RESUMEN
alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR >> alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).
Asunto(s)
Adenilil Ciclasas/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Clonidina/farmacología , AMP Cíclico/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Adenilil Ciclasas/genética , Animales , Cricetinae , Epinefrina/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Pruebas de Precipitina , Transfección , Células Tumorales CultivadasRESUMEN
Beta-adrenergic agonists are widely used for preterm labor treatment, but their effectiveness may be limited by desensitization. We thus investigated the effects of a beta-agonist, isoproterenol, on the myometrial beta-adrenergic receptor (beta-AR)/adenylyl cyclase pathway after administration in vivo to late-pregnant rats (8 mg/kg, twice-daily injections). One hour after the first injection, isoproterenol-stimulated adenylyl cyclase activity was reduced by 37%. This was associated with a rapid and transient uncoupling of the beta2-ARs (53% reduction of high-affinity receptors). After prolonged isoproterenol treatment (76 h), adenylyl cyclase activity was desensitized not only to isoproterenol but also to guanosine triphosphate and forskolin. Such treatment induced 1) a selective decrease of beta2-ARs as assessed by 125I-cyanopindolol binding, which was reversed by 5'-guanylylimidodiphosphate and thus probably did not involve irreversible loss of receptors, and 2) a rapid alteration of their transcript levels. Prolonged isoproterenol treatment also led to myometrial Gi2alpha and Gi3alpha increase (44% and 70%) as assessed by Western blotting. Furthermore, pertussis toxin pretreatment of membranes abolished the decrease in isoproterenol-stimulated adenylyl cyclase activity. Thus, we demonstrated that myometrial adenylyl cyclase desensitization after beta-agonist treatment results mainly from beta2-AR uncoupling and increase in Gi activity.
Asunto(s)
Adenilil Ciclasas/metabolismo , Agonistas Adrenérgicos beta/farmacología , Isoproterenol/farmacología , Miometrio/enzimología , Receptores Adrenérgicos beta/efectos de los fármacos , Antagonistas Adrenérgicos beta/metabolismo , Animales , Colforsina/farmacología , Tolerancia a Medicamentos , Femenino , Proteínas de Unión al GTP/fisiología , Guanosina Trifosfato/farmacología , Guanilil Imidodifosfato/farmacología , Isoproterenol/administración & dosificación , Miometrio/efectos de los fármacos , Pindolol/análogos & derivados , Pindolol/metabolismo , Embarazo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/fisiologíaRESUMEN
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.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Isoproterenol/farmacología , Miometrio/metabolismo , Receptores Adrenérgicos alfa 2/genética , Antagonistas Adrenérgicos beta/farmacología , Animales , Northern Blotting , AMP Cíclico/metabolismo , Femenino , Immunoblotting , Embarazo , Propranolol/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Estimulación QuímicaRESUMEN
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.
Asunto(s)
Trabajo de Parto/fisiología , Miometrio/fisiología , Receptores Adrenérgicos beta 1/metabolismo , Transducción de Señal/fisiología , Fosfolipasas de Tipo C/metabolismo , Agonistas alfa-Adrenérgicos/farmacocinética , Antagonistas Adrenérgicos alfa/farmacocinética , Antagonistas Adrenérgicos alfa/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Clonidina/análogos & derivados , Clonidina/farmacología , Dioxanos/farmacología , Femenino , Guanilil Imidodifosfato/farmacología , Miometrio/inervación , Embarazo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Miometrio/metabolismo , Receptores Adrenérgicos alfa 2/genética , 5'-Nucleotidasa/metabolismo , Animales , Unión Competitiva , Northern Blotting , Femenino , Humanos , Ligandos , Masculino , Embarazo , Ratas , Receptores Adrenérgicos alfa 2/metabolismo , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Adenilil Ciclasas/química , Miometrio/enzimología , Adenilil Ciclasas/metabolismo , Animales , Toxina del Cólera/farmacología , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Guanosina Trifosfato/farmacología , Humanos , Isoproterenol/farmacología , Miometrio/efectos de los fármacos , Embarazo , Segundo Trimestre del Embarazo , Tercer Trimestre del Embarazo , Ratas , Ratas Sprague-Dawley , Simpatomiméticos/farmacología , Transducina/farmacologíaRESUMEN
