Characterization of freshly dispersed porcine myometrial cells: evidence for voltage-dependent Ca2+ channels and regulatory receptors
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A patch-clamp study of Ca2+ currents and spectrofluorometric detection of the intracellular Ca2+ concentration [Ca2+]i was performed on porcine myometrial cells that had been isolated by collagenase. Isolated myometrial cells were the typical long cylinder shape. The main length and diameter of myometrial cells were 505 ± 20 and 11± 0.5 μm (n = 40), respectively, in the prepartum period and 265 ± 22 and 7 ± 0.4 μm (n = 40), respectively, in the luteal phase. Immunocytochemistry with an antibody against desmin stained 90% of the cells positively, and about 95% of the cells excluded Trypan blue dye. The basal [Ca2+]i of myometrial cells in the luteal phase and the prepartum period was 119 ± 12 (n = 30) and 154 ± 31 nmol l−1 (n = 48), respectively. In prepartum myometrial cells, oxytocin (10−7 mol l−1) and carbachol (10−4 mol l−1) increased [Ca2+]i in a biphasic pattern, with a sharp peak followed by a plateau. In cells in the luteal phase, adrenaline (10−7 mol l−1) plus propranolol (10−6 mol l−1) produced a biphasic increase in [Ca2+]i. However, in the absence of propranolol, the increase in [Ca2+]i by adrenaline was small. Prostaglandin F2α (10−6 mol l−1) induced a monophasic increase in the [Ca2+]i in cells in the luteal phase. By depolarizing the cells from −30 to +50 mV from a holding potential of −50 mV, Ca2+ currents were evoked with a threshold at −20 mV, reaching a maximum between 10 and 30 mV. BayK8644 (10−7 mol l−1), an L-type Ca2+ channel agonist, and oxytocin (10−7 mol l−1) enhanced Ca2+ currents by 166 ± 64% and 41 ± 10%, respectively, in prepartum cells. These results suggest that freshly dispersed porcine myometrial cells contain an intact membrane and possess functional voltage-dependent Ca2+ channels and receptors for major physiological regulators. Thus, porcine myometrial cells provide a useful model for the study of excitation–contraction coupling and the influence of physiological regulators in the myometrium.
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