Chantal Legrand

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 α2-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 βγ scavengers transducin-α 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 μm. Thus, the presence in myometrium of AC II family (types II, IV, VII) confers ability to G inhibitory proteins to stimulate enzyme activity via βγ 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.

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 α1‐adrenergic receptors (α1‐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 α1‐AR agonist phenylephrine (Phe) and oxytocin (OT). 2 Using the reverse transcription‐polymerase chain reaction RT‐PCR, we detected the α1a‐AR subtype in late pregnant mouse myometrium. We also detected, by immunoblotting studies, PLCβ1, PLCβ3 and different α‐subunits of pertussis toxin‐insensitive (Gαq/11) and ‐sensitive G proteins (Gαo/i3, Gαi1/2). 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 μm) recruited PLCβ1 and PLCβ3 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 Gαq 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 μm. 5 The results indicate that OT receptors (OTR) but not α1‐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 α1‐AR subtype expressed in myometrium at this time.

Heterogeneity of α2-adrenoceptors in human and rat myometrium and differential expression during pregnancy

1 The aim of this study was first, to characterize α2‐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 α2‐adrenoceptors (yohimbineclonidine>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 (α2A‐selective), ARC239, prazosin or chlorpromazine (α2B‐ and α2C‐selective) indicated that pregnant human and rat myometrium contain at least two pharmacologically distinct α2‐adrenoceptor subtypes (α2A, α2B and/or α2C). RNA blot analysis with probes specific for each cloned human and rat α2‐adrenoceptor subtype demonstrated that α2A‐ and α2B‐subtypes were present in both species but α2C seems to be expressed only in human tissues. 4 In the pregnant rat myometrium, subtype selective compounds competition curves revealed a predominant expression of α2A‐adrenoceptors at mid‐pregnancy whereas, at term, α2A‐ and α2B‐subtypes density reached approximately the same level (α2A : α2B ratio=73 : 27 at mid‐pregnancy and=43 : 57 at term). In addition, quantification of α2A‐ and α2B‐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 α2‐adrenoceptors in pregnant human and rat myometria and an alteration of the α2A‐/α2B‐subtypes expression pattern during rat pregnancy. Such observations lead us to suggest a multiple role for α2‐adrenoceptors in regulating specific functions of myometrium throughout the time course of pregnancy.

Characterization of α-adrenoceptors in myometrium of preparturient rats

Abstract We describe three methods for the quantitative analysis of the α-adrenoceptor subtypes in preparturient rat myometrial membrane fractions. A non-subtype-selective antagonist radioligand, [ 3 H]dihydroergocryptine ([ 3 H]DHE), was used to label all of the α-receptors. [ 3 H]DHE bound to both α 1 - and α 2 -receptors with indistinguishable affinity. Computer modelling of competition curves of unlabeled selective antagonists or agonists was then required in order to determine reliably α 1 and α 2 affinities and proportions: the α 1 -receptors represent 45% and the α 2 -receptors 55% of the entire α-receptor population in rat uterus. The second approach involved the administration of phenoxybenzamine (POB) that irreversibly blocks the α 1 -adrenoceptors. Myometrial membranes obtained from rats 1 h after the administration of varying amounts of POB showed a dose-dependent reduction in specific [ 3 H]DHE binding. This reduction was accompanied by a progressive increase of the value of the dissociation constant. Our data indicate that a dose of 1 mg of POB left the α 2 -receptors intact while entirely blocking the α 1 -receptors in rat myometrium. The third approach utilized the selective radioligand antagonists [ 3 H]prazosin ([ 3 H]PRAZ) and [ 3 H]rauwolscine ([ 3 H]RAUW). The results obtained with these radioligands confirmed our observations on the α-adrenoceptor subtypes in experiments with [ 3 H]DHE. The results obtained with the 3 methods are in good agreement. Each approach appears valid and applicable to the characterization of α 1 - and α 2 -receptors subtypes in rat uterus, but the method using [ 3 H]PRAZ and [ 3 H]RAUW demonstrates more directly the presence of the two receptor subtypes.

Autoradiographic visualization of alpha 1-adrenergic receptors in cervix of early pregnant rat.

alpha 1-Adrenergic receptors were identified, characterized, and localized in rat cervix on Day 6 of pregnancy by autoradiography. Autoradiographic study was performed in slide-mounted rat cervix sections using [3H]-prazosin ([3H]-PRAZ) as ligand. Binding was time dependent and specific. Pharmacological study indicated that specific [3H]-PRAZ binding was inhibited with high affinity by prazosin and phenylephrine and low affinity by yohimbine and clonidine. In cervix, the alpha 1-adrenergic receptors were localized mainly to the inner circular layer of the myometrium. Binding to the outer longitudinal layer of myometrium was moderate, and binding was absent in the endometrium. The regional distribution of alpha 1-adrenergic receptors strongly suggests that the circular layer of myometrium may function as an important modulator of contractile response of the cervix, probably involved in the retention of blastocysts at the utero-cervical end of the horn.