The possibility that progesterone or estradiol may regulate expression of G protein in the rat myometrium during the course of pregnancy has been investigated using 1) immunoblot analysis of Gi2 alpha, Gi3 alpha, and Gq alpha subunits and 2) hybridization blot analysis of subunit mRNA. Eighteen hours after administration, estradiol had significantly increased the levels of both Gi2 alpha subunit and Gi2 alpha mRNA (by 40% and 32%, respectively). In control pregnant rats, we observed similar changes at the end of pregnancy, when myometrial concentrations of estradiol had increased, i.e., a 41% increase in immunoreactive Gi2 alpha subunit that correlated with a parallel 45% increase in mRNA levels. In contrast, levels of immunoreactive Gi3 alpha subunit and mRNA, which decreased with advancing gestation, were not influenced by estradiol or progesterone administration. Progesterone administration resulted 30 h later in a significantly decreased level of Gq alpha immunoreactivity (32%) and Gq alpha mRNA (30%). In control rats, Gq alpha protein and mRNA were also significantly lower at midpregnancy under progesterone dominance vs. term. At this stage, a twofold increase in Gq alpha subunit correlated with a 40% increase in mRNA levels. These results demonstrate that myometrial Gi2 alpha and Gq alpha subunits are physiological targets for estradiol and progesterone, respectively, in vivo. Alterations of these G protein levels are discussed in relation to their mediating effects on adenylyl cyclase activity or the phospholipase C pathway during the course of pregnancy.
Asunto(s)
Estradiol/farmacología , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica , Miometrio/metabolismo , Progesterona/farmacología , Adenilil Ciclasas/metabolismo , Animales , Secuencia de Bases , Femenino , Datos de Secuencia Molecular , Miometrio/efectos de los fármacos , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Adenilil Ciclasas/metabolismo , Miometrio/fisiología , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/metabolismo , Toxina de Adenilato Ciclasa , Adenilil Ciclasas/genética , Animales , Secuencia de Bases , Membrana Celular/enzimología , Clonidina/farmacología , Cartilla de ADN/química , Femenino , Proteínas de Unión al GTP/metabolismo , Expresión Génica , Guanosina Trifosfato/farmacología , Isoproterenol/farmacología , Masculino , Datos de Secuencia Molecular , Embarazo , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factores de Virulencia de Bordetella/farmacología , Yohimbina/metabolismo , Yohimbina/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Miometrio/metabolismo , Preñez/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Secuencia de Bases , Toxina del Cólera/metabolismo , Sondas de ADN/genética , Estradiol/farmacología , Femenino , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/inmunología , Expresión Génica/efectos de los fármacos , Mifepristona/farmacología , Datos de Secuencia Molecular , Peso Molecular , Miometrio/efectos de los fármacos , Embarazo , Preñez/genética , Progesterona/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Miometrio/ultraestructura , Preñez/fisiología , Receptores Adrenérgicos alfa/análisis , Receptores Adrenérgicos alfa/clasificación , Antagonistas Adrenérgicos alfa/farmacología , Animales , Autorradiografía , Unión Competitiva , Dioxanos/farmacología , Femenino , Idazoxan , Cinética , Microscopía , Miometrio/metabolismo , Oxidopamina/farmacología , Embarazo , Preñez/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa/metabolismo , Tritio , Yohimbina/metabolismoRESUMEN
Administration of 5 mg of progesterone to late pregnant rats induced an increase in myometrial beta-adrenergic receptors density detected by 125I-cyanopindolol binding. This increase was significant after 24 h (1.4-fold; p less than 0.05) and reached 1.6-fold (p less than 0.05) after 36 h. The antiprogestin RU 486 or cycloheximide completely suppressed the effect of progesterone injection. Quantification of the beta 1- and beta 2-receptor subtypes revealed that progesterone selectively up-regulated the beta 2-subtype. The increase in beta 2-adrenoreceptors was preceded by an elevation of their mRNA (2.3 kilobases) levels as determined by Northern blot hybridization with a rat heart beta 2-adrenoreceptor cDNA probe. This increase was significant after 18 h of exposure to progesterone (2.1-fold; p less than 0.05) and reached a maximum after 24 h (3.4-fold; p less than 0.01). The rate of beta 2-adrenergic gene transcription evaluated by nuclear run-on transcription assays, increased by 2.5-fold in myometria exposed for 4 h to progesterone. This study indicates that progesterone regulates myometrial beta 2-adrenergic receptor expression by controlling the rate of transcription of the gene.
