Characterization of myometrial desensitization to beta-adrenergic agonists

Continuous exposure of ovine myometrial strips exposed to isoproterenol (10 microM) resulted in only transient inhibition with contractions returning within 60 min. Rechallenging these strips with isoproterenol failed to induce inhibition, confirming the occurrence of desensitization. In contrast, exposure of myometrial tissue to isoproterenol for only 5 min did not result in desensitization. Myometrial strips exposed to isoproterenol demonstrated a significant increase in cAMP content associated with inhibition of contractile activity and a subsequent fall in cAMP content upon desensitization. Elevation of endogenous cAMP levels by either inhibition of cAMP-dependent phosphodiesterase activity (0.5 mM isobutylmethylxanthine, in ovine strips) or direct activation of adenylyl cyclase (10 microM forskolin, in rat strips) induced a rapid and significant inhibition of myometrial contractile activity in desensitized tissue. Scatchard analysis of the binding of the beta-adrenoceptor antagonist, [125I]iodocyanopindolol, revealed a significant reduction in the concentration of beta-adrenergic receptors (but no change in binding affinity) in desensitized myometrial tissue. Incubation of desensitized tissue with fresh buffer for 3 h induced only a partial recovery in responsiveness to isoproterenol. These data suggest that prolonged, but not acute, exposure of the myometrium to beta-adrenergic agonists induces a state of desensitization that is associated with a down-regulation of beta-adrenoceptors but maintenance of postreceptor function.     

Relaxation of myometrium by calcitonin gene-related peptide is independent of nitric oxide synthase activity in mouse uterus

Calcitonin gene-related peptide (CGRP) inhibits myometrial contractile activity. However, the responsiveness of the mouse myometrium to CGRP is dependent on the hormonal and gestational stage. The inhibitory effect of CGRP in the myometrium is prominent during gestation and declines at parturition. The present study was undertaken to examine if nitric oxide (NO) production by nitric oxide synthase (NOS) isoforms mediates the inhibitory action of CGRP on uterine contractions as has been suggested earlier. Transgenic mice deficient in either of the three major NOS isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were used. Isometric force measurements on myometrial strips obtained from NOS-deficient mice were carried out and the inhibitory capacity of CGRP was monitored. CGRP inhibited KCl-induced contractions of the myometrial strips obtained from eNOS(-/-), iNOS(-/-), and nNOS(-/-) mice with equal efficiency as in wild-type animals. Additionally, NOS protein expression in the mouse uterus during gestation and during the estrous cycle was examined by means of Western immunoblot analysis. No correlation between NOS expression and inhibitory activity of CGRP was evident. The results suggest that the inhibitory action of CGRP in the mouse uterus is independent of the activity of these NOS isoforms.     

Increased MMPs expression and decreased contraction in the rat myometrium during pregnancy and in response to prolonged stretch and sex hormones

Normal pregnancy is associated with uterine relaxation to accommodate the stretch imposed by the growing fetus; however, the mechanisms underlying the relationship between pregnancy-associated uterine stretch and uterine relaxation are unclear. We hypothesized that increased uterine stretch during pregnancy is associated with upregulation of matrix metalloproteinases (MMPs), which in turn cause inhibition of myometrium contraction and promote uterine relaxation. Uteri from virgin, midpregnant (day 12), and late-pregnant rats (day 19) were isolated, and myometrium strips were prepared for measurement of isometric contraction and MMP expression and activity using RT-PCR, Western blot analysis, and gelatin zymography. Oxytocin caused concentration-dependent contraction of myometrium strips that was reduced in mid- and late-pregnant rats compared with virgin rats. Pretreatment with the MMP inhibitors SB-3CT (MMP-2/MMP-9 Inhibitor IV), BB-94 (batimastat), or Ro-28-2653 (cipemastat) enhanced contraction in myometrium of pregnant rats. RT-PCR, Western blot analysis, and gelatin zymography demonstrated increased mRNA expression, protein amount, and activity of MMP-2 and MMP-9 in myometrium of late-pregnant>midpregnant>virgin rats. Prolonged stretch of myometrium strips of virgin rats under 8 g basal tension for 18 h was associated with reduced contraction and enhanced expression and activity of MMP-2 and MMP-9, which were reversed by MMP inhibitors. Concomitant treatment of stretched myometrium of virgin rats with 17β-estradiol (E2), progesterone (P4), or E2+P4 was associated with further reduction in contraction and increased MMP expression and activity. MMP-2 and MMP-9 caused significant reduction of oxytocin-induced contraction of myometrium of virgin rat. Thus, normal pregnancy is associated with reduced myometrium contraction and increased MMPs expression and activity. The results are consistent with the possibility that myometrium stretch and concomitant increase in sex hormones during pregnancy are associated with increased expression/activity of specific MMPs, which in turn inhibit uterine contraction and promote uterine relaxation.     

