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We investigated whether beta-adrenergic receptors (beta-AR) regulate the phospholipase C (PLC) system in midpregnant rat myometrium. PLCbeta isoforms were characterized, and the effect of isoproterenol (beta-adrenergic agonist) was tested on myometrial inositol phosphate (InsP) production and uterine contraction. Using specific antibodies, we showed that rat myometrium expresses PLCbeta1, PLCbeta3, and PLCbeta4, and to a lesser degree PLCbeta2. Quantitative analysis revealed that PLCbeta isoforms are differentially expressed during pregnancy. Indeed, the amount of PLCbeta4 is increased at midpregnancy, whereas PLCbeta1, PLCbeta2, and PLCbeta3 are up-regulated at term. At midpregnancy, pretreatment of myometrial strips with isoproterenol significantly reduced basal and agonist-stimulated InsP production. Forskolin, a diterpene that increases cAMP accumulation by directly activating adenylyl cyclases, had no effect on InsP production. In contrast, two global potassium (K+) channel inhibitors, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), prevented attenuation of InsP production by isoproterenol. Isoproterenol also significantly decreased spontaneous and agonist-induced contraction of the longitudinal layer of midpregnant rat myometrium. Preincubation of uterine strips with TEA plus 4-AP prior to beta-AR activation blocked only partial uterine relaxation, whereas Forskolin was as potent as isoproterenol. This indicates that beta-AR operate through both K+ channels and cAMP to induce uterine relaxation. In conclusion, we show for the first time that three myometrial PLCbeta isoforms (PLCbeta1, PLCbeta2, and PLCbeta3) are down-regulated at midpregnancy. At this period, beta-AR reduce basal and agonist-stimulated InsP production through activation of K+ channels. Altogether, these mechanisms could act to decrease responsiveness of the longitudinal layer of myometrium to contractant factors.

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.

Pharmacological characterization of alpha-2 adrenoceptors of the pregnant rat myometrium was assessed using the ability of various alpha adrenoceptor agonists and antagonists to inhibit [3H]rauwolscine or [3H]idazoxan binding to myometrial 50,000 x g fraction or to slide-mounted sections of the whole pregnant uterus. Saturation binding studies with both radioligands showed that the number of myometrial alpha-2 adrenoceptors is greatly increased on days 10 to 12 of pregnancy vs. cyclic rats. It then decreased from midpregnancy to term (about -75%; P < .01) with no change of the equilibrium dissociation constant (between 7-11 nM). Chemical sympathectomy by 6-hydroxydopamine significantly decreased (P < .01) the density of alpha-2 adrenoceptors at days 8 and 12 of pregnancy. Later, 6-hydroxydopamine administration did not alter Bmax or Kd values suggesting that the pregnancy decrease of alpha-2 adrenoceptors may be related to a loss of presynaptic receptors. In order to identify myometrial postsynaptic alpha-2 adrenoceptor subtypes, the inhibition of [3H]rauwolscine or [3H]idazoxan binding by oxymetazoline, prazosin and chlorpromazine was studied on days 20 and 21 of pregnancy. All inhibition curves were consistent with a model of two classes of binding sites: about 55% of the myometrial alpha-2 adrenoceptors, which had a higher affinity for oxymetazoline, may represent the alpha-2A subtype whereas the other 45% of the sites, which had a higher affinity for prazosin and chlorpromazine, may represent the alpha-2B subtype. Autoradiographic studies using [3H]rauwolscine revealed that both subtypes are colocalized in the longitudinal muscle. A high density of alpha-2A and alpha-2B subtypes was also detected in the chorioallantoic and yolk sac placenta and in the embryonic nervous system.