Asunto(s)
Miometrio/efectos de los fármacos , Progesterona/farmacología , Receptores Adrenérgicos beta/genética , Transcripción Genética/efectos de los fármacos , Animales , Autorradiografía , Northern Blotting , Cicloheximida/farmacología , Femenino , Técnicas In Vitro , Mifepristona/farmacología , Miometrio/metabolismo , Embarazo , Progesterona/antagonistas & inhibidores , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , RatasRESUMEN
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)
Asunto(s)
Adenilil Ciclasas/metabolismo , Miometrio/metabolismo , Preñez/metabolismo , Progesterona/farmacología , Receptores Adrenérgicos beta/metabolismo , Animales , Membrana Celular/metabolismo , Femenino , Yodocianopindolol , Isoproterenol/farmacología , Cinética , Mifepristona/farmacología , Pindolol/análogos & derivados , Pindolol/metabolismo , Embarazo , Ratas , Ratas Endogámicas , Receptores Adrenérgicos beta/efectos de los fármacos , Valores de ReferenciaRESUMEN
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.
Asunto(s)
Preñez/metabolismo , Receptores Adrenérgicos alfa/análisis , Receptores Adrenérgicos beta/análisis , Útero/química , Animales , Autorradiografía , Femenino , Yodocianopindolol , Pindolol/análogos & derivados , Pindolol/metabolismo , Prazosina/metabolismo , Embarazo , Ratas , Ratas EndogámicasRESUMEN
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.
Asunto(s)
Cuello del Útero/ultraestructura , Receptores Adrenérgicos alfa/análisis , Animales , Autorradiografía/métodos , Cuello del Útero/análisis , Cuello del Útero/citología , Femenino , Miometrio/fisiología , Miometrio/ultraestructura , Prazosina , Embarazo , RatasRESUMEN
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.
Asunto(s)
Blastocisto/fisiología , Miometrio/fisiología , Prazosina/farmacología , Preñez/fisiología , Animales , Electrofisiología , Estradiol/análisis , Femenino , Miometrio/efectos de los fármacos , Embarazo , Progesterona/análisis , Ratas , Ratas Endogámicas , Útero/análisis , Útero/anatomía & histologíaRESUMEN
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.
Asunto(s)
Estradiol/fisiología , Miometrio/metabolismo , Preñez/efectos de los fármacos , Progesterona/fisiología , Receptores Adrenérgicos beta/fisiología , Animales , Estradiol/análisis , Estradiol/farmacología , Femenino , Miometrio/análisis , Ovariectomía , Embarazo , Progesterona/análisis , Progesterona/farmacología , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
The purpose of this study was to characterize the beta-adrenoceptor subtypes in pregnant rat myometrial membrane fractions and to determine the concentration of beta 2-adrenoceptors in uterus during late pregnancy. Two methods are compared. A non-subtype-selective antagonist radioligand [3H]dihydroalprenolol ([3H]DHA) was used to label all of the beta-adrenoceptors. [3H]DHA bound to both beta 1- and beta 2-adrenoceptors with indistinguishable affinity (KD = 1.31 nM, Bmax = 174 fmol/mg protein). Computer modelling of competition curves of unlabeled selective antagonists or agonists was then required in order to determine reliably beta 1- and beta 2-adrenoceptor affinities and proportions: the beta 1-adrenoceptors represent 35.5% and the beta 2-adrenoceptors 64.5% of the entire beta-adrenoceptor population in rat gravid myometrium at term. The second approach utilized the radioligand [3H]hydroxybenzylisoproterenol ([3H]HBI) which is a very high-affinity beta-adrenoceptor agonist. The characteristics of the [3H]HBI binding sites are essentially those expected of beta 2-adrenoceptors, but the [3H]HBI binding sites represent only 34% of [3H]DHA binding sites and may represent the fraction of beta 2-adrenoceptors that mediate adenylate cyclase stimulation and uterine relaxation. Between 21 d 09 h and 22 d 09 h of gestation, the number of beta 2-adrenoceptors was constant (mean = 225.6 +/- 20.2 fmol/mg protein). At term, the number of [3H]HBI binding sites dropped (-75%) during the last 7 h of pregnancy, suggesting a reduced ability to elicit relaxation through beta-adrenoceptor activation in parturient myometrium of rat.
