BMY, a pK(B), of 7.2 1 +/- 0.13 (n = 13, K-B, of 62 nM); for MDL72, a pK(B), of 7.95 +/- 0. 15 (n = 12, K-B, of 11.2 nM); and for SK&F 105854, a pK(B), of 5.82 +/ - 0.08 (n = 15, K-B, of 1.52 mu M). For MDL73, pK(B), values decreased with antagonist concentration: 7.88 +/- 0.06 at 10 nM, 7.56 +/- 0.28 at 100 nM and 6.92 +/- 0.18 at 1000 nM, which suggests the presence of more than one receptor subtype. CEC (10 and 100 mu M) almost complete ly inhibited responses to PE; lower concentrations had no significant effect. 5-MU (10-300 nM) and RS (3-300 nM) were ineffective antagonist s in this tissue. Because WE, a highly selective alpha-1D and alpha-1A adrenoceptor subtypes inhibitor, blocked PE responses (with less affi nity than for alpha-1A adrenoceptors), and 5-MU and RS, which are sele ctive blockers for alpha-1A adrenoceptor, were ineffective, we conclud e that alpha-1A adrenoceptors are absent in the dog aorta. The effects of the less selective MDL72 were inconsistent with actions at alpha-1 B or alpha-1D adrenoceptors. Although WE shifted the PE concentration- response curve to the right, the abilities of BMY, MDL73 and SKF to in hibit competitively PE contraction were of lower affinity compared wit h expectations for interaction with alpha-1D adrenoceptors; they are n ot the predominant subtype. The complete inhibition of PE responses by CEC suggests that the dog aorta contains the alpha-1B adrenoceptor su btype. In immunocytochemical studies of the expression of alpha-1B adr enoceptor, all cells apparently expressed this protein. Moreover, West ern blot studies of the microsomal fractions confirmed the presence of alpha-1B adrenoceptors. In the dog aorta, the alpha-1 adrenoceptors p redominantly resemble alpha-1B rather than alpha-1D adrenoceptors as r eported in the rat aorta.