Increased potency of alpha 1-adrenergic receptors to induce inositol phosphates production correlates with the up-regulation of alpha 1d/Gh alpha/phospholipase C delta 1 signaling pathway in term rat myometrium.

In the present study, we studied the potential regulation by rat myometrial alpha1-adrenergic receptors (alpha1-AR) of the newly identified Gh alpha protein/phospholipase C delta 1 (PLC delta 1) signaling pathway and compared myometrial inositol phosphates (InsP) production and activity of the uterine circular muscle in response to alpha1-AR activation between mid-pregnancy and term. For this, we quantified the level of rat myometrial alpha1-AR coupling to Gh alpha protein by photoaffinity-labeling, the cytosolic amount of PLC delta 1 enzyme by immunoblotting, and the expression level of alpha1-AR subtypes by RT-PCR. The results showed an increased level of alpha1-AR/Gh alpha protein coupling and the amount of PLC delta 1 at term (+147 and +65% respectively, versus mid-pregnancy). This was correlated with an up-regulation of alpha 1d-AR subtype (+70% versus mid-pregnancy). Incubation of myometrial strips with phenylephrine (Phe), a global alpha1-agonist, increased InsP production in a dose-dependent manner at both mid-pregnancy and term, but with an enhanced potency (tenfold decrease in EC(50) value) at term. Phe also dose-dependently induced contraction of the circular muscle at both mid-pregnancy and term. However, unlike InsP response, no amelioration of potency was observed at term. Similar results were obtained with the endogenous agonist norepinephrine. Our results show, for the first time, that rat myometrial alpha 1d-AR/Gh alpha/PLC delta 1 signaling pathway is up-regulated at term. This is associated with an increased potency of alpha1-AR to elicit InsP production but not uterine contraction at this period. It is thus hypothesized that alpha1-AR, through activation of Gh alpha/PLC delta 1 system, are not primarily involved in the initiation of labor but may rather regulate responses such as myometrial cell proliferation or hypertrophy.

Up-regulation of rat myometrial phospholipases C{beta}1 and C{beta}3 correlates with increased term sensitivity to carbachol and oxytocin.

In the present study, we compared rat uterine contractility and myometrial inositol phosphate (InsP) production in response to activation of muscarinic and oxytocin receptors during pregnancy and at term. The level of myometrial phospholipase (PL) Cbeta was also determined by Western blotting at different stages of pregnancy and following administration of oestradiol, progesterone or vehicle. The results showed an increased potency of carbachol (CCh), a cholinergic muscarinic agonist, and oxytocin (OT) to enhance myometrial InsP production at term. This correlated with an increased potency of both agonists to induce contraction of the circular but not the longitudinal muscle. For both InsP production and contractile activity, the maximal response of CCh was unaltered, while that of OT was significantly increased. Interestingly, the increased responsiveness to CCh and OT was associated with an up-regulation of PLCbeta1 and PLCbeta3 enzymes. Such regulation is under the control of oestradiol since administration of this steroid to pregnant rats increased the amount of both enzymes by 200-260%. In contrast, progesterone administration was without effect. The present study presents the first evidence that the expression of rat myometrial PLCbeta1 and PLCbeta3 is under the positive control of oestradiol. This could participate in the enhancement of myometrial InsP accumulation and uterine contraction at term in response to CCh and OT. Based on contraction studies, we also propose that the longitudinal and circular uterine muscles differ in the regulation of the PLC pathway during pregnancy.

A functional genomic study to identify differential gene expression in the preterm and term human myometrium.

The mechanisms that lead to the onset of human parturition are still unknown, although selected critical factors have been identified. To investigate the changes in myometrial gene expression associated with parturition, we used two macroarrays each containing 1176 different complementary human cDNA clones. Methods involving hierarchical clustering and conventional statistical analysis allowed us to generate a profile of genes expression at three stages of late pregnancy: preterm (29 wk amenorrhea); full term, not in labor (38 wk amenorrhea); and full term in labor (39 wk amenorrhea). Only 4% of the genes investigated were differentially expressed between the preterm and term groups (P < 0.05). These genes could be clustered as groups of either down-regulated or up-regulated transcripts. The changes in transcript abundance were particularly marked between the preterm and term stages of gestation, whereas the differences between term not in labor and term in labor were less pronounced. The parturition was characterized by a massive down-regulation of a large panel of developmental, cell adhesion molecule and proliferation-related genes, along with the up-regulation of inflammatory, contraction and apoptosis associated genes. We propose that the mechanisms of parturition consist primarily in the arrest of the processes of myometrial development, a step that might be essential to allow the uterus to recover appropriate contractile function before delivery.