CyPPA, a positive modulator of small-conductance Ca(2+)-activated K(+) channels, inhibits phasic uterine contractions and delays preterm birth in mice

Organized uterine contractions, including those necessary for parturition, are dependent on calcium entry through voltage-gated calcium channels in myometrial smooth muscle cells. Recent evidence suggests that small-conductance Ca(2+)-activated potassium channels (K(Ca)2), specifically isoforms K(Ca)2.2 and 2.3, may control these contractions through negative feedback regulation of Ca(2+) entry. We tested whether selective pharmacologic activation of K(Ca)2.2/2.3 channels might depress uterine contractions, providing a new strategy for preterm labor intervention. Western blot analysis and immunofluorescence microscopy revealed expression of both K(Ca)2.2 and K(Ca)2.3 in the myometrium of nonpregnant (NP) and pregnant (gestation day 10 and 16; D10 and D16, respectively) mice. Spontaneous phasic contractions of isolated NP, D10, and D16 uterine strips were all suppressed by the K(Ca)2.2/2.3-selective activator CyPPA in a concentration-dependent manner. This effect was antagonized by the selective K(Ca)2 inhibitor apamin. Whereas CyPPA sensitivity was reduced in D10 and D16 versus NP strips (pIC(50) 5.33 ± 0.09, 4.64 ± 0.03, 4.72 ± 0.10, respectively), all contractions were abolished between 30 and 60 μM. Blunted contractions were associated with CyPPA depression of spontaneous Ca(2+) events in myometrial smooth muscle bundles. Augmentation of uterine contractions with oxytocin or prostaglandin F(2α) did not reduce CyPPA sensitivity or efficacy. Finally, in an RU486-induced preterm labor model, CyPPA significantly delayed time to delivery by 3.4 h and caused a 2.5-fold increase in pup retention. These data indicate that pharmacologic stimulation of myometrial K(Ca)2.2/2.3 channels effectively suppresses Ca(2+)-mediated uterine contractions and delays preterm birth in mice, supporting the potential utility of this approach in tocolytic therapies.     

A novel signaling pathway for beta-adrenergic receptor-mediated activation of phosphoinositide 3-kinase in H9c2 cardiomyocytes

Stimulation of cardiac beta-adrenergic receptors (beta-AR) activates both the G(s)- and G(i)-coupled signaling cascades, including the phosphoinositide 3 kinase (PI3K) pathway, that have important physiological implications. Multiple isoforms of PI3K exist in the heart. The goals of this study were to examine the intracellular signaling pathways linking beta-AR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute beta-AR stimulation with isoproterenol resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Cotreatment with ICI-118,551, but not CGP-20712, abolished the increase in PI3K activity, suggesting a beta(2)-AR-mediated event. PI3K activation was also abrogated by cotreatment with pertussis toxin, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2, a selective Src-family tyrosine kinases inhibitor), or AG-1296 [selective platelet-derived growth factor receptor (PDGFR) inhibitor] but not with an inhibitor for protein kinase A, protein kinase C, Ras, adenylyl cyclase, epidermal growth factor receptor, or insulin-like growth factor-1 receptor. beta-AR stimulation induced an increase in tyrosine phosphorylation of PDGFR, which was abolished by inhibition of Src either by PP2 or small interfering RNA. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a Gbetagamma sequestrant peptide derived from the COOH-terminus of beta-AR kinase-1 failed to activate PI3K after beta-AR stimulation, suggesting Gbetagamma is required for the transactivation. Furthermore, acute beta-AR stimulation in vivo resulted in increases in PDGFR-associated PI3K and PI3Kalpha isoform activities but not the activities of other isoforms (PI3Kbeta, -delta, -gamma) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of beta-AR-mediated transactivation of cardiac PI3Kalpha via sequential involvement of Galpha(i)/Gbetagamma, Src, and PDGFR.     

