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.     

Platelet-activating factor acetylhydrolase isoforms I and II in human uterus. Comparisons with pregnant uterus and myoma

The concentrations of platelet-activating factor (PAF) that possesses the ability to stimulate myometrial contraction are partially regulated by intracellular type of platelet-activating factor acetylhydrolase (PAF-AH) in many tissues. Tissue cytosol contains at least two intracellular PAF-AH, isoforms I and II. To examine the relationship between the activity and isoforms of intracellular PAF-AH in human uterine myometrium and myoma, we assayed the PAF-AH activity and identified the PAF-AH isoforms I and II by Western blot analysis. The intense bands of the alpha2 and ss subunits of PAF-AH isoform I were detected in nonpregnant uterus; however, the specific bands of the alpha1 subunit of PAF-AH isoform I and the PAF-AH isoform II were extremely weak. The levels of the alpha2 and ss subunits and PAF-AH activity in pregnant uterus (37-39 wk gestation) were significantly lower than those in nonpregnant uterus. On the other hand, the level of ss subunit and the PAF-AH activity in myoma were significantly higher than those in nonpregnant uterus. No significant difference was found in the expression of the PAF-AH isoform II among three tissues. These results indicate that the change in the PAF-AH activity observed in pregnant uterus and myoma are due to the lower or higher protein expression of the PAF-AH isoform I, especially the alpha2 and/or ss subunits. The decrease of the uterine PAF-AH activity in the late stage of pregnancy may facilitate the action of PAF to stimulate myometrial contraction.     

Influence of 5alpha- and 5beta-reduced progestins on the contractility of isolated human myometrium at term.

It is very well known that progesterone induces uterine relaxation on myometrium contractile activity. However, little attention has been paid to the effect induced by its metabolites on human uterine contractility. Therefore, we set out to analyze the potential relaxing effect of some 5alpha- and 5beta-reduced progesterone derivatives on the spontaneous contractility of myometrium from pregnant women. Samples were obtained by caesarian section at 38-40 weeks of pregnancy. Spontaneous uterine contractions were recorded in vitro in the presence of progesterone, or progestins independently, at different non-cumulative microM concentrations. The progestins elicited an immediate relaxing effect that was concentration-dependent. With the exception of two 5alpha-reduced progestins (5alpha and 3beta,5alpha), the remaining progestins used in the present study were more potent than progesterone. The potency order with respect to their IC50 values was: 3alpha,5alpha (35 microM) > 5beta (81 microM) > 3beta,5beta (156 microM) > 3alpha,5beta (205 microM) > P4 (225 microM) > 5alpha (19 mM) > 3beta,5alpha (28 mM). When tissues were washed, the contractile activity was recovered. This rapid and reversible relaxing effect was not blocking by antiprogestin RU 486, suggesting that is not through receptor-mediated genomic action. The metabolites from progesterone may also determine the pattern of motility, ensuring the necessary quiescent environment to prevent abortion during gestation.     

Inhibition of tissue transglutaminase 2 attenuates contractility of pregnant human myometrium

Premature delivery remains a serious risk factor in pregnancy, with currently licensed tocolytics unable to offer significant improvement in neonatal outcome. Further understanding of the regulators of uterine contractility is required to enable the development of novel and more effective tocolytic therapies. The transglutaminase family is a class of calcium-dependent, transamidating enzymes, of which tissue transglutaminase 2 is a multifunctional enzyme with roles in cell survival, migration, adhesion, and contractility. The aim of the present study was to investigate the role of this enzyme in regulating the contractility of pregnant human myometrium. Tissue strips from biopsy samples obtained at elective cesarean section were either allowed to contract spontaneously or induced to contract with oxytocin, phenylephrine, or bradykinin. Activity integrals, used to measure contractile activity, were taken following cumulative additions of the reversible, polyamine transglutaminase inhibitors cystamine and mono-dansylcadaverine and the irreversible, site-specific transglutaminase inhibitors N-benzyloxycarbonyl-l-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine and 1,3-dimethyl-2[(oxopropyl)thio]imidazolium. The ability of cystamine and mono-dansylcadaverine to affect oxytocin-mediated calcium mobilization within primary cultured myometrial cells was also measured utilizing a calcium indicator. All inhibitors attenuated myometrial contractions in a concentration-dependent manner independent of the method of contraction stimulus. Similarly cultured myometrial cells preincubated with cystamine and mono-dansylcadaverine displayed an altered calcium response to oxytocin stimulation. Our findings demonstrate a potential role for tissue transglutaminase 2 in regulating uterine contractility in pregnant human myometrium that may be associated with the calcium signaling cascade required for contraction.     

