Alpha2-adrenergic receptor-mediated Ca2+ influx and release in porcine myometrial cells.

We studied the mechanisms underlying alpha2-adrenergic receptor (AR)-mediated increase in intracellular free calcium ([Ca2+]i) in freshly dispersed myometrial cells from sows in the luteal phase of the estrous cycle. After the blockade of beta-ARs with propranolol, epinephrine increased [Ca2+]i dose-dependently in both the presence and absence of extracellular Ca2+. The rank order of alpha antagonists in inhibiting [Ca2+]i response to epinephrine was yohimbine > WB4101 >> prazosin in both the presence and absence of extracellular Ca2+, suggesting that epinephrine acts on alpha(2A)-ARs to increase Ca2+ influx as well as Ca2+ release from intracellular stores. Thapsigargin, the blocker of the Ca2+ pump in the sarcoplasmic reticulum, abolished the release but did not affect the influx. Pertussis toxin (PTX) inhibited the influx but failed to change the release. Nimodipine, an L-type Ca2+ channel blocker, nearly abolished the influx. The peak increase in [Ca2+]i caused by epinephrine was reached within 20 sec of administration. Intracellular cAMP concentrations were also decreased at 20 sec post-epinephrine. Epinephrine enhanced the L-type Ca2+ channel current, whereas forskolin suppressed it. Maximization of intracellular cAMP content by applying 8-bromo-cAMP (100 microM) blocked the effect of epinephrine on the current. U-73122, a phospholipase C inhibitor, reduced the Ca2+ release by epinephrine and oxytocin. Our results suggested that 1) activation of alpha2-ARs induces Ca2+ influx through opening L-type Ca2+ channels as well as inducing Ca2+ release from intracellular stores, and 2) a PTX-sensitive G protein couples negatively to adenylyl cyclase, leading to a decrease in cAMP formation which may be involved in the activation of Ca2+ channels. In addition, our results are consistent with the coupling of alpha2-ARs to a PTX-insensitive G protein (G(q)) to release Ca2+ from intracellular stores.