Intracellular free Ca2+ concentration ([Ca2+]i) was monitored using the fluorescence from the dye Fura-2-AM in single myometrial cells from pregnant rats. Oxytocin and acetylcholine applied to the cell evoked an initial peak in [Ca2+]i followed by a smaller sustained rise which was rapidly terminated upon removal of acetylcholine or persisted after oxytocin removal. A Ca2+ channel blocker (oxodipine) and external Ca2+ removal decreased both the transient and sustained rises in [Ca2+]i suggesting that Ca2+ influx through L-type Ca2+ channels participated in the global Ca2+ response induced by oxytocin. However, the initial peak in [Ca2+]i produced by oxytocin was mainly due to Ca2+ store release: it was abolished by inclusion of heparin [which blocks inositol 1,4,5-trisphosphate (InsP3) receptors] in the pipette (whole-cell recording mode of patch-clamp) and external application of thapsigargin (which blocks sarcoplasmic reticulum Ca(2+)-ATPases). In contrast, the transient Ca2+ response induced by oxytocin was unaffected by ryanodine. Moreover, caffeine failed to induce a rise in [Ca2+]i but reduced the oxytocin-induced transient Ca2+ response. The later sustained rise in [Ca2+]i produced by oxytocin was due to the entry of Ca2+ into the cell as it was suppressed in external Ca(2+)-free solution. The Ca2+ entry pathway is permeable to Mn2+ ions, in contrast to that described in various vascular and visceral smooth muscle cells. Oxytocin-induced Ca2+ release is blocked by the oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT. The prolonged increase in [Ca2+]i after oxytocin removal is rapidly terminated by addition of the oxytocin antagonist suggesting that oxytocin dissociation from its receptor is very slow.(ABSTRACT TRUNCATED AT 250 WORDS)
The presence of functional alpha 2-adrenoceptors was investigated in isolated smooth muscle cells from rat portal vein using the nystatin-perforated patch-clamp technique. The free cytoplasmic calcium concentration ([Ca2+]i) was estimated using emission from the dye Fura-2. Activation of alpha 2-adrenoceptors by clonidine (an alpha 2-adrenoceptor agonist) or noradrenaline (a non-selective alpha-adrenoceptor agonist), both in the presence of 0.1 microM prazosin to block alpha 1-adrenoceptors, caused a slow and sustained increase in [Ca2+]i which was inhibited by 0.1 microM rauwolscine (an alpha 2-adrenoceptor antagonist). A similar Ca2+ response was obtained with oxymetazoline (a selective alpha 2A-adrenoceptor agonist) suggesting that the increase in [Ca2+]i resulted from activation of the alpha 2A-adrenoceptor subtype. The increase in [Ca2+]i did not occur in calcium-free solution or in the presence of oxodipine (a voltage-dependent calcium channel blocker), indicating that it depended on a calcium influx. The alpha 2A-adrenoceptor-activated calcium influx was unchanged after complete release of the stored calcium induced by applications of ryanodine and caffeine. In addition, no accumulation of inositol trisphosphate was detected in the presence of 0.1 microM prazosin. Taken together, these results indicate that alpha 2A-adrenoceptor activation does not stimulate phosphoinositide turnover and subsequent calcium release from intracellular stores. Whole-cell patch-clamp experiments showed that alpha 2A-adrenoceptor activation promoted calcium influx through voltage-dependent L-type channels.(ABSTRACT TRUNCATED AT 250 WORDS)
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