Endogenous prostaglandins regulate spontaneous contractile activity of uterine strips isolated from non-pregnant pigs

Myometrial strips isolated from non-pregnant pigs show spontaneous contractile activity. In the present study, the involvement of endogenous prostaglandins in regulation of uterine spontaneous contraction was investigated using mechanical, immunohistochemical and biochemical approaches. Immunohistochemical study and Western blot analysis for immunoreactive cyclooxygenase (COX) indicated that COX-1 but not COX-2 was expressed predominantly in the myometrium of non-pregnant pigs in a muscle layer-dependent manner (longitudinal muscle>circular muscle). Pretreatment of uterine strips with indomethacin and selective COX-1 inhibitors (SC-560 and FR122047) significantly reduced both the amplitude and frequency of spontaneous contraction in the longitudinal muscle, but inhibition by COX inhibitors was negligible in the circular muscle. On the other hand, CAY10404, a COX-2 inhibitor, did not change the spontaneous contraction in either of the muscle layers. Pretreatment with SC-560 reduced myometrial PGF(2alpha) and PGE(2) levels. Contractile FP and EP(3) receptors were expressed in a muscle layer-dependent manner (longitudinal muscle>circular muscle), similar to the expression pattern of COX-1. In conclusion, endogenous prostaglandins produced by COX-1 regulate spontaneous contractile activity of non-pregnant porcine uterine longitudinal muscle selectively due to the heterogeneous expression of contractile prostanoid receptors and COX-1.     

Functional innervation of the myometrium of Myotis lucifugus, the little brown bat

Myometria of pregnant and nonpregnant Myotis lucifugus were studied in vitro by using electrical field stimulation as well as autonomic agonists and antagonists to determine whether functional responses corresponded with structural evidence showing abundant adrenergic and sparse cholinergic innervation, which uniquely does not disappear during pregnancy. Field stimulation (70 V, 0.6 ms, 5.0-s pulse train, 2.5 - 60 Hz) of myometria from nonpregnant (hibernating) bats produced graded responses consisting of an initial alpha-adrenergic contraction and a subsequent beta-adrenergic relaxation phase. Responses were sensitive to both the nerve poison tetrodotoxin and the adrenergic antagonist guanethidine, demonstrating that they resulted from stimulation of intrinsic adrenergic nerves. Field stimulation responses were unaffected by atropine indicating that there was no functional cholinergic innervation, even though carbachol-induced contraction showed that muscarinic receptors were present. In contrast, functional innervation of cervical tissue was cholinergic and nonadrenergic-non-cholinergic, but not adrenergic. At the beginning of active gestation, some myometrial preparations exhibited little of no response to field stimulation. However, as uterine size increased, the biphasic response to field stimulation was enhanced, particularly the inhibitory (beta-adrenergic) phase. Moreover, the contractile phases, though reduced, was not abolished by alpha-adrenergic antagonists. The residual contractile response was also tetrodotoxin-resistant, suggesting that the myometrium was sensitive to direct electrical stimulation. Near the end of pregnancy, myometrial tissue became nonresponsive to both field stimulation and autonomic agonists, suggesting an absence of available receptor sites on muscle cells.     