In vivo adaptive control of beta-receptors and adenylate cyclase during short-term heat exposure in rat parotid glands.

1. Adaptation of beta-adrenergic receptors (beta-AR) and adenylate cyclase (AC) in rat parotid glands during short-term heat exposure (33 degrees C) were studied. 2. Heat exposure reduced AC activity in response to isoproterenol (IPR). 3. The number of beta-AR on the cell surface significantly increased after 24 hr but returned to control level after 48 hr. 4. IPR-induced [3H]GDP release was significantly reduced throughout exposure. 5. The data suggest that the major factor which results in the desensitization of AC during short-term heat exposure is a blunted coupling between beta-AR and GTP binding protein(s).     

Anti-inflammatory and utero-relaxant effects in human myometrium of new generation phosphodiesterase 4 inhibitors

The anti-inflammatory and utero-relaxant effects of two potent phosphodiesterase 4 (PDE4) inhibitors of the latest generation: cilomilast (one of the most advanced PDE4 inhibitors in clinical development, reportedly more selective for PDE4D) and compound A (which displays 12-fold greater selectivity toward PDE4B and/or PDE4A than toward PDE4D) were evaluated in human uterine smooth muscle. We first established that these compounds exhibit greater efficacy in inhibiting total cAMP-PDE activity in pregnant versus nonpregnant myometrium (E(max) = 78.0% +/- 3.6% and 80.3% +/- 2.2% in pregnant versus 57% +/- 4.7% and 70.5% +/- 5.9% in nonpregnant women for compound A and cilomilast, respectively; P < 0.05 for both compounds), confirming the prominent participation of PDE4 isoforms in cAMP hydrolysis in the near-term pregnant myometrium. Using pregnant myometrial explants, we have shown that both these drugs and also rolipram, the prototype PDE4 inhibitor, produce concentration-dependent inhibition of lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNFalpha) release with similar potency in each case (pD2 = 8.0 +/- 0.5, 7.9 +/- 0.2, and 7.6 +/- 0.2 for compound A, cilomilast, and rolipram, respectively). The maximum inhibition produced is 65%. Pretreatment with forskolin or 8-bromo-cAMP mimics the PDE4 inhibitor effect. Furthermore, compound A and cilomilast both produce concentration-dependent inhibition of the spontaneous contractions of myometrial strips and are more potent in pregnant than in nonpregnant myometrium (pD2 = 7.3 +/- 0.7 and 8.1 +/- 0.3 in pregnant versus 6.2 +/- 0.9 and 6.6 +/- 0.1 in nonpregnant myometrium for compound A and cilomilast, respectively; P < 0.05 for both compounds). This demonstrates that the PDE4 isoforms involved in the mechanism of contraction are different in the pregnant and nonpregnant myometrium. Our study highlights the importance of developing PDE4 inhibitors for the pharmacological management of infection-induced preterm labor.     

The presence of Na+ channels in myometrial smooth muscle cells is revealed by specific neurotoxins

This paper shows the presence, in rat myometrial smooth muscles, of low affinity binding sites for tetrodotoxin with a K0.5 value of 2 microM. Electrophysiological experiments using both intact strips and single isolated myometrial cells in culture have shown that veratridine and sea anemone toxins reveal functional Na+ channels. The activity of these channels was blocked by tetrodotoxin (10 microM) or by removal of Na+ ions. Results presented here are the first direct demonstration of the existence in rat myometrium of Na+ channels of the tetrodotoxin-resistant type.     

Prostacyclin formation by myometrial & decidual fractions of the pregnant rat uterus

Chopped samples of myometrium, decidua and extrinsic blood vessels from the pregnant rat uterus when incubated at room temperature generated a prostacyclin-like substance. Activity in the incubation mixtures was compared against authentic prostacyclin in two assay systems: relaxation of strips of bovine coronary artery and inhibition of ADP-induced aggregation of rabbit platelet-rich plasma. Results estimated from inhibition of platelet aggregation showed that activity generated by all samples was low on day 12 of pregnancy (less than 0.25 ng/mg). However at the time of delivery (day 22) myometrial synthesis had increased 18.5 fold to over 3 ng/mg of prostacyclin whereas decidual production had only increased 5 times. As there was no increase in synthesis by the extrinsic uterine blood vessels over this period it is proposed that the myometrial muscle cells are the probable source of the prostacyclin-like material.     

