Development of a rat myometrial cell culture system to study hormonal and toxicant effects on gap junctional communication.

Abstract

Disorders of gestational length result in increased perinatal mortality and morbidity yet mechanisms regulating labor onset and toxicant effects on this process are not clear. Gap junctions (GJs), which are intercellular channels allowing diffusion of small molecules and ions between adjacent cells, increase in the myometrium immediately prior to labor onset. GJs are believed to function in parturition by providing a low-resistance pathway for signal propagation, producing synchronized uterine contractions. This research established an in vitro rat uterine smooth muscle cell system for investigating gap junctional communication (GJC). If functional GJs are critical for parturition, then hormones and reproductive toxicants known to alter labor onset or progression may do so by modulating myometrial GJC. Initially, rat myometrial cells were isolated from pregnant, midgestation (RMC-P cells) Sprague-Dawley rats. These cells had stable GJC levels under a variety of culture conditions. In these cells, 2-methoxyethanol (2-ME), a reproductive toxicant in rodents in vivo, decreased GJC through a mechanism which may involve increased membrane fluidity. Pretreatment of RMC-P cells with cholesterol, a membrane stabilizing agent, alleviated the changes seen with 2-ME alone. Although the mechanism of GJC inhibition was interesting, it is unlikely to be the sole mechanism whereby 2-ME alters labor onset, because effective doses of 2-ME in vitro were higher than those altering gestational length in vivo. To more closely mimic the in vivo situation, myometrial cell cultures (RMC-I) were obtained from immature Sprague-Dawley rats that maintained estrogen receptors (ERs) in vitro. These RMC-I cells responded as predicted to in vitro progesterone treatment by decreasing ERs; however, progesterone treatment produced only limited, although statistically significant, inhibition of GJC. Additional experiments showed that arachidonic acid, prostaglandin (PG) E$\sb2$, and PGF$\sb{2\alpha}$ were all inhibitory to GJC in RMC-I cell cultures. Although differing in some ways from the in vivo situation, in vitro test systems described in this work lend themselves to more carefully controlled studies of mechanisms regulating GJ function in the myometrium.