The presence of prostacyclin binding sites in nonpregnant bovine uterine tissue

Myometrium of various animal species makes a considerable amount of prostacyclin (PGI2) which is a potent myometrial and uterine vascular smooth muscle relaxing agent. This action of PGI2 is perhaps mediated by binding to specific receptors, which have never been demonstrated in uterine tissue of any animal species until very recently. The quantitative light microscopic autoradiographic approach used in the present studies demonstrated that while bovine myometrial smooth muscle and uterine vascular smooth muscle contained PGI2 specific binding sites, endometrial and perimetrial cells contained few or no binding sites. The number of binding sites in circular and elongated myometrial smooth muscle and in arteriolar smooth muscle were similar (P greater than 0.05). The PGI2 binding to the uterine cells was greatly reduced (P less than 0.001) following coincubation with excess unlabeled PGI2, but not with its stable metabolite, 6-keto PGF1 alpha, PGE2, PGF2 alpha and leukotriene C4 which bind to nonpregnant bovine uterine tissue, also had no effect of PGI2 binding. In conclusion, nonpregnant bovine uterine tissue contain specific PGI2 binding sites which may mediate its potent relaxing effect on myometrium and uterine vasculature.     

Distension of the uterus induces HspB1 expression in rat uterine smooth muscle

The uterine musculature, or myometrium, demonstrates tremendous plasticity during pregnancy under the influences of the endocrine environment and mechanical stresses. Expression of the small stress protein heat shock protein B1 (HspB1) has been reported to increase dramatically during late pregnancy, a period marked by myometrial hypertrophy caused by fetal growth-induced uterine distension. Thus, using unilaterally pregnant rat models and ovariectomized nonpregnant rats with uteri containing laminaria tents to induce uterine distension, we examined the effect of uterine distension on myometrial HspB1 expression. In unilaterally pregnant rats, HspB1 mRNA and Ser(15)-phosphorylated HspB1 (pSer(15) HspB1) protein expression were significantly elevated in distended gravid uterine horns at days 19 and 23 (labor) of gestation compared with nongravid horns. Similarly, pSer(15) HspB1 protein in situ was only readily detectable in the distended horns compared with the nongravid horns at days 19 and 23; however, pSer(15) HspB1 was primarily detectable in situ at day 19 in membrane-associated regions, while it had primarily a cytoplasmic localization in myometrial cells at day 23. HspB1 mRNA and pSer(15) HspB1 protein expression were also markedly increased in ovariectomized nonpregnant rat myometrium distended for 24 h with laminaria tents compared with empty horns. Therefore, uterine distension plays a major role in the stimulation of myometrial HspB1 expression, and increased expression of this small stress protein could be a mechanoadaptive response to the increasing uterine distension that occurs during pregnancy.     

Evaluation of tocolytic efficacy of selective beta2 adrenoceptor agonists on buffalo uterus

Present study was conducted on prostaglandin F2alpha (PGF2alpha), oxytocin, (OT), potassium chloride (KCI) and barium chloride (BaCl2) pre-contracted perimetrial uterine strips of dioestrus and pregnant buffaloes to evaluate the tocolytic efficacy of selective beta2 adrenoceptor agonists-albuterol (salbutamol) and terbutaline. Cumulative concentration-response curves of both the beta2 adrenoceptor agonists were constructed and the mean effective concentration (EC50) values determined and compared statistically. Based on the comparative EC50 values in relaxing the pre-contracted uterine strips with different spasmogens, the rank order potency of albuterol was found to be--PGF2alpha > BaCl2 > OT > KCl on uterine strips from dioestrus animals, while OT> BaCl2> PGF2alpha >KCl on the uterine strips of pregnant buffaloes. The rank order potency of terbutaline on uterine strips from dioestrus stage animals was- BaCl2 > OT > KCl > PGF2alpha, while BaCl2 > PGF2alpha > KCl > OT on uterine tissues of pregnant animals. Thus, irrespective of the state of uterus, whether gravid or non-gravid, KCl-depolarized uterine tissues required comparatively higher concentrations of albuterol or terbutaline to produce tocolytic effect. High concentrations of K+ in biophase may have interfered with the beta2 adrenoceptor agonists-induced outward K+ current and hyperpolarization. From the results of present study, it was evident that selective beta2 adrenergic agonists had good tocolytic efficacy on the uterus of buffaloes. Further, indirectly the possibility of existence and activation of K(Ca) channels by selective beta2 adrenoceptor agonists in mediating tocolysis of buffalo myometrium can not be ruled out, however, detailed studies using specific K(Ca) channel blockers are required for characterizing the nature of such channels in buffalo uterus.     