Catecholamines are not linked to myometrial phospholipase C and uterine contraction in late pregnant and parturient mouse.

1. We investigated whether catecholamines through activation of alpha(1)-adrenergic receptors (alpha(1)-AR) are involved in mouse uterine contraction at parturition. Myometrial phospholipase C (PLC) activity and uterine contraction were measured in response to noradrenaline (NA), the specific alpha(1)-AR agonist phenylephrine (Phe) and oxytocin (OT). 2. Using the reverse transcription-polymerase chain reaction RT-PCR, we detected the alpha(1a)-AR subtype in late pregnant mouse myometrium. We also detected, by immunoblotting studies, PLCbeta(1), PLCbeta(3) and different alpha-subunits of pertussis toxin-insensitive (Galpha(q/11)) and -sensitive G proteins (Galpha(o/i3), Galpha(i1/2)). 3. Phenylephrine and NA did not alter the myometrial inositol phosphate (InsP) production of late pregnant or parturient mouse. In similar conditions, OT increased InsP production in a dose-dependent manner. Consistent with these results, only OT (10 microM) recruited PLCbeta(1) and PLCbeta(3) to myometrial plasma membranes. The OT-induced InsP response was not altered by pertussis toxin (300 ng ml(-1), 2 h pretreatment), suggesting the involvement of a member of the Galpha(q) family. 4. Noradrenaline and Phe failed to increase uterine contraction at late pregnancy and at parturition. In contrast, OT induced uterine contraction in a dose-dependent manner with maximal increase (400 %) at a concentration of 1 microM. 5. The results indicate that OT receptors (OTR) but not alpha(1)-AR are linked to myometrial PLC activation and uterine contraction in late pregnant and parturient mouse. This discrepancy between mouse and other mammals could be attributed to the alpha(1)-AR subtype expressed in myometrium at this time.

Concomitant increase of G protein-coupled receptor kinase activity and uncoupling of beta-adrenergic receptors in rat myometrium at parturition.

The myometrial beta-adrenergic receptor (beta-AR)-adenylyl cyclase pathway is markedly desensitized at the end of pregnancy in the rat. We have investigated whether changes in the amount and/or the activity of G protein-coupled receptor kinase (GRK) occurred at the same period of pregnancy. Using Northern and Western blotting, we have identified GRK2, GRK5, GRK6, and a small amount of GRK3 in late pregnant rat myometrium. GRK activity, as measured by in vitro phosphorylation of rhodopsin, was detected in both cytosolic and plasma membrane fractions. Interestingly, in the 6-10 h preceding parturition, there was a substantial increase (+190%) of myometrial membrane-associated GRK activity. This was associated with an increase in membrane GRK2 immunoreactivity. Such alterations occurred concomitantly with uncoupling of beta-AR, as assessed by quantification of high-affinity binding receptors. These data suggest that GRK activity increase may be one of the mechanisms underlying alterations in the coupling between beta-AR and adenylyl cyclase and may thus contribute to the initiation of myometrial contractions at term.

Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation.

We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the alpha(1b)-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the alpha(1b)-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the alpha(1b)-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type alpha(1b)-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the alpha(1b)-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.

Constitutively active alpha-1b adrenergic receptor mutants display different phosphorylation and internalization features.

