The long term objective of the present proposal is to define the physiological role of myometrial ionic channels in relation to the excitability and contractile function of the uterus. We propose that ionic channels expression and their regulatory mechanism(s) are influenced by hormones. Single cells freshly dissociated or maintained in tissue culture and purified plasmalemma membrane fractions from uterine smooth muscle will be used. Freshly dissociated cells will reflect the hormonal condition of the animal while primary culture will be used as a model to study the action of hormones. Macroscopic currents will be explored using both the whole cell patch clamp and the intracellular microelectrode voltage clamp techniques. Single channels will be made in conjunction with binding studies of adrenergic and dihydropyridine ligands to determine changes in number or type(s) of channels or receptors related to the hormonal conditions. In initial experiments macroscopic currents will be characterized in freshly dissociated cells from animals at diestrus. Our study will be mainly focused on voltage dependent K, Cl and Ca channels (voltage dependency, kinetics, pharmacology and regulation). Parallel studies will be performed on single channel in intact cells and in bilayers. Furthermore, the affinity and number of sites for adrenergic and dihydropyridine receptors will be measured. To test the hypothesis that the expression of ionic channels and their regulatory mechanism(s) may be affected by hormones, the next procedures will be followed: I. Animals at estrus and diestrus phases of the estrus cycle will be comparatively studied. Acutely dissociated cells and membrane vesicles incorporated into bilayers will be used. The characteristics of membrane K, Ca and Cl currents and the corresponding single channels will be analyzed. To explain the differences in activity of the uterus during these phases, the actions of adrenergic agents, oxytocin and prostaglandins on Ca and K channels and their mechanism(s) will be studied. These studies will be correlated with binding studies of adrenergic and dihydropyridine ligands; II. Hormonal changes will be induced in animals or in cultured cells. Ionic channels will be studied in acutely dissociated cells from: a) spayed animals treated with estradiol or progesterone; and b) in cultured cells from animals at estrus treated with estradiol or progesterone. Ionic channels from rats at mid- and late- pregnancy will be studied following the same experimental plan as for cycling animals. The knowledge of the functional role and regulation of ionic channels will allow a comprehension of the excitability of the uterus in relation to hormonal effects. This will be important in the medical sciences to determine the role of hormones in the reproductive cycle and to design or improve therapeutic treatment(s) for pathological situations such as premature labor and dysmenorrhea.
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