Long-term hypoxia changes myometrial responsiveness and oxytocin receptors in the pregnant ewe: differential effects on longitudinal versus circular smooth muscle

Previous studies from our laboratory demonstrated that long-term hypoxia (LTH) altered in vitro contractile responses to oxytocin in full-thickness myometrial strips from pregnant sheep. The present study was designed to determine, first, if the reduced contractile response to oxytocin following LTH is the result of combined effects on longitudinal and circular smooth muscle or if the effect is specific to a single muscle layer and, second, if the reduced contractile response to oxytocin following LTH is caused by changes in oxytocin-receptor protein. Pregnant ewes were maintained at high altitude (3820 m) from Day 30 to Days 137-142 of gestation, when the ewes were killed for collection of myometrial tissue. Tissue was also collected from age-matched, normoxic controls. Longitudinal and circular layers were separated, length-tension curves generated to determine optimal resting tension, and all strips exposed to increasing half-log doses of oxytocin ranging from 10-12 to 10-6.5 M. The expression of oxytocin-receptor protein was measured using Western blot analysis. We found that LTH did not affect KCl-induced contraction of either smooth muscle layer, whereas the sensitivity of both myometrial layers to oxytocin was altered. A decreased maximum contractile response of the circular layer to oxytocin was also observed. Additionally, LTH decreased expression of oxytocin-receptor protein in the circular layer and increased levels in the longitudinal layer. Results from the present study indicate that LTH alters contractile responses and oxytocin-receptor protein expression in a layer-specific manner in the pregnant sheep myometrium.     

Regulation of myometrial phospholipase C system and uterine contraction by beta-adrenergic receptors in midpregnant rat

We investigated whether beta-adrenergic receptors (beta-AR) regulate the phospholipase C (PLC) system in midpregnant rat myometrium. PLCbeta isoforms were characterized, and the effect of isoproterenol (beta-adrenergic agonist) was tested on myometrial inositol phosphate (InsP) production and uterine contraction. Using specific antibodies, we showed that rat myometrium expresses PLCbeta1, PLCbeta3, and PLCbeta4, and to a lesser degree PLCbeta2. Quantitative analysis revealed that PLCbeta isoforms are differentially expressed during pregnancy. Indeed, the amount of PLCbeta4 is increased at midpregnancy, whereas PLCbeta1, PLCbeta2, and PLCbeta3 are up-regulated at term. At midpregnancy, pretreatment of myometrial strips with isoproterenol significantly reduced basal and agonist-stimulated InsP production. Forskolin, a diterpene that increases cAMP accumulation by directly activating adenylyl cyclases, had no effect on InsP production. In contrast, two global potassium (K+) channel inhibitors, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), prevented attenuation of InsP production by isoproterenol. Isoproterenol also significantly decreased spontaneous and agonist-induced contraction of the longitudinal layer of midpregnant rat myometrium. Preincubation of uterine strips with TEA plus 4-AP prior to beta-AR activation blocked only partial uterine relaxation, whereas Forskolin was as potent as isoproterenol. This indicates that beta-AR operate through both K+ channels and cAMP to induce uterine relaxation. In conclusion, we show for the first time that three myometrial PLCbeta isoforms (PLCbeta1, PLCbeta2, and PLCbeta3) are down-regulated at midpregnancy. At this period, beta-AR reduce basal and agonist-stimulated InsP production through activation of K+ channels. Altogether, these mechanisms could act to decrease responsiveness of the longitudinal layer of myometrium to contractant factors.     

