Excitation—contraction coupling in smooth muscle cells of the guinea‐pig mesenteric artery

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Abstract
The excitation-contraction coupling mechanism in the smooth muscle of the guinea pig mesenteric artery was studied using intact and chemically skinned muscle cells. The mean membrane potential of the intact smooth muscle was -65.8 .+-. 2.4 mV. It was electrically quiescent. Caffeine (5 mM), procaine (> 1 mM) and TEA [tetraethylammonium chloride] (> 1 mM) depolarized the membrane, increased the membrane resistance and in their presence, outward current pulses evoked action potentials with overshoot. These potential changes were still observed in Na-deficient solution but were abolished in the presence of 3 mM MnCl2. Caffeine (5 mM) and TEA (1 mM) produced contractions in the intact muscle which were suppressed by procaine (5-10 mM). Caffeine (5 mM) continued to produce contraction even after prolonged exposure to Ca-free solution (containing 2 mM EGTA [ethylene glycol bis(.beta.-aminoethyl ether) N,N,N'',N''-tetraacetic acid]); this contraction was suppressed by procaine (5 mM). The K-induced contraction was rapidly abolished in 0 mM Ca. Electrical stimulation (1 s) in the presence of TTX [tetrodotoxin] (10-7 M) evoked a contraction. Caffeine (5 mM) and TEA (5 mM) enhanced but procaine (5 mM) suppressed the contraction. Chemically skinned smooth muscle cells were prepared by adding saponin, 50 .mu.g/ml, to the relaxing solution. The minimum concentration of free Ca required to evoke contraction in skinned muscle cells was 1-2 .times. 10-7 M and the maximum contraction was produced at 10-5 M. When Ca was replaced with Sr, this relationship also shifted to the right (ED50 for Ca is 4.4 .times. 10-7 M and that for Sr is 1.5 .times. 10-5). Treatment with high concentrations of caffeine and procaine had no effect on the pCa[-log[Ca]]-tension relationship. Caffeine induced contraction in skinned muscle cells preloaded with Ca, and this contraction was markedly suppressed by procaine (5-10 mM). In skinned muscles, depolarization of the internal membrane by replacement of K with choline (116 mM) in the relaxing solution produced contraction, but the amplitude was much smaller than the caffeine-induced contraction. The relationship between the amplitude of caffeine-induced contraction and the duration of preincubation in various Ca concentrations was observed in skinned muscles. The minimum concentration of Ca required to produce a subsequent caffeine-induced contraction was below threshold for contraction. The Ca-induced Ca release mechanism appears to modify the amount of Ca stored by preincubation in over 3 .times. 10-7 M free Ca. When the amount of Ca stored in intact cells was estimated from the caffeine-induced contraction evoked in Ca-free solution following preincubation with Ca, Ca applied simultaneously with procaine increased and Ca with caffeine reduced the Ca stored in the cell. After preincubation in 2.5 mM [Ca]o with 1 mM-procaine for 5 min, the amplitude of the subsequently generated caffeine-induced contraction (5 mM) in Ca-free solution (2 min) was much the same as that observed in 118 mM [K]o. The excitation-contraction coupling mechanism in the mesenteric artery may be as follows: the Ca inward current generated at the myoplasmic membrane may not directly provide the free Ca required to activate the Ca-receptor of the contractile protein, but the Ca carrying the inward current may first be sequestered inside the cell and activate a Ca release mechanism which in turn leads to contraction.