Total cytoplasmic calcium in relaxed and maximally contracted rabbit portal vein smooth muscle.

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Abstract
The concentration of total cytoplasmic Ca in vascular smooth muscle was measured by electron probe microanalysis of strips of rabbit portal anterior mesenteric vein that were rapidly frozen either when relaxed or during a maintained (30 min) maximal contraction stimulated with high K and noradrenaline [norepinephrine]. Strips were also frozen and analyzed after incubation in Ca-free, high-Mg2+ solution. Probe diameters of 0.1-0.2 .mu.m and 1.0-1.5 .mu.m were used to measure, respectively, cytoplasmic and cellular (including stored) Ca. There was a highly significant increase (P < 0.0005) in cytoplasmic Ca of 1.0 .+-. 0.2 (SD) mmol Ca/kg dry wt from 0.8 .+-. 0.2 (SE of mean) mmol/kg dry wt (n = 262 spectra, 6 animals) to 1.8 .+-. 0.2 (SE of mean) mmol Ca/kg dry wt (n = 296 spectra, 6 animals) during maximal contraction. This increase was greater than can be accounted for by Ca binding to calmodulin and to myosin, suggesting the presence of other Ca-binding proteins in smooth muscle. A small amount (0.4-0.6 mmol/kg dry wt) of cytoplasmic Ca remained after incubation in Ca-free, high-Mg2+ EGTA [ethyleneglycol-bis(.beta.-aminoethylether)-N,N''-tetraacetic acid] solution. This tightly bound, cytoplasmic Ca was insufficient to account for the total amount of divalent cation known to be bound to F-actin. Mg is the major inexchangeably bound cation in F-actin in smooth as in striated muscle. In the contracted muscles, the cellular Ca concentration, measured with the large probes that include Ca stored in the sarcoplasmic reticulum (s.r.), was 3.2 .+-. 0.3 (SE of mean) mmol Ca/kg dry wt (n = 93), significantly higher than the cytoplasmic Ca concentration measured with small probes. This value of cellular Ca was probably an underestimate, as the large-diameter probes did not cover all of the peripheral s.r. The cellular Ca (measured with large probes) was highest in the contracted and lower in the relaxed tissue, and was significantly reduced in the muscles incubated in Ca-free solution. In contracted muscle, cytoplasmic Mg significantly decreased and mitochondrial Mg increased. In 0 Ca, high-Mg2+ solution, the cytoplasmic Mg increased significantly. Mitochondrial Ca did not significantly change during a maintained contraction, but was significantly lower (0.0 .+-. 0.2 (SE of mean) mmol Ca/kg dry wt) after incubation in Ca-free, high-Mg2+ solution than in the relaxed tissue (1.6 .+-. 0.2 mmol Ca/kg dry wt) in normal Ca-containing solution.