Differences in the gestational pattern of mRNA expression of the Rnd family in rat and human myometria

Uterine myometrial contractility remains a poorly characterized area of research in reproductive physiology. Rnd1, a novel member of the GTP-binding Rho protein family, inhibits Ca(2+)-sensitization by specifically interfering with a RhoA/Rho-activated kinases-dependent mechanism in smooth muscle. In addition to Rnd1, there are two other members, Rnd2 and Rnd3, in the Rnd family of Rho proteins. In the present comparative study of myometrial contractility in rats and humans, we found that all three Rnd mRNAs were expressed in nonpregnant rat myometrium and in nonpregnant human myometrial tissues. Although all three mRNA levels increased significantly after gestation in rat myometria, only Rnd1 expression was significantly greater after gestation in human samples. In the ovariectomized rat, administration of estrogen and/or progesterone increased the expression of all Rnd mRNAs. These results suggest that universal Rnd family up-regulation during pregnancy in rats may have an important role for negative-feedback control of uterine contraction during gestation by inhibiting RhoA-mediated increase in Ca(2+) sensitivity of contractile elements. Such increases in Rnd levels may be due to augmented levels of reproductive steroids in rats. Our data also point to gestational differences between rats and humans in Rnd isoform patterns.     

The effect of prostaglandin I on the contractility of the term pregnant human myometrium

Small myometrial strips were dissected from the upper and lower segments of the term pregnant human uterus. The specimens were superfused in organ chambers and contractile activity was recorded isometrically. In strips from the upper segment, prostacyclin (PGI2), induced an initial excitatory response followed in the majority of experiments by transient inhibition. In the lower segment the response was generally the same although direct inhibition without initial stimulation occurred in some cases. During the period of inhibition the specimens were refractory to iterated exposure to PGI2. Furthermore, during this period of PGI2-induced inhibition the muscle strip was also refractory to PGE2 but responded to PGF2 alpha and oxytocin by stimulation. After inhibition of spontaneous contractile activity induced by indomethacin PGI2 induced an excitatory response. The results do not indicate any critical change in the myometrial responsiveness of the upper uterine segment to PGI2 during labor. In strips from the lower segment obtained before labor there tended to be a dominance of non-responders and inhibition only as compared to the results during labor. Nevertheless, whether or not PGI2 under physiological or pharmacological conditions has any significant influence on the contractility of the term pregnant human uterus, still remains obscure. As judged from earlier reports from our laboratory and the present study it is evident that the uterine vessels are considerably more sensitive to the action of PGI2 than the myometrium.     

Stretch Activates Human Myometrium via ERK,Caldesmon and Focal Adhesion Signaling