Prostacyclin formation by myometrial & decidual fractions of the pregnant rat uterus.

Chopped samples of myometrium, decidua and extrinsic blood vessels from the pregnant rat uterus when incubated at room temperature generated a prostacyclin-like substance. Activity in the incubation mixtures was compared against authentic prostacyclin in two assay systems: relaxation of strips of bovine coronary artery and inhibition of ADP-induced aggregation of rabbit platelet-rich plasma. Results estimated from inhibition of platelet aggregation showed that activity generated by all samples was low on day 12 of pregnancy (less than 0.25 ng/mg). However at the time of delivery (day 22) myometrial synthesis had increased 18.5 fold to over 3 ng/mg of prostacyclin whereas decidual production had only increased 5 times. As there was no increase in synthesis by the extrinsic uterine blood vessels over this period it is proposed that the myometrial muscle cells are the probable source of the prostacyclin-like material.     

Mares with delayed uterine clearance have an intrinsic defect in myometrial function

Persistent, postmating endometritis affects approximately 15% of mares and results in reduced fertility and sizable economic losses to the horse-breeding industry. Mares that are susceptible to postmating endometritis have delayed uterine clearance associated with reduced uterine contractility. Unfortunately, the mechanism for reduced uterine contractility remains an enigma. The present study examined the hypothesis that mares with delayed uterine clearance have an intrinsic contractile defect of the myometrium. Myometrial contractility was evaluated in vitro by measuring isometric tension generated by longitudinal and circular uterine muscle strips in response to KCl, oxytocin, and prostaglandin F(2alpha) (PGF(2alpha)) for young nulliparous mares, older reproductively normal mares, and older mares with delayed uterine clearance. In addition, intracellular Ca(2+) regulation was evaluated using laser cytometry to measure oxytocin-stimulated intracellular Ca(2+) transients of myometrial cells loaded with a Ca(2+)-sensitive fluorescent dye, fluo-4. For all contractile agonists, myometrium from mares with delayed uterine clearance failed to generate as much tension as myometrium from older normal mares. Oxytocin-stimulated intracellular Ca(2+) transients were similar for myometrial cells from mares with delayed uterine clearance and from older normal mares, suggesting that the contractile defect did not result from altered regulation of intracellular Ca(2+) concentration. Furthermore, no apparent age-dependent decline was observed in myometrial contractility; KCl-depolarized and oxytocin-stimulated longitudinal myometrium from young normal mares and older normal mares generated similar responses. However, circular myometrium from young normal mares failed to generate as much tension as myometrium from older normal mares when stimulated with oxytocin or PGF(2alpha), suggesting possible age-related alterations in receptor-second messenger signaling mechanisms downstream of intracellular Ca(2+) release. In summary, for mares with delayed uterine clearance, an intrinsic contractile defect of the myometrium may contribute to reduced uterine contractility following breeding.     

Intracellular cyclic AMP concentration modulates gap junction permeability in parturient rat myometrium.

We investigated whether cell-to-cell coupling between myometrial cells of parturient rats is influenced by intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration. To evaluate the coupling, we measured input resistance (Ro) and injected Lucifer Yellow (LY) using microelectrode techniques. The intercellular spread of the dye was then observed. Longitudinal muscle strips from rat myometrium were exposed to isoproterenol, forskolin, or dibutyryl cAMP (DB-cAMP) to elevate cAMP. Isoproterenol (10(-11)-10(-6) M) and DB-cAMP (10(-5)-10(-3) M) hyperpolarized the resting membrane potential (Em) and increased Ro in a dose-dependent fashion. Forskolin (10(-6) M) also hyperpolarized Em and increased Ro. When LY was injected into a single cell, LY spread rapidly and extensively to neighboring cells in parturient control tissues, while LY transfer was completely blocked by any of the three agents at high concentrations. The increased Ro and blocked transfer of LY owing to these agents indicate that the cell-to-cell coupling was decreased both electrically and metabolically. Myometrial cells of parturient rats show increased number and size of gap junctions (GJs). The rapid and reversible decrease in coupling is interpreted to reflect the reduced permeability of GJs between the muscle cells because of an elevation of cAMP. Control of GJ permeability by this second messenger may be important for the physiological regulation of intercellular coupling and the extent of synchronizing and coordinating electrical, metabolic, and contractile activity in the uterine wall during pregnancy and parturition.     

