Pregnancy-related modifications of rat myometrial Gs proteins: ADP ribosylation, immunoreactivity and gene expression studies

Abstract

Previous studies from our laboratory have suggested that post-receptor events at the level of beta-adrenergic receptor-adenylate cyclase interaction could be altered in myometrium by steroid hormones or pregnancy. In this study, we have addressed this question by performing a direct evaluation of rat myometrial Gs proteins at various stages of pregnancy or 24 h after administration of progesterone. In the 50,000 g myometrial plasma membrane fraction, in the presence of 32P-labelled NAD, cholera toxin ribosylated three predominant proteins with apparent molecular masses of 42, 47 and 55 kDa. Western blot analysis using the RM/1 antibody recognized the 42 and 47 kDa cholera toxin ADP-ribosylated bands but not the 55 kDa band. Thus, the 42 and 47 kDa immunoreactive bands were interpreted as being the small (Gs alpha-S) and large (Gs alpha-L) forms of Gs respectively. With a more purified myometrial plasma membrane fraction (105,000 g) an additional minor band of 44 kDa could be observed with both techniques. Treatment of late pregnant rats with 5 mg progesterone resulted in a significant increase in both Gs alpha subunits: +25% and +30% after ADP-ribosylation, +50% and +60% after Western blot analysis for Gs alpha-L and Gs alpha-S respectively. Pretreatment with the antiprogestin RU 486 completely suppressed the effect of progesterone, suggesting that the expression of Gs alpha subunits may be under the control of progesterone. However, changes in the myometrial content of Gs in progesterone-treated rats were not associated with concomitant variations in the steady-state levels of mRNA as demonstrated by Northern blot analysis. These data suggest a post-translational regulation of Gs expression by progesterone. Amounts of ADP-ribosylated Gs showed characteristic changes during the course of pregnancy with a fourfold or threefold increase (P < 0.05) on day 15 versus day 12 or delivery respectively. During pregnancy, or after progesterone administration, myometrial alterations of Gs strongly correlated (r = 0.913, P < 0.01) with the cholera toxin-stimulated adenylate cyclase activity. These findings provide evidence that changes in myometrial amounts of functional Gs i) are controlled by the hormonal status of pregnancy and progesterone and ii) play an important role in the transduction pattern of adenylate cyclase activity during the course of pregnancy.

Molecular Diversity of Adenylyl Cyclases in Human and Rat Myometrium

“…Furthermore, the reduced ability of guanine nucleotides or cholera toxin (acting at the G s level) to stimulate myometrial AC activity at term and the fact that Mn 2ϩ /GDP␤S (acting solely at the catalytic unit of the AC, 34) has no different regulatory effects, points to a defect in the availability or functionality of regulatory G s proteins rather than a reduction in the catalytic subunits of AC. This conclusion supports previous data of our laboratory establishing that amounts of cholera toxin-catalyzed ADP-ribosylated G s ␣ subunits diminished by 3-fold at delivery versus pregnancy (35). In addition, as shown by radioligand binding experiments, the reduced response to isoproterenol at term is not caused by changes in total ␤-AR number (28) but is the consequence of G s protein uncoupling (24).…”

“…At this latter period, no substantial modification in the amounts of specific types of AC transcripts as well as no alteration in the basal activity of the AC system have been observed. So, in the parturient rat myometrium, when the balance among G i3 , G i2 , and G s proteins was changed (35,37) we suggest the possibility that distinct patterns of responsiveness of the myometrial AC population may account for the switch between the two types of input (positive and negative) initiated by ␣ 2 -AR activation. Interestingly, a decline of immunodetected ␤ subunits of G proteins which is expected to reflect the status of the ␤␥ complexes (38) paralleled the 1.7-fold decrease of Gi 3 ␣ subunit in the late pregnant rat myometrium (37,38).…”

In vivo stimulation of the beta(2)-adrenergic pathway increases expression of the Gi proteins and the alpha(2)A-adrenergic receptor genes in the pregnant rat myometrium

“…At the end of pregnancy, the stimulabilities of the cAMP generating system by isoproterenol (Litime et al 1989, Cohen-Tannoudji et al 1991) and 2 agonists decline. Concomitantly, a reduction in the myometrial levels of ADP-ribosylated G s and G i3 as well as in the 2A -AR subtype expression is observed (Elwardy-Mérézak et al 1994.…”

“…Preparation of myometrial membrane fractions, SDS-PAGE and immunoblotting with AS/7 (anti-G i 2 /G i 1 ) or EC/2 (anti-G i 3 /G o ) antisera were performed according to the methods previously described (Elwardy-Mérézak et al 1994. Antibody complexes were detected using a chemiluminescent method (ECL; Amersham Corp.) and quantified by scanning densitometry on a GS300 densitometer (Hoefer Scientific Instruments, San Francisco, CA, USA).…”

Pregnancy-induced decrease in the relaxant effect of terbutaline in the late-pregnant rat myometrium: role of G-protein activation and progesterone

“…On the other hand, progesterone treatment inverted the dose-dependent decrease in the amount of activated G-protein of b 2 -ARs by terbutaline on day 22. Earlier findings suggested that sex hormones play a role in the regulation of G-proteins in the myometrium (Elwardy-Merezak et al 1994, Cohen-Tannoudji et al 1995. It was also revealed that an estrogen predominance decreases the b-AR-mediated Gs-proteins and the cAMP level and progesterone treatment increases the number of G-protein coupled receptors (Riemer et al 1988, Nimmo et al 1995.…”