The effects of pregnancy or progesterone dominance on the beta-adrenergic responsiveness of the uterus were studied in myometrial membranes from mid- and late-pregnant rats (day 15 and on the 16th h of day 22 of pregnancy respectively) or 24 h after administration of progesterone. Levels of the high (RH)- and low (RL)-affinity states of the beta-adrenergic receptor were determined by competition experiments between 125I-labelled cyanopindolol binding and the selective beta-agonist isoproterenol. The ratio KL/KH (respective dissociation constants) was determined since it also reflects the degree of formation of the high-affinity state of the beta-adrenergic receptor. From day 15 to the 10th h of day 22 of pregnancy, two distinct affinity states were apparent: 80-55% RH (KH = 0.31-0.21 microM) and 45-20% RL (KL = 14-5 microM) with a ratio of KL/KH of 55-34. In the last 6 h before birth, beta-adrenergic receptors underwent uncoupling which was paralleled by decreased responsiveness of myometrial adenylate cyclase to isoproterenol (maximum velocity (Vmax) = 17 +/- 3 vs 44 +/- 3 fmol cyclic AMP/10 min per mg protein on day 15). At this stage of pregnancy, previous exposure to progesterone resulted in a 1.8-fold increase in 125I-labelled cyanopindolol-binding sites (Bmax) and the reappearance of the high-affinity state (67% RH, KH = 0.19 +/- 0.04 (S.E.M.) microM, ratio KL/KH = 81.1 +/- 16.9).(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor autoradiography using (-)-3-[125I]cyanopindolol and [3H]prazosin was used to study the distribution of beta and alpha-1 adrenoceptors in the rat uterus at early and midpregnancy. The binding of [3H]prazosin to slide-mounted sections at 25 degrees C was time dependent (K1 = 3.01 x 10(7) M-1 min-1, K2 = -0.0116 min-1) and saturable (1 nM). Competition binding curves with the selective alpha-1 and alpha-2 antagonists (prazosin, yohimbine) or alpha-1 and alpha-2 agonists (phenylephrine, clonidine) showed the presence of alpha-1 adrenoceptors; autoradiographic studies revealed that this subtype is highly localized in the circular layer of the myometrium during pregnancy. (-)-3-[125I]Cyanopindolol binding to slide-mounted sections of the pregnant uterus at 25 degrees C was time dependent (K1 = 4.68 x 10(8) M-1 min-1, K2 = -0.0117 min-1) and saturable (200 pM). Competition binding curves with beta-1 or beta-2 selective agonists (dobutamine, metaproterenol) and antagonists (atenolol, ICI 118,551) revealed the presence of beta adrenoceptors in the proportion of 67% beta-2 to 33% beta-1. Hyperfilm exposed to sections of the whole pregnant uterus incubated with (-)-3-[125I]cyanopindolol with or without ICI 118,551 or atenolol showed a high density of beta-2 adrenoceptors in the longitudinal layer of the myometrium and in the placenta. A small density of beta-2 adrenoceptors was also located in the decidua basalis on day 8 of pregnancy.

Testosterone (T), dihydrotestosterone (DHT), androstenedione (delta 4) and dehydroepiandrosterone (DHA) were measured by radioimmunoassay in peripheral plasma, uterine vein plasma, ovary and placentae within the last 3-4 days of pregnancy in Sprague-Dawley rats. In the uterine vein, T and delta 4 levels were significantly higher than those reported in the peripheral plasma at any particular day of late pregnancy. In peripheral plasma as in uterine vein plasma delta 4 and T gradually decreased from day 20 to term (-63% for delta 4 and approximately -45% for T) whereas DHT concentrations were unchanged. This decline is concomitant with a similar pattern of change in the placentae; indeed placental concentrations of delta 4 and T decreased significantly from day 19 to 22 (-63 and -79%, respectively) and were positively correlated with the levels of both androgens in uterine venous and peripheral plasma. Thus it was concluded that in the late phase of pregnancy in the rat circulating delta 4 and T originate from placentae. In contrast, ovarian androgen concentrations showed a sharp increase from day 21 to term: 58% for DHA, 49% for delta 4, 87% for T and 24% for DHT. It is suggested that these increasing concentrations of ovarian androgens could be related to the next ovulatory cycle.

