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

Abstract

1. The excitation-contraction coupling mechanism in the smooth muscle of the guinea-pig mesenteric artery was studied using intact and chemically skinned muscle cells.2. 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 (> 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-MnCl(2).3. 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) and this contraction was suppressed by procaine (5 mm). On the other hand, the K-induced contraction was rapidly abolished in 0-Ca.4. Electrical stimulation (1 sec) in the presence of TTX (10(-7)m) evoked a contraction. Caffeine (5 mm) and TEA (5 mm) enhanced but procaine (5 mm) suppressed the contraction.5. Chemically skinned smooth muscle cells were prepared by adding saponin, 50 mug/ml., to the relaxing solution. The minimum concentration of free Ca required to evoke contraction in skinned muscle cells was 1-2 x 10(-7)m and the maximum contraction was produced at 10(-5)m. When Ca was replaced with Sr, the above relationship also shifted to the right (ED(50) for Ca is 4.4 x 10(-7)m and that for Sr is 1.5 x 10(-5)m). Treatment with high concentrations of caffeine and procaine had no effect on the pCa-tension relationship.6. Caffeine induced contraction in skinned muscle cells preloaded with Ca, and this contraction was markedly suppressed by procaine (5-10 mm).7. 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.8. 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 itself below threshold for contraction. The results also indicate that the Ca-induced Ca release mechanism appears to modify the amount of Ca stored by preincubation in over 3 x 10(-7)m free Ca.9. 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).10. The results support the view that 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.