Divergence in Murine Myometrium Spontaneous and Oxytocin-Stimulated Contractile Responses to Serine/Threonine Protein Phosphatase-1 Inhibition1

Abstract

Reversible phosphorylation is essential in regulating uterine contractions. Identification, characterization, and functional understanding of myometrium protein phosphatase(s) are lacking. Okadaic acid (OA), which inhibits protein phosphatase-1 (PP1) and PP2A, has been shown to alter uterine contractions. Experiments were conducted to determine the 1) identity of the myometrial OA-sensitive PP, 2) influence of OA on spontaneous and oxytocin (OT)-stimulated myometrial contractions, and 3) expression of uterine PPs during sexual development. Western blot analysis indicated the presence of PP1(alpha) and PP2A in immature and mature mice. As determined by immunohistochemistry, gonadotropin-stimulated adult mouse uteri contain PP1(alpha) in longitudinal and circular myometrial layers and endometrial epithelium. Conversely, PP2A was localized to the endometrial stroma. Cumulative addition of OA (n = 9; 10, 100, 250, 500, 1000 nM) did not significantly alter spontaneous contractions of mouse uterine horns in comparison to vehicle-treated controls (n = 9). By the end of the test period OA- and vehicle-treated uteri displayed a comparable decline in uterine contractions to 79.2% and 63.7%, respectively, of basal contractile activity. Pretreatment of uterine tissue with OA (1 microM; n = 7) significantly reduced contractile response to increasing concentrations of OT (8, 16, 32, 64 nM) in comparison to vehicle pretreatment (dimethyl sulfoxide; n = 7). At the end of the OT-administration period, contractile activity was 160.4% and 67.3% of basal contractile activity for vehicle (no OA) and OA-pretreated groups, respectively. During the early prepubertal period PP1(alpha) was expressed in longitudinal myometrium and absent in circular myometrium; whereas, during the transition to sexual maturity PP1(alpha) was observed in both the longitudinal and circular myometrium. In summary, these studies have indicated 1) that PP1 is the primary myometrial OA-sensitive PP; 2) that inhibition of PP1 had no effect on spontaneous contractions, whereas it markedly inhibited OT-stimulated uterine contractions; and 3) that PP1 is differentially expressed in the circular and longitudinal myometrium in relation to sexual development.

Expression and Intracellular Localization of Protein Phosphatases 2A and 2B, Protein Kinase A, A-Kinase Anchoring Protein (AKAP79), and Binding of the Regulatory (RII) Subunit of Protein Kinase A to AKAP79 in Human Myometrium

“…Band densities of PP2A were quantified and compared; no significant differences were found between the study groups ( immunohistochemistry that PP2A expression is localized to the endometrial stroma and the cytoplasm of endometrial epithelial cells but not the myometrium. 26 Similarly, in this study, PP2A expression was absent in the myometrial cytoplasm in all three study groups, yet present in the blood vessel endothelium (Figure 2). PP2A activity in rat uteri has been shown to be positively correlated to serum estradiol and progesterone ratios and appears to be under hormonal control.…”

Multiple Signals Regulate Phospholipase CBeta3 in Human Myometrial Cells1

“…Consistent with these observations, okadaic acid inhibited OXT-induced PI turnover, whereas cypermethrin had no effect. Although PRKCs have multiple effects on the contractile apparatus as well as on the signaling pathways, the fact that okadaic acid does not affect basal contractile activity, but significantly impairs the ability of OXT to induce mouse myometrial contraction [39], is at least consistent with a negative effect of PP1/PP2A on OXT action. PP1 regulates many cell functions, with substrate targeting and specificity determined by its interaction with different regulatory units [40,41].…”

Oxytocin-induced phasic and tonic contractions are modulated by the contractile machinery rather than the quantity of oxytocin receptor

“…The influences of gestation (27), phosphatase inhibition (41), and intracellular Ca 2ϩ concentration (30) have subsequently been reported. In this study, we analyzed the OT-induced phasic and tonic contractions in relation to the concentrations of OT to initiate the contractions, the pD 2 values, and maximum responses in nonpregnant and pregnant mouse myometrium.…”