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Morphological and Functional Characterization of Vascular Muscle Cells Enzymatically Dispersed from Dog Mesenteric Arteries
C.Y. Kwan , V. Gaspar , I. Berezin , A.M. Low , E.E. Daniel
23 September 2008
Vascular smooth muscle, Calcium, Contraction, Mesenteric artery
Abstract
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<sup>2+</sup>-free medium for about 1 -2 h after exposure to 1 mM Ca<sup>2+</sup> 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 Ca<sup>2+</sup> and KCl-induced cell shortening was prevented by 10<sup>–7</sup> M nifedipine indicating the presence of functional voltage-operated Ca<sup>2+</sup> 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<sup>2+</sup>-free medium. Isolated muscle shortened in the presence of sarcoplasmic reticulum selective Ca<sup>2+</sup> 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<sup>+</sup>, implying an altered membrane potential or voltage dependence of ion channels. Their impaired ability to contract to phenylephrine in Ca<sup>2+</sup>-free medium implies some alteration in intracellular Ca<sup>2+</sup> 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.
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