Isoproterenol opens K+(ATP) channels via a beta2-adrenoceptor-linked mechanism in Sertoli cells from immature rats.

In the present study, we investigated the mechanism by which isoproterenol hyperpolarises membrane potential (MP) in Sertoli cells from seminiferous tubules of 15-day-old rat testes. Modification of MP and resistance (R0) was analysed using conventional intracellular glass microelectrodes. Isoproterenol (2 x 10(-6) M) induced an immediate and significant hyperpolarisation in the Sertoli-cell membrane. The beta2-AR antagonist, butoxamine (1 x 10(-6) M), nullified isoproterenol action. The effect of the beta1 antagonist, metoprolol (1 x 10(-6) M), was light and non-significant. Sulphonylurea glibenclamide inhibition of the K+(ATP) channels suppressed isoproterenol action, and testosterone, while depolarising Sertoli-cell MP closing the K+(ATP) channels through the PLC/PIP2 pathway, reduced beta-AR agonist-induced hyperpolarisation. Also, polycations LaCl3 and spermine reversed isoproterenol's hyperpolarisation effect, probably depolarising the membrane potential through ionic interaction neutralising the action of isoproterenol on K+(ATP) channels. Adenylate cyclase agonist forskolin (0.1 microM) rapidly hyperpolarised Sertoli-cell MP, mimicking the isoproterenol effect. These effects indicate that isoproterenol's action on K+(ATP) channel probably involves the known signalling cascade beta-AR/Gs/AC/cAMP/PKA. These results suggest that the isoproterenol-induced hyperpolarisation is mediated by the opening of K+(ATP) channels in Sertoli cells. This beta-adrenergic hyperpolarisation might play a physiological role in the modulation of MP.     

Beta-adrenergic receptor-mediated phospholipase C activation independent of cAMP formation in turkey erythrocyte membranes.

The effect of the beta-adrenergic receptor agonist isoproterenol on guanine nucleotide-dependent phospholipase C (PLC) activity was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes. In the presence of guanosine 5'-(gamma-thiotriphosphate) (GTP[S]) isoproterenol caused a dose-dependent stimulation of [3H]inositol phosphate ([3H]InsP) formation. The activation of PLC by GTP[S] occurred after an initial lag period of 1-2 min and was followed by a sustained rate of [3H]InsP formation which remained linear for 4-5 min. Isoproterenol decreased the lag period for GTP[S]-induced [3H]InsP formation and increased PLC activity at all time points following this lag. Consequently, isoproterenol shifted the dose-response curve for GTP[S] to the left (10-fold) and increased the maximal response. The EC50 value for isoproterenol-induced activation of PLC was 104 +/- 17 nM. Isoproterenol also potentiated GTP-dependent PLC activity but was ineffective in stimulating the enzyme in the presence of AIF4-. The PLC activation by isoproterenol was completely inhibited by propanolol and atenolol but was unaffected by prazosin or yohimbine. Although GTP[S] and isoproterenol could increase cAMP formation in this membrane preparation, the isoproterenol-induced stimulation of PLC occurred in the absence of ATP and was independent of cAMP formation. Furthermore, addition of cAMP, 8-bromo-cAMP, forskolin, or either the regulatory or catalytic subunits of cAMP-dependent protein kinase failed to stimulate [3H]InsP formation and had no effect on the responses elicited by GTP[S] and isoproterenol. Isoproterenol also stimulated [3H]InsP2 and [3H]InsP3 production in intact erythrocytes. Cholera toxin had no effect on [3H]InsP formation in the intact cells under conditions where it stimulated cAMP accumulation. In addition, the activation of PLC by GTP[S] and isoproterenol was unaffected in membranes prepared from cholera toxin-treated erythrocytes. These data demonstrate that stimulation of turkey erythrocyte beta-adrenergic receptors by isoproterenol results in a direct activation of guanine nucleotide-dependent PLC.     

