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Vascular tone and Ca(2+) signaling in murine cremaster muscle arterioles in vivo.

ABSTRACT: We sought to determine some of the molecular requirements for basal state "tone" of skeletal muscle arterioles in vivo, and whether asynchronous Ca(2+) waves are involved or not.Cremaster muscles of anesthetized exMLCK and smGCaMP2 biosensor mice were exteriorized, and the fluorescent arterioles were visualized with wide-field, confocal or multiphoton microscopy to observe Ca(2+) signaling and arteriolar diameter.Basal state tone of the arterioles was ~50%. Local block of Ang-II receptors (AT1 ) or ?1 -adrenoceptors (?1 -AR) had no effect on diameter, nor did complete block of sympathetic nerve activity (SNA). Inhibition of phospholipase C caused dilation nearly to the Ca(2+) -free (passive) diameter, as did exposure to nifedipine or 2-APB. Arterioles were also dilated when treated with SKF96365. High-resolution imaging of exMLCK fluorescence (ratio) or GCaMP2 fluorescence in smooth muscle cells failed to reveal Ca(2+) waves (although Ca(2+) waves/transients were readily detected by both biosensors in small arteries, ex vivo).Arterioles of cremaster muscle have vascular tone of ~ 50%, which is not due to ?1 -AR, AT1 R, or SNA. PLC activity, L-type Ca(2+) channels, 2-APB- and SKF96365-sensitive channels are required. Propagating Ca(2+) waves are not present. A key role for PLC and InsP3 R in vascular tone in vivo, other than producing Ca(2+) waves, is suggested.

SUBMITTER: Mauban JR

PROVIDER: S-EPMC4019383 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Vascular tone and Ca(2+) signaling in murine cremaster muscle arterioles in vivo.

Mauban Joseph R H JR Zacharia Joseph J Zhang Jin J Wier Withrow Gil WG

Microcirculation (New York, N.Y. : 1994) 20130401 3

<h4>Objectives</h4>We sought to determine some of the molecular requirements for basal state "tone" of skeletal muscle arterioles in vivo, and whether asynchronous Ca(2+) waves are involved or not.<h4>Methods</h4>Cremaster muscles of anesthetized exMLCK and smGCaMP2 biosensor mice were exteriorized, and the fluorescent arterioles were visualized with wide-field, confocal or multiphoton microscopy to observe Ca(2+) signaling and arteriolar diameter.<h4>Results</h4>Basal state tone of the arteriol ...[more]

PMID: 23140521

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Project description:Background and purposealpha(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 approachEC 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 resultsPhenylephrine-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 and implicationsWe 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+).

Project description:Arteriolar myogenic tone shows a marked dependency on extracellular Ca(2+). The contribution played by mechanisms such as intracellular Ca(2+) release and capacitative entry, however, are less certain. The present studies aimed to demonstrate functional evidence for involvement of such mechanisms in myogenic tone and reactivity. Single cremaster arterioles were denuded of endothelium, pressurized under no-flow conditions and loaded with fura 2-AM for measurement of changes in intracellular Ca(2+) [Ca(2+)](i). The cell permeable, putative, IP(3) receptor antagonist 2APB (2 aminoethoxydiphenyl borate) was used to determine the possible role of IP(3) receptor-mediated mechanisms in arteriolar myogenic tone and reactivity. Arterioles dilated in response to increasing concentrations of 2APB (1 - 300 microM) without a concomitant change in global [Ca(2+)](i). Also 2APB (50 microM) completely inhibited the myogenic constriction in response to a step change in luminal pressure (50 - 120 mmHg) with no apparent effect on pressure-mediated increases in [Ca(2+)](i). 2APB markedly attenuated the constrictor response and [Ca(2+)](i) increase stimulated by phenylephrine but not KCl. Capacitative Ca(2+) influx in arterioles was demonstrated either by re-addition of extracellular [Ca(2+)] following pre-treatment with 1 or 10 microM nifedipine in Ca(2+) free buffer or exposure of vessels to thapsigargin (1 microM) to induce store depletion. In both cases 2APB inhibited the increase in [Ca(2+)](i). Capacitative Ca(2+) entry showed an inverse relationship with intraluminal pressure over the range 10 - 120 mmHg. Consistent with an effect on a Ca(2+) entry pathway, 2APB had no effect on intracellular (caffeine releasable) Ca(2+) stores while decreasing the rate of Mn(2+) quench of fura 2 fluorescence. The results provide functional evidence for capacitative Ca(2+) entry in intact arteriolar smooth muscle. The effectiveness of 2APB in inhibiting both non-voltage gated Ca(2+) entry and responsiveness to an acute pressure step is consistent with the involvement of an axis involving IP(3)-mediated and or capacitative Ca(2+) entry mechanisms in myogenic reactivity. Given the lack of effect of 2APB on pressure-induced changes in global [Ca(2+)](i) it is suggested that such mechanisms participate on a localized level to couple the myogenic stimulus to contraction.

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Vascular tone and Ca(2+) signaling in murine cremaster muscle arterioles in vivo.

Publications

Vascular tone and Ca(2+) signaling in murine cremaster muscle arterioles in vivo.

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