This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca2+ concentrations ([Ca2+]i) in acutely dispersed myometrial cells from prepartum sows. A dose-dependent increase in [Ca2+]i was induced by OT (0.1 nM to 1 microM) in the presence and absence of extracellular Ca2+ ([Ca2+]e). [Ca2+]i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca2+]e. However, in the absence of [Ca2+]e, the [Ca2+]i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca2+ ionophore. Administration of OT (1 microM) for 15 sec increased inositol 1,4,5-trisphosphate (IP3) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 microgram/ml) for 2 hr failed to alter the OT-induced increase in [Ca2+]i and IP3 formation. U-73122 (30 nM to 3 microM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca2+]i by OT dose dependently. U-73122 (3 microM) also abolished the OT-induced IP3 formation. Thapsigargin (2 microM), an inhibitor of Ca(2+)-ATPase in the endoplasmic reticulum, did not increase [Ca2+]i. However, it did time-dependently inhibit the OT-induced increase in [Ca2+]i. Nimodipine (1 microM), a voltage-dependent Ca2+ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La3+ (1 mM), a nonspecific Ca2+ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 microM) increased Ca2+ current (ICa) by 40% with no apparent changes in the current-voltage relationship.(ABSTRACT TRUNCATED AT 250 WORDS)

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.