The long-range goal of the research described in this project is to identify and characterize the pregnancy-induced biochemical modifications of myometrial cells that contribute to the maintenance of uterine quiescence during 95% of pregnancy (phase O of parturition). The intracellular concentration of free Ca2+ ({Ca2+]i) and the phosphorylation/dephosphorylation state of myosin light chain are the principal determinants of smooth muscle contraction. We will evaluate pregnancy-induced modifications of these fundamental processes, which govern uterine smooth muscle contraction/relaxation (viz., [Ca2+]i, Ca2+ - signal processing, and myosin light chain phosphorylation). The rate and extent of myosin light chain phosphorylation during contraction of myometrial tissue obtained from pregnant women (compared with that of myometrial tissue from nonpregnant women)is markedly attenuated; and, the [Ca2+]i in myometrial smooth muscle cells isolated from uterine tissues of pregnant women is low. We will investigate the possibility that there are alterations in the regulation of myometrial cell Ca2+ homeostasis during pregnancy that serve to decrease the availability of free cytoplasmic Ca2= and thereby attenuate the Ca2+/calmodulin-dependent phosphorylation of myosin light chain. We will quantify cytoplasmic free calcium in strips of myometrial tissue and in myometrial smooth muscle cells from nonpregnant women and from pregnant women at various stages of gestation before and after the onset of labor. We will ascertain whether myosin light chain kinase (MLCK), the enzyme that catalyzes the phosphorylation of myosin light chain, is phosphorylated and thereby desensitized to Ca2+ activation in the myometrium of pregnant women. Such a phenomenon could constitute yet another mechanism by which phosphorylation of myosin light chain is attenuated. We will investigate three cellular processes that may serve to limit the availability of free cytoplasmic Ca2+: (o) Ca2+ extrusion by the plasma membrane, (ii) Ca2+ sequestration by the sarcoplasmic reticulum, and (iii) Ca2+ buffering by cytoplasmic Ca2+ pumps (Ca2+ pumps (Ca2+-ATPases, i.e., number and function) facilitate extrusion and sequestration of cytoplasmic Ca2+ in myometrium of pregnant women. Calbindins may act to modify cellular responses to cytoplasmic Ca2+ by modifications in Ca2+ by modifications in Ca2+ signal transduction or by way of the Ca2+=buffering capacity of these proteins. We will assess the functional consequences of increased/decreased levels of calbindin-9K (induced in vitro by hormone treatment) in human myometrial cells in culture. We will investigate the role of estrogen and progesterone in effecting modifications in Ca2+ homeostasis and MLCK phosphorylation. Knowledge of the mechanisms by which myometrial cell refractoriness to contraction is effected is crucial to an understanding of phase O of parturition and thence the transitions of the uterus to phases 1 and 2.
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