Role of ATP -sensitive potassium channels in nitric oxide and biological thiol modulation of myometrial plasma membrane potential and uterine contractility.

Abstract

During a normal pregnancy, a well orchestrated series of biochemical mechanisms maintain uterine tone while preventing the propagation of forceful, coordinated uterine contractions. Nitric oxide (NO) and activators of ATP-sensitive potassium (KATP) channels inhibit spontaneous rat and human myometrial contractions during pregnancy. The mechanisms responsible for NO-induced relaxation of uterine smooth muscle are only partially understood. Several reports suggest that NO activates potassium channels, including K_ATP channels, resulting in inhibition of smooth muscle contraction. <italic>In vitro</italic> application of spermine NONOate, a NO generating compound, to late gestation (day 20) rat uterine strips inhibited spontaneous contractions. Spermine NONOates's inhibition of was blocked by glibenclamide, a K_ATP channel blocker. This is the first report that inhibition of late gestation rat uterine contractions by exogenous NO may be mediated K_ATP channel activation. Functional activity of K_ATP channels was measured in myometrial cells cultured from late gestation uteri by evaluating membrane potential using the bis-oxonol dye DIBAC₄(3) and confocal microscopy. As indicated by a decrease in fluorescence, the K_ATP channel opener pinacidil hyperpolarized myometrial cells and this decrease in fluorescence was blocked by glibenclamide. Spermine NONOate also hyperpolarized myometrial cells and glibenclamide pretreatment blocked this response, indicating a role for K_ATP channels in NO-induced hyperpolarization of myometrial plasma membrane potential. Glibenclamide itself caused a significant depolarization suggesting that K_ATP channels contribute to the resting membrane potential of myometrial cells. The biological thiols L-cysteine and homocysteine inhibited pinacidil-induced plasma membrane hyperpolarization of myometrial cells and relaxation of uterine strips. L-Cysteine and homocysteine alone significantly stimulated uterine contractility. In the case of homocysteine, pinacidil inhibited homocysteine's stimulation of uterine contraction frequency, supporting a role for K_ATP channels in homocysteine's effects on uterine contractility. Both thiols suppressed L-arginine-induced increase of NO in myometrial cells, as measured by DAF-FM fluorescence changes, and blocked L-arginine-stimulated relaxation of uterine strips, suggesting that L-cysteine and homocysteine may interfere with NO-mediated relaxation of pregnant uterus by inhibiting myometrial NO synthase. In summary, K_ATP channel activity, NO, and biological thiols may modulate quiescence of the pregnant uterus <italic>in vivo</italic>.