Clarithromycin inhibits myometrial contractions in isolated human myometrium independent of stimulus

Erythromycin has a well-known dual effect on the contractility of the gastrointestinal system and recently has also been shown to inhibit contractions of the rat myometrium. The aim of the present study was to investigate the effects of clarithromycin on oxytocin, prostaglandin F2alpha (PGF2alpha) and KCl-induced contractions of human myometrium in vitro. Myometrial strips were obtained from pregnant women undergoing elective Cesarean section and the strips were suspended in a jacketed organ bath filled with Krebs solution at 37 degrees C (pH 7.4) and continuously aired with 95% oxygen and 5% carbon dioxide. Isometric contractions were measured using a force displacement transducer. Oxytocin, PGF2alpha, KCl and clarithromycin were applied to the tissue bath and the amplitude and frequency of contractions were evaluated at 20-min intervals. Freidmann analysis of variance, Kruskal Wallis and Wilcoxon Rank tests were used for statistical analysis of the data. Clarithromycin dose dependently inhibited the amplitude of contractions independent of the stimulus. Pre-treatment with apamin prevented clarithromycin-induced effects on amplitude and frequency of contractions. We conclude that the macrolide antibiotic clarithromycin may have a direct inhibitory effect on contractions of human myometrium.     

Antioxidants prevent gamma-hexachlorocyclohexane-induced inhibition of rat myometrial gap junctions and contractions

Lindane (gamma-hexachlorocyclohexane) is a commonly used pesticide that bioaccumulates in mammalian adipose tissue. Lindane inhibits gap junctional intercellular communication and oscillatory contractions of pregnant rat myometrium in vitro. The present study investigated the role of oxidative stress in lindane's inhibition of myometrial function in mid-gestation pregnant rat uteri. Lucifer yellow dye was microinjected into cultured myocytes to assess gap junctional intercellular communication. Lindane exposure (100 microM) resulted in a time-dependent, biphasic inhibition of dye transfer. This pattern of inhibition was also seen upon cell exposure to the pro-oxidant, tert-butyl hydroperoxide (100 microM). Lindane's initial and secondary-onset dye transfer inhibitions were reversed by cotreatment and pretreatment with the antioxidants, alpha-tocopherol (25-100 microM), diphenyl-1,4-phenylene diamine (10-30 microM), and superoxide dismutase (100-400 U/ml). D-mannitol (100-300 mM) also reversed lindane's initial dye transfer inhibition. Nitro blue tetrazolium reduction to formazan (measured spectrophotometrically) was elevated upon exposure of cultured cells to lindane or tert-butyl hydroperoxide, indicating the presence of reducing agents. Lipid peroxidation, assessed as thiobarbituric acid-reactive substances, was also elevated in lindane-exposed cell cultures. alpha-Tocopherol reversed this elevation. Finally, uterine contractility was assessed by measuring isometric contractions of uterine strips hung in standard muscle baths. Pretreatment with alpha-tocopherol prevented lindane's abolishment of uterine contractions in vitro. These data support the hypothesis that lindane inhibits uterine contractility and myometrial gap junctions by establishing an oxidative stress environment.     

Effects of simvastatin treatment on oxidant/antioxidant state and ultrastructure of diabetic rat myocardium

In the present study we investigated the effects of simvastatin treatment on lipid metabolism and peroxidation, antioxidant enzyme activities and ultrastructure of the diabetic rat myocardium. Diabetes was induced by single injection of streptozotocin (45 mg/kg i.p.). Eight weeks after induction of diabetes, a subgroup of control and of diabetic rats was treated with simvastatin for 4 weeks (10 mg/kg/day, orally). Blood glucose, plasma cholesterol and triacylglycerol, as well as levels of cardiac thiobarbituric acid reactive substances (TBARS) were significantly increased in diabetic rats. The activities of antioxidant enzymes, catalase (CAT) and glutathione peroxidase (GSHPx), were also elevated in the diabetic myocardium. Treatment with simvastatin markedly reduced serum triacylglycerol and cholesterol, and partially controlled hyperglycemia in diabetic animals. The increased activation of antioxidant enzymes and the excess of lipid peroxidation measured by TBARS were completely reversed by simvastatin treatment. Diabetic rats displayed ultrastructural ischemia-like alterations of cardiomyocytes and capillaries, which support oxidative stress-induced tissue remodelling. In the diabetic myocardium simvastatin treatment partly attenuated angiopathic and atherogenic processes, detected by electron microscopy. These results suggest that simvastatin, known as a lipid-lowering drug, may positively affect diabetes induced cardiovascular complications via reducing risks of atherosclerotic pathological processes, such as imbalance between oxidant and antioxidant state.     

