alpha 2A- and alpha 2B-adrenoreceptors (AR), identified by Northern blotting in rat myometrium, showed a differential expression during the course of pregnancy. Indeed, the alpha 2A-AR transcript was present at mid-pregnancy, whereas high levels of alpha 2B-AR mRNA could be detected at term. The role of these subtypes in modulating beta 2-AR-stimulated adenylyl cyclase activity was investigated on myometrial membranes from mid-pregnancy and term. At nanomolar concentrations of clonidine (full alpha 2-AR agonist) or oxymetazoline (partial alpha 2A-AR agonist), adenylyl cyclase activity was inhibited by up to 50 +/- 7% at mid-pregnancy or 75 +/- 7% at term, whereas at micromolar concentrations, alpha 2-AR agonists potentiate adenylyl cyclase activity by 140-170% at mid-pregnancy. Both inhibitory and stimulatory components of this biphasic response were blocked by yohimbine, a selective alpha 2-AR antagonist. Preincubation of myometrial membranes with Gi2 and/or Gi3 antisera eliminated alpha 2-AR mediated attenuation or potentiation of isoproterenol-stimulated adenylyl cyclase, thus indicating that both the inhibitory and stimulatory components are mediated via Gi2 and Gi3. In addition, type II and IV adenylyl cyclases were identified by Northern blotting in the pregnant rat myometrium. Altogether these data strongly suggest that the alpha 2A-AR at mid-pregnancy potentiates adenylyl cyclase types II and IV through beta gamma released from Gi2 and Gi3 proteins, whereas the alpha 2B-AR expression at term may be related to persistent inhibition.

To define the integration of multiple signals by different types of adenylyl cyclase (AC) within the cell, we altered the population of enzymes expressed in the cell and determined the subsequent processing of stimulatory and inhibitory input. DDT 1 -MF2 cells expressed AC VI-IX and were stably transfected with AC II, III, or IV. Enzyme expression was confirmed by RNA blot analysis and functional assays. Basal enzyme activity was only increased in AC II transfectants (6-fold). Maximum stimulation of enzyme activity was increased in each of the AC transfectants to varying extents. ␣ 2A/D -AR activation elicited enzyme type-specific responses. ␣ 2 -AR activation inhibited the effect of isoproterenol in control transfectants, and this action was magnified in AC III transfectants. In AC II and AC IV transfectants, ␣ 2 -AR activation initiated both positive (G␤␥) and negative signals (G i ␣) to the G s ␣-stimulated enzyme, and both types of signals were blocked by cell pretreatment with pertussis toxin. The negative input to AC II from the ␣ 2 -AR was blocked by protein kinase C activation in AC II transfectants, but it was the positive input to AC IV that was compromised by protein kinase C activation. These data indicate that the integration of multiple signals by adenylyl cyclases is a dynamic process depending upon the enzyme type and phosphorylation status.

We demonstrate in the present work that ␤␥ scavengers transducin-␣ and QEHA peptide abolished this positive input. On the other hand, increasing submicromolar concentrations of free Ca 2؉ , a situation that mimics late term, reduced the forskolin-stimulated AC activity with an IC 50 of 3.9 M. Thus, the presence in myometrium of AC II family (types II, IV, VII) confers ability to G inhibitory proteins to stimulate enzyme activity via ␤␥ 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 Ca 2؉ -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.Data on hormonal regulation of myometrial contractility during the course of pregnancy implicate adenylyl cyclase (AC) 1 stimulatory pathways as a key component that may affect the degree of intracellular cAMP generation and consequently the contractile state of the uterus. Because one of the major sites of control of the biochemical events leading to uterine relaxation during normal pregnancy lies at the AC/ cAMP system, the identification of AC isoforms in the pregnant myometrium is essential in understanding the influence exerted by the regulatory external signals (neurotransmitters and hormones) acting via G protein-coupled receptors. Hormonal control of AC activity is brought about by receptor-catalyzed activation of heterotrimeric G proteins that in turn regulate the cyclases by the release of ␣ or ␤␥ subunits or kinase activation. Recent studies have revealed an unexpected diversity of G protein-regulated AC by identifying nine distinct AC cDNA from various mammalian tissues (1-5). All of these isoforms of AC differ in their tissue distribution and their regulatory properties, providing a mode for different cells to respond diversely to similar external stimuli. Among all of the AC identified so far, the highly similar types II, IV, and VII form the largest known subfamily. Types II and IV share the property of being highly stimulated by ␤␥ subunits of G i /G o inhibitory proteins in the presence of activated G s ␣ (6, 7). These AC are also influenced by phosphorylation with protein kinase C (8 -11). Types V and VI AC, a two-member subfamily, are inhibited directly by low levels of Ca 2ϩ (2, 12), whereas AC I and VIII are regulated positively by Ca 2ϩ -calmodulin (13, 14). On the other hand, AC III can be phosphorylated by a calmodulindependent protein kinase II in response to the elevation of intracellular Ca 2ϩ which results, in vivo, in a 50% inhibition of hormone-stimulated enzyme (15). The novel ninth AC is quite distinct from all of the other known AC subfamilies, and it is not affected by G ␤␥ proteins or Ca 2ϩ (5). Thus, in vivo, when a cell type or tissue expresses various isoforms of AC one may expect that...

1 The aim of this study was ®rst, to characterize a 2 -adrenoceptor subtypes in human and rat pregnant myometrium and second, to investigate the possibility of a di erential expression of the putative subtypes according to the stage of pregnancy. 2 In both species, speci®c [ 3 H]-rauwolscine binding was inhibited by ®ve di erent compounds with an order of a nity characteristic of the one described for a 2 -adrenoceptors (yohimbine5clonidine4nor-adrenaline4phenylephrine4propranolol). Binding a nities (pK i ) 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 ®tting procedures and by ®tting the data to a two-site model, analysis of [ 3 H]-rauwolscine inhibition binding curves performed in the presence of oxymetazoline (a 2A -selective), ARC239, prazosin or chlorpromazine (a 2B -and a 2C -selective) indicated that pregnant human and rat myometrium contain at least two pharmacologically distinct a 2 -adrenoceptor subtypes (a 2A , a 2B and/or a 2C ). RNA blot analysis with probes speci®c for each cloned human and rat a 2 -adrenoceptor subtype demonstrated that a 2A -and a 2B -subtypes were present in both species but a 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 a 2A -adrenoceptors at mid-pregnancy whereas, at term, a 2A -and a 2B -subtypes density reached approximately the same level (a 2A : a 2B ratio=73 : 27 at mid-pregnancy and=43 : 57 at term). In addition, quanti®cation of a 2A -and a 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 a 2 -adrenoceptors in pregnant human and rat myometria and an alteration of the a 2A -/a 2B -subtypes expression pattern during rat pregnancy. Such observations lead us to suggest a multiple role for a 2 -adrenoceptors in regulating speci®c functions of myometrium throughout the time course of pregnancy.