Asunto(s)
Trabajo de Parto/metabolismo , Miometrio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Dihidroalprenolol/metabolismo , Femenino , Técnicas In Vitro , Isoproterenol/análogos & derivados , Isoproterenol/metabolismo , Cinética , Embarazo , Ratas , Ratas Endogámicas , Contracción UterinaRESUMEN
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.
Asunto(s)
Miometrio/metabolismo , Preñez/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/metabolismo , 5'-Nucleotidasa , Animales , Femenino , Isoproterenol/análogos & derivados , Isoproterenol/metabolismo , Nucleotidasas/metabolismo , Prazosina/metabolismo , Embarazo , Ensayo de Unión Radioligante , Ratas , Ratas EndogámicasRESUMEN
Sympathetic nerve terminals were destroyed by administration of 6-hydroxydopamine (2 x 50 mg/kg) at 10:00 h on Days 4 and 5 of pregnancy in the rat. In the myometrium, this treatment markedly decreased noradrenaline concentrations (by 99%, P less than 0.001), demonstrating that myometrial noradrenaline is mainly originated from sympathetic nerves; therefore after 6-hydroxydopamine, the distribution and spacing of blastocysts remain unaffected throughout the uterus. Administration of phenoxybenzamine (2 x 6 mg/kg) in the morning of Days 4 and 5, or prazosin (4 x 3 mg/kg) from 12:00 h on Day 4 until 12:00 h on Day 5 disorganized the even distribution of blastocysts from the tubal end to the cervical end of the uterine horns. These results provide evidence that a noradrenergic transmission via action on myometrial post-synaptic alpha 1-adrenoceptors is involved as a regulatory mechanism of uterine motility for distribution and spacing of blastocysts in the rat uterus.
Asunto(s)
Implantación del Embrión/efectos de los fármacos , Hidroxidopaminas/farmacología , Fenoxibenzamina/farmacología , Prazosina/farmacología , Animales , Blastocisto , Femenino , Miometrio/análisis , Miometrio/efectos de los fármacos , Miometrio/inervación , Norepinefrina/análisis , Oxidopamina , Embarazo , Ratas , Ratas Endogámicas , Simpatectomía QuímicaRESUMEN
6-Hydroxydopamine, when injected at 14:00 h on Days 21 and 22 of pregnancy in the rat (2 X 50 mg/kg), markedly decreased plasma and uterine noradrenaline concentrations (-60% and -82% respectively; P less than 0.001). As a consequence of this treatment, there was severe disturbance in the distribution pattern of parturitions: 61% of rats had suppressed parturition and 31% of rats displayed a lengthened or interrupted labour. A bolus dose of prazosin (3 mg/kg) administered at 12:00 h on Day 22 completely blocked the normal process of parturition throughout the next 6 h, a result which is compatible with the half-life of the drug (2.9 +/- 0.8 h). Administration of phentolamine (3 mg/kg) at term induced a significant decrease of uterine activity (frequency X duration of bursts of spike potentials) as revealed by electromyographic recordings in vivo. These results suggest that noradrenaline released from sympathetic nerve terminals interacts with alpha-adrenoceptors located post-synaptically to improve the overall excitability of the myometrium at the onset of labour.