We compared the phosphorylation and internalization properties of constitutively active alpha-1b adrenergic receptor (AR) mutants carrying mutations in two distant receptor domains, i.e., at A293 in the distal part of the third intracellular loop and at D142 of the DRY motif lying at the end of the third transmembrane domain. For the A293E and A293I mutants the levels of agonist-independent phosphorylation were 150% and 50% higher than those of the wild-type alpha-1b AR, respectively. On the other hand, for the constitutively active D142A and D142T mutants, the basal levels of phosphorylation were similar to those of the wild-type alpha-1b AR and did not appear to be further stimulated by epinephrine. Overexpression of the guanyl nucleotide binding regulatory protein-coupled receptor kinase GRK2 further increases the basal phosphorylation of the A293E mutant, but not that of D142A mutant. Both the wild-type alpha-1b AR and the A293E mutant could undergo beta-arrestin-mediated internalization. The epinephrine-induced internalization of the constitutively active A293E mutant was significantly higher than that of the wild-type alpha-1b AR. In contrast, the D142A mutant was impaired in its ability to interact with beta-arrestin and to undergo agonist-induced internalization. Interestingly, a double mutant A293E/D142A retained very high constitutive activity and regulatory properties of both the A293E and D142A receptors. These findings demonstrate that two constitutively activating mutations occurring in distant receptor domains of the alpha-1b AR have divergent effects on the regulatory properties of the receptor.

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.

Cross-regulations between Gs and Gi mediated pathways controlling the adenylyl cyclase activity have been clearly demonstrated in vitro. To elucidate whether activation of the beta-adrenergic pathway in the pregnant myometrium might affect Gi proteins and alpha(2)-adrenergic receptors (ARs), we treated late pregnant rats from day 18 to day 21 with twice-daily administration of isoproterenol (8 mg/kg). This treatment increased myometrial cAMP levels and led after 76 h to a significant and maximal rise in the immunoreactive amount of myometrial Gi alpha 2 and Gi alpha 3 proteins (1.4- and 1.7-fold respectively) associated with a parallel increase of the steady-state levels of both Gi alpha 2 and Gi alpha 3 mRNA (1.6- and 1.9-fold respectively). Propranolol antagonized this response indicating the implication of the beta-adrenergic pathway. Nuclear run-on assays demonstrated that isoproterenol enhanced respectively by 1.3- and 1.2-fold the transcription rate of the Gi alpha 2 and Gi alpha 3 genes. Quantification of myometrial alpha(2)-ARs by [3H]rauwolscine binding revealed that the total number of receptors was also increased at 76 h by 1.7-fold when compared with controls, with no change in the affinity of the alpha(2)-ARs for the ligand. This effect was antagonized by propranolol. Quantification of both alpha(2A)- and alpha(2B)-subtypes by Northern blotting analysis demonstrated that this elevation was due to a selective increase of the alpha(2A)-subtype mRNAs. The present results indicate that in vivo stimulation of the beta-adrenergic pathway by isoproterenol increases both Gi alpha 2/Gi alpha 3 and alpha(2A)-AR expression in the pregnant rat myometrium. The possible contribution of such a mechanism in pregnancy-related changes of both entities is discussed.

The alpha1B-adrenergic receptor subtype activates the phospholipase C signaling pathway in rat myometrium at parturition.

This study demonstrates that alpha1-adrenergic receptors previously identified in the pregnant rat myometrium are heterogeneous. They can be subtyped alpha1A- and alpha1B-adrenergic receptors on the basis of their affinity for the antagonists WB4101 (alpha1A > alpha1B) and chloroethylclonidine (alpha1B selective). Between Day 21 of pregnancy and term, the proportion of [3H]prazosin binding sites with low affinity for WB4101 and sensitive to inactivation by 10(-5) M chloroethylclonidine under hypotonic conditions (alpha1B subtype) remained constant. In contrast, the number of [3H]prazosin binding sites with a high affinity for WB4101 and insensitive to chloroethylclonidine (alpha1A subtype) increased by 88% at term. The effect of 5'-guanylylimidodiphosphate (Gpp[NH]p) on competition of the agonist phenylephrine for [3H]prazosin binding in the presence of WB4101 or after chloroethylclonidine pretreatment indicates that the alpha1A-adrenergic receptor underwent uncoupling whereas the alpha1B-adrenergic receptor-G protein coupled state was increased (+ 63%). Phenylephrine consistently stimulated phospholipase C activity on membrane fractions prepared from term myometria. This stimulation was completely inhibited after 10(-5) M chloroethylclonidine but was not consistently decreased with 5-methylurapidyl, a selective alpha1A-antagonist. Furthermore QL antibody (anti-G alpha(q)/G alpha11) also specifically blocked the phenylephrine-stimulated phospholipase C activity. Altogether these results strongly suggest that activation of the alpha1B-adrenergic receptor subtype in the pregnant myometrium at term may contribute to the stimulation of the G alpha(q)/G alpha11/phospholipase C signaling pathway.

