Ca2+ signaling in porcine myometrial cells: Ca2+ channels, intracellular Ca2+ stores and guanine nucleotide-binding protein-coupled receptors

Fura-2 spectrofluorometry and imaging, and whole-cell patch-clamp techniques were used to characterize Ca[superscript]2+ channels and intracellular Ca[superscript]2+ stores for exploring signal transduction of the oxytocin receptor and the [alpha][subscript] 2A-adrenergic receptor in freshly dispersed porcine myometrial cells. Two types of voltage-dependent Ca[superscript]2+ channels, i.e., L-type and T-type, were found in 8% of myometrial cells while in 92% of the cells, only L-type Ca[superscript]2+ channels were detected. The analysis of occurrence, voltage dependence and kinetics of Ca[superscript]2+ channels suggested that L-type channels were responsible for the delivery of Ca[superscript]2+ into cell upon membrane depolarization and T-type channel may be involved in uterine pacemaking. In addition to the Ins(1,4,5)P[subscript]3- sensitive Ca[superscript]2+ store, the myometrial cells contained the caffeine- and ryanodine-sensitive store. Ca[superscript]2+ release from the caffeine- and ryanodine-sensitive store in a fashion of quanta, in which quanta was defined as part of stores in 70% and the entire stores in 30% of cells. Activation of oxytocin receptors and [alpha][subscript] 2A-adrenergic receptors induced an increase in the intracellular Ca[superscript]2+ concentration ((Ca[superscript]2+][subscript] i) through Ca[superscript]2+ release from intracellular stores and influx from extracellular environment. The contribution of influx and release was variable between two receptors: the release was a predominate component for oxytocin receptors while the influx is the major one for [alpha][subscript] 2A-adrenergic receptors. The release process posed by activation of oxytocin receptors and [alpha][subscript] 2A-adrenergic receptors was mediated by activation of Ins(1,4,5)P[subscript]3 receptors via a pertussis toxin (PTX)-insensitive G protein-phospholipase C-Ins (1,4,5)P[subscript]3 cascade. Oxytocin receptor-mediated influx was predominately attributed to opening of receptor-operated Ca[superscript]2+ channels, and to a lesser extent by L-type Ca[superscript]2+ channels. The capacitative Ca[superscript]2+ entry mechanism could not account for activation of these channels, instead, it required a concomitant formation of Ins(1,4,5)P[subscript]3 and depletion of Ca[superscript]2+ stores. On the other hand, the depletion of Ca[superscript]2+ stores was not involved in [alpha][subscript] 2A-adrenergic receptor-induced Ca[superscript]2+ influx. Instead this influx was primarily mediated by opening of L-type Ca[superscript]2+ channels which resulted from a decrease in cAMP through a PTX-sensitive G protein-adenlylyl cyclase mechanism, or a direct coupling of the channels with a PTX-sensitive G protein