Smooth muscle α1D-adrenoceptors mediate phenylephrine-induced vasoconstriction and increases in endothelial cell Ca2+ in hamster cremaster arterioles

Background and purpose: alpha(1)-Adrenoceptor agonists induce Ca(2+)-transients in endothelial cells (ECs) of arterioles. However, the presence of alpha(1)-adrenoceptors on arteriolar ECs has not been excluded, and the identity of alpha(1)-adrenoceptor subtypes in arterioles only has been inferred from pharmacology. Therefore, we determined which subtypes were expressed by vascular smooth muscle cells (VSMCs) and ECs, and which subtype mediated alpha(1)-adrenoceptor-induced constriction. Experimental approach: EC Ca(2+)-transients in isolated, cannulated hamster cremasteric arterioles or freshly isolated ECs were studied using Fura 2. Arteriolar diameter was measured by video microscopy. alpha(1)-Adrenoceptor expression was assessed by western blot of whole-arteriolar homogenates and real-time RT-PCR on enzymatically isolated VSMCs and ECs. Key results: Phenylephrine-induced constriction and EC Ca(2+)- transients were abolished by the alpha(1)-adrenoceptor antagonist prazosin (30 nM) in arterioles. Phenylephrine-induced constriction was inhibited by the alpha(1D)-adrenoceptor antagonist BMY 7378 (K(B) = 2.96 nM) and the alpha(1A)-adrenoceptor antagonist 5-methylurapidil (K(B) = 4.08 nM), suggesting a significant role for alpha(1D)-adrenoceptors. Western blots confirmed alpha(1D)-adrenoceptor expression, but did not detect alpha(1A)-adrenoceptors. VSMCs expressed alpha(1D)- and alpha(1A)-, but not alpha(1B)-, adrenoceptor transcripts. No alpha(1)-adrenoceptor transcripts were detected in ECs. Neither phenylephrine (10 mu M) nor noradrenaline (0.1-1 mu M) elicited Ca(2+)- transients in freshly isolated ECs, whereas the endothelium-dependent vasodilators methacholine (1 mu M) and substance P (100 nM) consistently increased Ca(2+). Conclusions and implications: We reject the hypothesis that hamster cremasteric arteriolar ECs express alpha(1)-adrenoceptors and conclude that alpha(1)-adrenoceptor agonists predominantly act on VSMC alpha(1D)-adrenoceptors to cause vasoconstriction and a subsequent rise in EC Ca(2+).