Enhancement of the recycling and activation of beta-adrenergic receptor by Rab4 GTPase in cardiac myocytes

We investigate the role of Rab4, a Ras-like small GTPase coordinating protein transport from the endosome to the plasma membrane, on the recycling and activation of endogenous beta-adrenergic receptor (beta-AR) in HL-1 cardiac myocytes in vitro and transgenic mouse hearts in vivo. Beta1-AR, the predominant subtype of beta-AR in HL-1 cardiac myocytes, was internalized after stimulation with isoproterenol (ISO) and fully recycled at 4 h upon ISO removal. Transient expression of Rab4 markedly facilitated recycling of internalized beta-AR to the cell surface and enhanced beta-AR signaling as measured by ISO-stimulated cAMP production. Transgenic overexpression of Rab4 in the mouse myocardium significantly increased the number of beta-AR in the plasma membrane and augmented cAMP production at the basal level and in response to ISO stimulation. Rab4 overexpression induced concentric cardiac hypertrophy with a moderate increase in ventricle/body weight ratio and posterior wall thickness and a selective up-regulation of the beta-myosin heavy chain gene. These data provide the first evidence indicating that Rab4 is a rate-limiting factor for the recycling of endogenous beta-AR and augmentation of Rab4-mediated traffic enhances beta-AR function in cardiac myocytes.     

The role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium

Cells growing in culture with previously described properties of rat uterine smooth muscle accumulated 45Ca2+ from the medium. Ca2+ uptake by these cells was stimulated by the addition to the medium of 8-bromo-cGMP but not by 8-bromo-cAMP. Ca2+ uptake was also stimulated by carbachol and by the nitro-vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor-mediated functions that reflected some neurotransmitter-sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor. The cells were also sensitive to the beta-adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca2+ uptake. Carbachol failed to inhibit isoproterenol-dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine-sensitive properties of uterine smooth muscle may be retained in cell culture.     

Identification of beta-N-acetylhexosaminidase A in mouse tissues with the fluorigenic substrate 4-methylumbelliferyl-beta-N-acetylglucosamine 6-sulphate.

The ionic mechanism of inositol trisphosphate (InsP3)-induced Ca2+ release was investigated in microsomes (microsomal fractions) isolated from rat brain. InsP3 stimulated Ca2+ release from microsomes incubated in media containing 100 mM-KCl. The InsP3-induced Ca2+ release was insensitive to a variety of Ca2+-channel blockers; however, the K+-channel blockers tetraethylammonium chloride (TEA; 1 mM) and 9-tetraethylammonium chloride (9-TEA; 1 mM) blocked InsP3-induced Ca2+ release. Moreover, addition of InsP3 increased 86Rb+ influx into the microsomes. The influx of 86Rb+ also was sensitive to TEA and 9-TEA. The above results suggest that InsP3-induced Ca2+ release requires an opposite flow of K+ ions, and modulation of K+ channels by TEA and 9-TEA may underlie the inhibition of InsP3-induced Ca2+ release from brain microsomes by these agents.     

Myometrial adenylyl cyclase protein decreases on the last day of pregnancy in the rat

To determine whether gestation-related changes in responsiveness of the rat uterus to beta-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12-14) and then late (Days 18-20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the beta-adrenergic receptor (isoproterenol, 10(-4) M), the G protein (GTP, 10(-5) M), or the adenylyl cyclase enzyme (MnCl(2), 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [(3)H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor.     

Corticotropin-releasing hormone effects on human pregnant vs. nonpregnant myometrium explants estimated from a mathematical model of uterine contraction.

In this paper, we applied a new theoretical model of uterine contraction to a large panel of human pregnant and nonpregnant myometrial strips, treated or not by corticotrophin-releasing hormone (CRH). This model is based on a fine analysis of the contraction curves. This analysis yielded four mathematical parameters (beta, theta, tau 1, and tau 2) related to excitability, duration of plateau phase, and time constants for relaxation describing, respectively, the different portions of the contraction cycle. This leads to specific differences in spontaneous contractile activity between pregnant and nonpregnant states. The relaxing effect of CRH in the pregnant state is presumably correlated with the origin of the strips (the lower uterine segment). Besides our observation of a specific receptor-dependent relaxing effect of CRH in both pregnant and nonpregnant myometrium, we could identify highly significant effects at given CRH concentration for beta in nonpregnant myometrium and for theta, tau 1, and tau 2 in pregnant myometrium. In addition, highly significant differences were found between pregnant and nonpregnant myometrium. Also, we discovered a strong correlation between theta and tau 1, specifically in the pregnant state. Although the biochemical signification of these results remains to be elucidated, they contribute to emphasize the complex network of CRH action at the myometrial level. Furthermore, our approach could pave the way toward a better analysis of the efficacy of the uterine contractile behavior.