An incomplete understanding of the molecular mechanisms responsible for myometrial activation from the quiescent pregnant state to the active contractile state during labor has hindered the development of effective therapies for preterm labor. Myometrial stretch has been implicated clinically in the initiation of labor and the etiology of preterm labor, but the molecular mechanisms involved in the human have not been determined. We investigated the mechanisms by which gestation-dependent stretch contributes to myometrial activation, by using human uterine samples from gynecologic hysterectomies and Cesarean sections. Here we demonstrate that the Ca requirement for activation of the contractile filaments in human myometrium increases with caldesmon protein content during gestation and that an increase in caldesmon phosphorylation can reverse this inhibitory effect during labor. By using phosphotyrosine screening and mass spectrometry of stretched human myometrial samples, we identify 3 stretch-activated focal adhesion proteins, FAK, p130Cas, and alpha actinin. FAK-Y397, which signals integrin engagement, is constitutively phosphorylated in term human myometrium whereas FAK-Y925, which signals downstream ERK activation, is phosphorylated during stretch. We have recently identified smooth muscle Archvillin (SmAV) as an ERK regulator. A newly produced SmAV-specific antibody demonstrates gestation-specific increases in SmAV protein levels and stretch-specific increases in SmAV association with focal adhesion proteins. Thus, whereas increases in caldesmon levels suppress human myometrium contractility during pregnancy, stretch-dependent focal adhesion signaling, facilitated by the ERK activator SmAV, can contribute to myometrial activation. These results suggest that focal adhesion proteins may present new targets for drug discovery programs aimed at regulation of uterine contractility.     

Effect of Indomethacin on cyclic AMP phosphodiesterase activity in myometrium from pregnant rhesus monkeys.

Our results indicate that indomethacin inhibits cyclic AMP phosphodiesterase in the myometrium of the pregnant rhesus monkey under in vitro as well as in vivo conditions. Kinetic data on extracts of myometrium from pregnant rhesus monkeys indicated two cyclic AMP phosphodiesterase activities. The apparent Km value for the high affinity enzyme averaged 3.9 muM and for the low affinity enzyme 23 muM; the Vmax values averaged 0.56 and 1.4 nmoles cyclic AMP hydrolized per mg protein min-1 respectively. When indomethacin was added to the myometrial extracts, the activity of the high Km phosphodiesterase was competitively inhibited, with an average Ki of 200 muM; the low Km enzyme was noncompetitively inhibited with an average Ki of 110 muM. Experiments on myometrial slices demonstrated that 10 muM indomethsacin potentiated the effect of PGE1 and epinephrine on cyclic AMP levels, presumably by inhibiting the phophodiesterase activity. The uterine relaxing effect of indomethacin is generally attributed to the inhibition of prostaglandin synthetase activity. However, treatment of pregnant rhesus monkeys with therapeutic doses of indomethacin resulted in a significant inhibition of myometrial cyclic AMP phosphodiesterase activity in association with uterine relaxation and prolongation of gestation.     

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.     

Effects of calcium channel blocker, mibefradil, and potassium channel opener, pinacidil, on the contractile response of mid-pregnant goat myometrium

The present study was undertaken to investigate the in vitro influence of mibefradil, a calcium channel blocker, and pinacidil, a potassium channel opener, on pregnant goat myometrial spontaneous rhythmic contractility and contractions induced with the agonist, oxytocin. Longitudinal strips from the distal region of uterus, collected from goats at midgestation, were mounted in an organ bath for recording isometric contractions. Mibefradil (10(-8)-10(-4) M) or pinacidil (10(-10)-10(-4) M), added cumulatively to the bath at an increment of 1 log unit, caused concentration-dependent inhibition of the spontaneous rhythmic contractions of isolated uterine strips. The rhythmic contraction was, respectively, abolished at 100 and 10 microM concentrations of mibefradil and pinacidil. In a concentration-dependent manner, mibefradil (1 and 10 microM) antagonized the contractions elicited with oxytocin (10(-5)-10(-2) IU). Pretreatment of uterine strips with glibenclamide (10 microM), a selective KATP channel blocker, caused a rightward shift of the concentration-response curve of pinacidil with a concomitant decrease in its pD2 value. Pinacidil (0.3, 1 and 3 microM), in a concentration-related manner, antagonized the oxytocin (10(-5)-10(-2) IU)-induced contractile response. The inhibition of spontaneous rhythmic contractions and antagonism of oxytocin-induced contraction by mibefradil in the pregnant goat myometrium may be related to the antagonism of voltage-dependent Ca2+ channels, while by pinacidil suggests that KATP channel could be a therapeutic target for tocolysis.     

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.