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 + + 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 + + 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 + + Mg +)-ATPase in the myometrium had a Vm of about 0.2 mol/min/mg of protein, a K0.5 of about 50 ,tM Mg-ATP, and a Hill coefficient of 1.85. Maximal inhibition by oxytocin occurred with the lowest [Ca2+ ] tested, 60 nM. Oxytocin only partially inhibited the enzyme at [Ca2 +] of 5 plM or less, but complete inhibition was seen at higher [Ca2+]. These results indicate that (Ca2 + + Mg2+)-ATP activity in the myometrium is composed of more than one enzyme, only one of which is inhibited by oxytocin. Oxytocin-inhibited (Ca2 + + 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 (Ca2+ + 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.