Background and purpose: a 1 -Adrenoceptor agonists induce Ca 2 þ -transients in endothelial cells (ECs) of arterioles. However, the presence of a 1 -adrenoceptors on arteriolar ECs has not been excluded, and the identity of a 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 a 1 -adrenoceptor-induced constriction. Experimental approach: EC Ca 2 þ -transients in isolated, cannulated hamster cremasteric arterioles or freshly isolated ECs were studied using Fura 2. Arteriolar diameter was measured by video microscopy. a 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 þ -transients were abolished by the a 1 -adrenoceptor antagonist prazosin (30 nM) in arterioles. Phenylephrine-induced constriction was inhibited by the a 1D -adrenoceptor antagonist BMY 7378 (K B ¼ 2.96 nM) and the a 1A -adrenoceptor antagonist 5-methylurapidil (K B ¼ 4.08 nM), suggesting a significant role for a 1D -adrenoceptors. Western blots confirmed a 1D -adrenoceptor expression, but did not detect a 1A -adrenoceptors. VSMCs expressed a 1D -and a 1A -, but not a 1B -, adrenoceptor transcripts. No a 1 -adrenoceptor transcripts were detected in ECs. Neither phenylephrine (10 mM) nor noradrenaline (0.1-1 mM) elicited Ca 2 þ -transients in freshly isolated ECs, whereas the endotheliumdependent vasodilators methacholine (1 mM) and substance P (100 nM) consistently increased Ca 2 þ . Conclusions and implications:We reject the hypothesis that hamster cremasteric arteriolar ECs express a 1 -adrenoceptors and conclude that a 1 -adrenoceptor agonists predominantly act on VSMC a 1D -adrenoceptors to cause vasoconstriction and a subsequent rise in EC Ca 2 þ .
The α1‐adrenoreceptor (α1‐AR) agonist, phenylephrine (PE) induces Ca2+ transients in endothelial cells (EC) of arterioles, possibly due to movement of Ca2+ or IP3 from smooth muscle (SMC) into EC via gap junctions. However, the presence of α1‐AR on EC has not been excluded, and their identity in arterioles only has been inferred from pharmacology. Therefore, we determined which α1‐AR subtypes are expressed by SMC and EC, and which subtype mediates PE‐induced EC Ca2+ transients. Cell specific expression was assessed by real time RT‐PCR in samples of 50 cells from dissociated hamster cremaster arterioles using primers for α1A‐, α1B‐ and α1D‐AR with α‐SMC actin and eNOS as respective SMC and EC markers. SMC expressed α1A‐ and α1D‐AR, but not α1B‐AR, and no α1‐AR transcripts were detected in EC expressing eNOS. Western blots of vessel homogenates confirmed α1D‐AR expression, but surprisingly, did not detect α1A‐AR in up to 100 μg protein, despite positive results in heart. In cannulated vessels, the Ki for inhibition of PE‐induced constriction was 2.95 nM for the α1D‐AR antagonist, BMY 7378 and 4.11 nM for the α1A‐AR antagonist, 5‐methylurapadil, data consistent with PE acting through α1D‐AR. In vessels with Fura‐2‐loaded EC, PE‐induced constriction and EC Ca2+ transients were abolished by the α1‐AR antagonist, prazosin (30 nM). Thus, PE‐induced EC Ca2+ transients are mediated by SMC α1D‐AR. Supported by HL32469 to WFJ.
Ryanodine receptors (RyR) in smooth muscle cells (SMC) underlie Ca2+ sparks and Ca2+‐induced‐Ca2+ release, contributing to the regulation of myogenic tone in arteries. In contrast, RyR are silent and are not involved in Ca2+ signals in SMC or myogenic tone in hamster arterioles. The purpose of the present study was to investigate the role of RyR in vessels of male C57BL/6 mice. We tested the hypothesis that differences in RyR isoform expression contribute to differences in RyR function in iliac feed arteries (FA) vs. cremaster arterioles (CA). In cannulated pressurized (80 cm H2O) vessels (34 – 37 °C), ryanodine (10 μM) constricted FA (from 53 ± 4 to 40 ± 3 μm; n = 5, p < 0.05) but not CA (24 ± 2 μm vs. 22 ± 2 μm; n = 5, p > 0.05). Transcript levels for RyR2 in freshly isolated SMC assessed by q‐RT‐PCR were 2.1 ± 0.3 ‐fold higher in FA vs. CA (n = 7, p < 0.05), while those for RyR3 were 0.6 ± 0.1 in FA vs. CA (n = 6, p < 0.05). RyR1 transcripts were not detected in SMC from FA or CA. Immunofluorescence with an anti‐RyR1/2 antibody showed clustered labeling in FA SMC (n = 6 SMC) suggestive of RyR2 expression in endoplasmic reticulum. In contrast, more uniform cytoplasmic staining in CA SMC (n = 13 SMC) suggested non‐organelle‐specific staining. These data support our hypothesis and indicate fundamental differences in SMC RyR expression and function between arteries and arterioles across rodent species. Supported by NIH HL086483 & AHA Fellowship 0815778G.
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