Smooth muscle alpha1D-adrenoceptors mediate phenylephrine-induced vasoconstriction and increases in endothelial cell Ca2+ in hamster cremaster arterioles.
OBJECTIVE
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.


METHODS
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.


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 microM) nor noradrenaline (0.1-1 microM) elicited Ca(2+)-transients in freshly isolated ECs, whereas the endothelium-dependent vasodilators methacholine (1 microM) and substance P (100 nM) consistently increased Ca(2+).


CONCLUSIONS
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+).
Jackson
W F
WF
Department of Pharmacology & Toxicology, Michigan State University, East Lansing, MI 48824, USA. jacks783@msu.edu
Boerman
E M
EM
Lange
E J
EJ
Lundback
S S
SS
Cohen
K D
KD
D058646:Adrenergic alpha-1 Receptor Agonists; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D001160:Arterioles / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology*; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014661:Vasoconstriction / Q000187:drug effects; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
D058646:Adrenergic alpha-1 Receptor Agonists; D000316:Adrenergic alpha-Agonists; D018340:Receptors, Adrenergic, alpha-1; D014662:Vasoconstrictor Agents; D010656:Phenylephrine; D002118:Calcium