Oxytocin inhibition of (Ca2+ + Mg2+)-ATPase activity in rat myometrial plasma membranes.

Enriched plasma membranes from uterine smooth muscle of estrogen-treated rats were prepared by discontinuous sucrose gradient centrifugation.This specific fraction contained oxytocin receptors and oxytocin-inhibited calcium-stimulated and magnesium-dependent adenosine-triphosphatase ((Ca 2 + + Mg2+)-ATPase) activity.Membranes from myometria of rats treated with progesterone lacked both basal and oxytocin-inhibited (Ca 2+ + Mg2+)-ATPase activities.The oxytocin-inhibited enzyme also was found in plasma membranes from rat adipocytes, which are oxytocin target cells, but not from a nontarget tissue like duodenal smooth muscle.Half-maximal inhibition of (Ca 2 + + Mg2+)-ATPase activity in myometrial membranes occurred with about 1 nM oxytocin, corresponding to the apparent Kd of oxytocin-receptor interaction.Several synthetic oxytocin analogues inhibited myometrial (Ca 2 ' + Mg2+)-ATPase activity in proportion to their ability to stimulate uterine contractions.Oxytocin-inhibited (Ca 2 + + Mg 2 +)-ATPase in the myometrium had a Vm of about 0.2 mol/min/mg of protein, a K 0 .5 of about 50 ,tM Mg-ATP, and a Hill coefficient of 1.85.Maximal inhibition by oxytocin occurred with the lowest [Ca 2+ ] tested, 60 nM.Oxytocin only partially inhibited the enzyme at [Ca 2 +] of 5 plM or less, but complete inhibition was seen at higher [Ca2+].These results indicate that (Ca 2 + + Mg 2 +)-ATP activity in the myometrium is composed of more than one enzyme, only one of which is inhibited by oxytocin.Oxytocin-inhibited (Ca 2 + + Mg2+)-ATPase was not affected by [Na +] or [K + ] ranging from 10 to 200 m, suggesting that counter-ion transport is not necessary for activity.Oxytocin action may be regulated by calmodulin because 2 M trifluoperazine inhibited the effect of oxytocin on (Ca 2+ + Mg2+)-ATPase activity.These studies provide a basis for postulating a mechanism of induction of uterine contraction by enzymatic regulation of intracellular calcium concentrations in response to oxytocin-receptor interaction.Despite extensive studies on the characterization of oxytocin receptors in the rat myometrium (1), little is known of the molecular actions of oxytocin beyond its interaction with receptor sites.It is likely that Ca 2+ is necessary for coupling excitation and contraction of uterine smooth muscle (2), but the origin of Ca 2+ activating uterine contractions is unknown.Because the myometrial cell membrane maintains a large electrochemical gradient for ionized calcium between the cy-