Changes in rat myometrial plasma membrane protein kinase A are confined to parturition

Abstract

We have previously shown that pregnant rat myometrial plasma membrane-associated cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) decreases prior to delivery, coincident with a decline in the inhibitory effect of cAMP on contractant-stimulated parameters. We now find that rat myometrial membrane-associated PKA concentrations in early to mid-pregnancy are equivalent to those in cycling rats. Following the decline associated with parturition, membrane PKA recovers within 1 to 2 days postpartum. Treatment with the antiprogestin onapristone caused a decrease in myometrial membrane PKA catalytic and regulatory subunits compared to untreated controls by 12 hours. This coincided temporally with recently reported increases in electrical and contractile activity. In unilaterally pregnant rats, the decline in plasma membrane PKA was observed in both nonpregnant and pregnant horns but was more rapid in the pregnant horns. These data indicate that the myometrial plasma membrane PKA pattern before and during most of pregnancy is not consistent with progesterone exerting a primary influence on PKA membrane localization. Rather, the fall in membrane PKA associated with parturition may contribute to or be influenced by the increased contractile and electrical activity of labor that is a consequence of the loss of progesterone influence and is not absolutely dependent on the presence of fetuses.

Figure 1

Expression of membrane PKA-catalytic subunit in cycling and pregnant rat myometrial plasma membranes. Individual measurements were normalized to G-beta protein in the same blot as loading control, and data were expressed relative to day 19 of pregnancy (solid circles). There were no detectable changes in G-beta/μg protein over this interval. Data represent mean ± SEM of data from 2–6 animals. The black bar represents the period over which delivery occurred. Significance by ANOVA analysis of log-transformed data between day 16 and days 19, 21, 22 and 23 are indicated (*P<0.05, **P<0.01, *** P<0.001). The plasma progesterone concentrations during the rat estrus cycle and during rat pregnancy (open triangles), as reported by Smith et al. and by Pepe and Rothchild, respectively, are inserted in the figure for comparison.

Figure 2

A progressive decline in rat myometrial plasma membrane (A) PKA-catalytic (PKA-cat) (diamonds) and (B) regulatory (PKA-RII) subunit (squares) concentrations occurred following administration of the antiprogestin onapristone (3mg) on day 16 of gestation (indicated by the arrow), where the day the vaginal plug was observed was defined as day 0. Closed symbols represent onapristone treatment and open symbols represent vehicle controls. Individual data points were normalized to caveolin in the same blots as loading control; there were no detectable changes in caveolin/μg protein between samples in this study. Data were expressed relative to the 6 hour vehicle control; significant differences (P<0.05) between groups are designated in the text. Superimposed on these data are the changes in contractile activity (IUP intensity) (open triangles) and electrical activity (EMG energy) (open circles) in instrumented rats injected with the same concentration of onapristone. The dating for Shi’s study has been adjusted to correspond with the convention used by our supplier (day of vaginal plug = day 0). Corresponding Western blots for PKA regulatory and catalytic PKA subunits and caveolin for one set of samples are shown above the respective graphs. The bands for PKA RII (52 kDa) and catalytic (40 kDa) subunits and caveolin (22 kDa) were the expected sizes.

Figure 3

Myometrial rat plasma membrane PKA catalytic (PKA-cat) in the nongravid (black bars) and gravid (grey bars) horns of unilaterally pregnant rats, measured between day 16 and day 21 of pregnancy. Individual data points were normalized to caveolin in the same blot as loading control and expressed relative to the value in the day 16 nonpregnant horn (mean ± SEM, n=3). Significant differences (P<0.05) between groups are designated with different lowercase letters. Western blots for one set of samples are shown above the graph.