Oxytocin mobilizes calcium from a unique heparin-sensitive and thapsigargin-sensitive store in single myometrial cells from pregnant rats

Abstract

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 (InsP ₃) receptors] in the pipette (whole-cell recording mode of patch-clamp) and external application of thapsigargin (which blocks sarcoplasmic reticulum Ca²⁺-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 Ca²⁺-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-NH ⁹₂ ]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. The oxytocin stimulation of [Ca²⁺]_i was insensitive to incubation with pertussis toxin, and blocked by a pipette solution containing anti-αq/α₁₁ antibody. These data show that myometrial cells possess an unique heparin-sensitive and thapsigargin-sensitive store that can be mobilized by activation of oxytocin receptors which couples with a Gq/G₁₁-protein to activate phospholipase C.