Potocnik S J, Hill M A
Microvascular Biology Group, School of Medical Sciences, Division of Biosciences, RMIT University, Plenty Road, Bundoora, Victoria 3083, Australia.
Br J Pharmacol. 2001 Sep;134(2):247-56. doi: 10.1038/sj.bjp.0704270.
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+). 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+). 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+). 2APB markedly attenuated the constrictor response and Ca(2+) 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+). 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+) it is suggested that such mechanisms participate on a localized level to couple the myogenic stimulus to contraction.
小动脉肌源性张力对细胞外Ca(2+)有显著依赖性。然而，细胞内Ca(2+)释放和容量性钙内流等机制所起的作用尚不太明确。本研究旨在为这些机制参与肌源性张力和反应性提供功能证据。分离出单个提睾肌小动脉的内皮，在无血流条件下加压，并加载fura 2-AM以测量细胞内Ca(2+)Ca(2+)的变化。使用可透过细胞的、假定的IP(3)受体拮抗剂2-APB（2-氨基乙氧基二苯硼酸盐）来确定IP(3)受体介导的机制在小动脉肌源性张力和反应性中的可能作用。随着2-APB浓度增加（1 - 300 microM），小动脉舒张，而整体Ca(2+)没有相应变化。同样，2-APB（50 microM）完全抑制了因管腔内压力阶跃变化（50 - 120 mmHg）引起的肌源性收缩，对压力介导的Ca(2+)增加没有明显影响。2-APB显著减弱了去氧肾上腺素刺激的收缩反应和Ca(2+)增加，但对氯化钾没有影响。在无钙缓冲液中用1或10 microM硝苯地平预处理后重新添加细胞外[Ca(2+)]，或使血管暴露于毒胡萝卜素（1 microM）以诱导钙库耗竭，均可证明小动脉中的容量性Ca(2+)内流。在这两种情况下，2-APB均抑制了Ca(2+)的增加。在10 - 120 mmHg范围内，容量性Ca(2+)内流与管腔内压力呈负相关。与对Ca(2+)内流途径的作用一致，2-APB对细胞内（咖啡因可释放的）Ca(2+)储存没有影响，同时降低了fura 2荧光的Mn(2+)淬灭速率。这些结果为完整小动脉平滑肌中的容量性Ca(2+)内流提供了功能证据。2-APB在抑制非电压门控Ca(2+)内流和对急性压力阶跃的反应性方面的有效性，与涉及IP(3)介导和/或容量性Ca(2+)内流机制的轴参与肌源性反应性一致。鉴于2-APB对压力诱导的整体Ca(2+)变化没有影响，提示这些机制在局部水平参与将肌源性刺激与收缩相偶联。