The catecholamines adrenaline (A) and noradrenaline (NA) were determined in plasma samples of late pregnant rats and rat foetuses using a radioenzymatic procedure. In maternal plasma of control rats, values of A were stable during the last three days of gestation (between 5.64 and 6.90 ng/ml) whereas NA values increased just before parturition (+ 70%) and on day 23 post-coitum NA was the main plasma catecholamine. In foetal plasma of control rats, values of A and NA were constant during the last three days of gestation and A was always higher than NA. For both catecholamines, values in foetal plasma were lower than in maternal plasma. Blood of rats injected with isotonic saline into the uterus on day 21 of gestation contained 24 h and 48 h later about 2-3 times more A, and no more NA, than blood of control rats. In foetal plasma the levels of both catecholamines especially of NA were greatly increased after the handling of the uterus or the intra-uterine isotonic saline injection. Both treatments had the same prolonged effect upon A and NA values in foetal plasma. There is no increase of A and NA in maternal and foetal plasma after sham-operation. Although the values of plasma A and NA were high in control rats because of blood sampling was carried out under stressful conditions, these results showed that handling of the uterus or injection of intrauterine isotonic saline was a severe aggression for the mother and foetus.

In the early pregnant rat, electrical activity of the myometrium consisted of regular bursts of spike potential, which appeared well propagated on Day 2 of pregnancy. During Day 3, there was a gradual disappearance of propagated activity. Concomitantly, there was a 7-fold increase (P less than 0.001) of uterine progesterone concentrations. At this stage, mean duration of bursts was 15.2 +/- 0.9 sec and intervals of complete quiescence between bursts were 84.2 +/- 7.0 sec. At 10:00 h on Day 4, there were peaks in the uterine concentrations of oestradiol and progesterone, +36% and +654%, respectively, compared with values on Day 2 (P less than 0.05). Between 10:00 and 20:00 h on Day 4, EMG activity exhibited a rapid and transient rise: bursts were of longer duration at the utero-tubal end of the horn (+60%, P less than 0.05) with an increased amplitude of spike potentials (+67% and +90% respectively at the tubal and cervical ends of the uterus, P less than 0.05). The administration of prazosin depressed EMG activity reversibly in a dose-dependent manner with maximal inhibition at about 2-3 h later. It is concluded that the changes observed during EMG recordings are relevant to the intrauterine distribution of blastocysts and related to changes in the steroidal environment and/or to catecholamine effects via alpha 1-adrenoceptors.

The myometrium of the rat has been found to contain both alpha 1- and beta-adrenergic receptors. To investigate the implication of these adrenergic receptors in uterine reactivity near term delivery, we have measured the number and the affinity of alpha 1-adrenergic antagonist [( 3H]prazosin: [3H]PRAZ)-binding sites and of very high affinity beta 2-adrenergic agonist [( 3H]hydroxybenzylisoproterenol: [3H]HBI)-binding sites in myometrial membranes throughout the last 5 days of pregnancy and at delivery. The number of specific binding sites was constant from Day 18 of pregnancy up to 6 h prior to birth. In the last 6 h of pregnancy, there was a sharp increase in the number of alpha 1-receptors (+70%, p less than 0.05). Simultaneously, the number of beta 2-receptors coupled to the adenylate cyclase system dropped (-75%; p less than 0.001). These results indicate that with the approach of parturition, there is a regulation of uterine reactivity by a modulation of the concentrations of myometrial adrenergic receptors during the last 6 h of gestation.

Rat myometrium exhibited a marked decrease in the concentration of beta 2-adrenergic receptors immediately before parturition, i.e., in the last 6 h of pregnancy. This phenomenon continued until the withdrawal of myometrial progesterone (-94% from Day 18 of pregnancy to term) and coincided with the sharp increase (+200%) of the myometrial concentration of estradiol. A linear positive correlation was found (r2 = 0.645) between the concentration of beta 2-adrenergic receptors and the log ratio of myometrial concentration of progesterone/myometrial concentration of estradiol (P/E2), suggesting a modulation of beta 2-adrenergic receptors by steroids. In rats with estrogen-dominated uteri (intact of ovariectomized late pregnant rats injected with estradiol), there was no change either in concentration or affinity of beta 2-adrenergic receptors relative to untreated control pregnant rats. In contrast, rats with progesterone-dominated uteri (intact or ovariectomized late pregnant rats treated with progesterone or ovariectomized rats) have an increased number of beta 2-adrenergic receptors, with a decreased affinity of these receptors compared to untreated control pregnant rats or to estrogen-treated rats. These results suggest that progesterone regulates the number of beta 2-adrenergic receptors in myometrium of late pregnant rats. The mechanisms by which progesterone exerts this regulation remains to be elucidated.