Kwan C Y, Gaspar V, Berezin I, Low A M, Daniel E E
Department of Biomedical Sciences, McMaster University, Hamilton, Ont., Canada.
J Vasc Res. 1992 Nov-Dec;29(6):450-60. doi: 10.1159/000158964.
The objective of this study was to compare the properties of single smooth muscle cells enzymatically dispersed from the dog mesenteric arteries to the properties of similar cells functioning in tissue strips. The isolated cells remained relaxed in nominally Ca(2+)-free medium for about 1-2 h after exposure to 1 mM Ca2+ and like intact mesenteric artery rings did not contract spontaneously. Enzymatically dispersed cells maintained all the characteristic morphological features observed in strips of muscle prior to isolation except that the amorphous materials covering the smooth muscle cell surfaces (basal lamina) were absent after enzymatic dispersion. Addition of 100 mM KCl to these vascular muscle cells elicited maximal shortening in the presence but not in the absence of extracellular Ca2+ and KCl-induced cell shortening was prevented by 10(-7) M nifedipine indicating the presence of functional voltage-operated Ca2+ channels. However, in contrast to the vascular muscle strips, in which graded contractile responses were observed with increasing KCl concentrations, isolated vascular muscle cells underwent nearly maximal contraction at concentrations as low as 15 mM KCl. Both intact tissue and isolated cell preparations responded similarly to phenylephrine in a concentration-dependent manner and the responses were blocked by prazosin. In contrast to muscle strips, the isolated cells did not shorten in response to phenylephrine in Ca(2+)-free medium. Isolated muscle shortened in the presence of sarcoplasmic reticulum selective Ca2+ transport ATPase inhibitors, cyclopiazonic acid or thapsigargin. Ryanodine also caused contraction. We conclude that enzymatically dispersed smooth muscle cells from dog mesenteric arteries are potentially useful for studies of the regulation of smooth muscle contractility, but have significantly increased sensitivity to external K+, implying an altered membrane potential or voltage dependence of ion channels. Their impaired ability to contract to phenylephrine in Ca(2+)-free medium implies some alteration in intracellular Ca2+ stores of their coupling to cellular activation. These differences will affect how the data obtained from freshly isolated enzymatically dispersed vascular muscle cells may be extrapolated to cell studies in intact tissues.
本研究的目的是比较从犬肠系膜动脉酶解分散得到的单个平滑肌细胞的特性与在组织条中发挥功能的类似细胞的特性。分离出的细胞在暴露于1 mM Ca2+后，在名义上无Ca(2+)的培养基中保持松弛约1 - 2小时，并且与完整的肠系膜动脉环一样不会自发收缩。酶解分散的细胞保持了分离前在肌肉条中观察到的所有特征性形态特征，只是酶解分散后覆盖平滑肌细胞表面的无定形物质（基膜）消失了。向这些血管平滑肌细胞中加入100 mM KCl在有细胞外Ca2+存在时引发最大程度的缩短，但在无Ca2+时则不会，并且10(-7) M硝苯地平可阻止KCl诱导的细胞缩短，这表明存在功能性电压门控Ca2+通道。然而，与血管肌肉条不同，在血管肌肉条中随着KCl浓度增加观察到分级收缩反应，而分离的血管平滑肌细胞在低至15 mM KCl的浓度下就经历了几乎最大程度的收缩。完整组织和分离细胞制剂对去氧肾上腺素的反应相似，呈浓度依赖性，且这些反应被哌唑嗪阻断。与肌肉条不同，分离的细胞在无Ca(2+)的培养基中对去氧肾上腺素不发生缩短反应。在存在肌浆网选择性Ca2+转运ATP酶抑制剂环匹阿尼酸或毒胡萝卜素的情况下，分离的肌肉发生缩短。ryanodine也会引起收缩。我们得出结论，从犬肠系膜动脉酶解分散得到的平滑肌细胞对于平滑肌收缩性调节的研究可能是有用的，但对外部K+的敏感性显著增加，这意味着膜电位或离子通道的电压依赖性发生了改变。它们在无Ca(2+)的培养基中对去氧肾上腺素收缩能力的受损意味着其细胞内Ca2+储存与细胞激活偶联存在某种改变。这些差异将影响从新鲜分离的酶解分散血管平滑肌细胞获得的数据如何外推至完整组织中的细胞研究。