Molecular diversity of adenylyl cyclases in human and rat myometrium. Correlation with global adenylyl cyclase activity during mid- and term pregnancy.

Expression and regulation of myometrial adenylyl cyclases (AC) were studied during pregnancy. Hybridization of poly(A)+ RNA with specific cDNA probes for enzyme types I-IX indicated 1) the presence of transcripts encoding types II-VI and type IX in rat and human, and type VII in rat and 2) the absence of detectable mRNA for types I and VIII in both species. No substantial change was observed in the amount of specific mRNA and basal AC activity from mid-pregnancy to term. However, activation of the alpha2-adrenergic receptor/Gi protein pathway resulted in potentiation of Gs-stimulated AC activity at mid-pregnancy but not at term (Mhaouty, S., Cohen-Tannoudji, J., Bouet-Alard, R., Limon-Boulez, I., Maltier, J. P., and Legrand, C. (1995) J. Biol. Chem. 270, 11012-11016). We demonstrate in the present work that betagamma scavengers transducin-alpha and QEHA peptide abolished this positive input. On the other hand, increasing submicromolar concentrations of free Ca2+, a situation that mimics late term, reduced the forskolin-stimulated AC activity with an IC50 of 3.9 microM. Thus, the presence in myometrium of AC II family (types II, IV, VII) confers ability to G inhibitory proteins to stimulate enzyme activity via betagamma complexes at mid-pregnancy, whereas expression of AC III, V, and VI isoforms confers to the myometrial AC system a high sensitivity to inhibition by Ca2+-dependent processes at term. These data suggest that in the pregnant myometrium, the expression of different species of AC with distinct regulatory properties provides a mechanism for integrating positively or negatively the responses to various hormonal inputs existing either during pregnancy or in late term.

Heterogeneity of alpha 2-adrenoceptors in human and rat myometrium and differential expression during pregnancy.

1. The aim of this study was first, to characterize alpha 2-adrenoceptor subtypes in human and rat pregnant myometrium and second, to investigate the possibility of a differential expression of the putative subtypes according to the stage of pregnancy. 2. In both species, specific [3H]-rauwolscine binding was inhibited by five different compounds with an order of affinity characteristic of the one described for alpha 2-adrenoceptors (yohimbine > or = clonidine > noradrenaline > phenylephrine > propranolol). Binding affinities (pKi) for the compounds tested were, in human and rat, respectively: 7.63 and 8.93 for yohimbine, 6.91 and 8.71 for clonidine, 6.23 and 6.09 for noradrenaline, 5.37 and 5.73 for phenylephrine, 4.64 and 4.72 for propranolol. 3. By use of non-linear iterative curve fitting procedures and by fitting the data to a two-site model, analysis of [3H]-rauwolscine inhibition binding curves performed in the presence of oxymetazoline (alpha 2A-selective), ARC239, prazosin or chlorpromazine (alpha 2B- and alpha 2C-selective) indicated that pregnant human and rat myometrium contain at least two pharmacologically distinct alpha 2-adrenoceptor subtypes (alpha 2A, alpha 2B and/or alpha 2C). RNA blot analysis with probes specific for each cloned human and rat alpha 2-adrenoceptor subtype demonstrated that alpha 2A- and alpha 2B-subtypes were present in both species but alpha 2C seems to be expressed only in human tissues. 4. In the pregnant rat myometrium, subtype selective compounds competition curves revealed a predominant expression of alpha 2A-adrenoceptors at mid-pregnancy whereas, at term, alpha 2A- and alpha 2B-subtypes density reached approximately the same level (alpha 2A:alpha 2B ratio = 73:27 at mid-pregnancy and = 43:57 at term). In addition, quantification of alpha 2A- and alpha 2B-transcripts by densitometry, following data normalization with an oligo(dT)12-18 probe, showed a pattern of expression comparable to the one characterized by pharmacological studies. 5. In conclusion, these data demonstrate heterogeneity of alpha 2-adrenoceptors in pregnant human and rat myometria and an alteration of the alpha 2A-/alpha 2B-subtypes expression pattern during rat pregnancy. Such observations lead us to suggest a multiple role for alpha 2-adrenoceptors in regulating specific functions of myometrium throughout the time course of pregnancy.