Involvement of lipoxygenase products in myometrial contractions

Studies with an in situ preparation of guinea pig uterus suggest the possible involvement of the lipoxygenase pathway of arachidonic acid (AA) metabolism in myometrial contractions. Female guinea pigs were sensitized to ovalbumin (OA) on day one of their estrous cycle. On day 14, these pigs were anesthetized and the uterus was cannulated for measuring contractions. OA challenge, with histamine antagonism (methapyrilene) resulted in uterine contractions which significantly raised myometrial tonus, presumably due to AA metabolites. Pretreatment with high doses of indomethacin resulted in only 60% inhibition of the OA induced contraction, suggesting the remaining contraction was due to something other than cyclooxygenase products. In the presence of indomethacin and methapyrilene, the addition of AA to increase available substrate caused increased myometrial tone following antigen challenge. This increase in uterine tone was inhibited in a dose dependent fashion by FPL-55712 demonstrating that leukotrienes can contract the uterus and that antigen challenge may provide a means for studying leukotriene involvement in uterine pathophysiology.     

Involvement of lipoxygenase products in myometrial contractions

Studies with an preparation of guinea pig uterus suggest the possible involvement of the lipoxygenase pathway of arachidonic acid (AA) metabolism in myometrial contractions. Female guinea pigs were sensitized to ovalbumin (OA) on day one of their estrous cycle. On day 14, these pigs were anesthetized and the uterus was cannulated for measuring contractions. OA challenge, with histamine antagonism (methpyrilene) resulted in utering contractions which significantly raised myometrial tonus, presumably due to AA metabolites. Pretreatment with high doses of indomethacin resulted. in only 60% inhibition of the OA induced contraction, suggesting the remaining contraction was due to something other than cyclooxygenase products. In the presence of indomethacin and methapyrilene, the addition of AA to increase available substrate caused increased myometrial tone following antigen challenge. This increase in uterine tone was inhibited in a dose dependent fashion by FPL-55712 demonstrating that leukotrienes can contract the uterus and that antigen challenge may provide a means for studying leukotriene involvement in uterine pathophysiology.     

Effect of cortisol on norepinephrine-mediated contractions in ovine uterine arteries

Cortisol potentiated norepinephrine (NE)-mediated contractions in ovine uterine arteries (UA). We tested the hypothesis that cortisol regulated alpha(1)-adrenoceptor-mediated pharmacomechanical coupling differentially in nonpregnant UA (NUA) and pregnant UA (PUA). Cortisol (10 ng/ml for 24 h) significantly increased contractile coupling efficiency of alpha(1)-adrenoceptors in NUA, but increased alpha(1)-adrenoceptor density in PUA. Cortisol potentiated NE-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] synthesis in both NUA and PUA, but increased coupling efficiency of alpha(1)-adrenoceptors to Ins(1,4,5)P(3) synthesis only in NUA. Carbenoxolone alone did not affect NE-mediated Ins(1,4,5)P(3) production, but significantly enhanced cortisol-mediated potentiation of NE-stimulated Ins(1,4,5)P(3) synthesis in PUA. In addition, cortisol potentiated the NE-induced increase in Ca(2+) concentration in PUA, but increased NE-mediated contraction for a given amount of Ca(2+) concentration in NUA. Collectively, the results indicate that cortisol potentiates NE-mediated contractions differentially in NUA and PUA, i.e., by upregulating alpha(1)-adrenoceptor density leading to increased Ca(2+) mobilization in PUA while increasing alpha(1)-adrenoceptor coupling efficiency and myofilament Ca(2+) sensitivity in NUA. In addition, the results suggest that pregnancy increases type 2 11 beta-hydroxysteroid dehydrogenase activity in the UA.     

Effect of proteinase inhibitors on rabbit uterine ultrastructure

Rabbit uterine epithelium was examined by electron microscopy after treatment with two proteinase inhibitors, aprotinin and antipain, administered as a single injection. Both compounds had similar effects, those of antipain being slighter. After treatment, uterine epithelium showed an increased number of very enlarged cells. Lateral cell membranes were often brocken off; basal cell membrane and basement membrane integrities are impaired. The possible interference of the system proteinase-proteinase inhibitors with the implantation process was discussed.     

Effect of brief vascular perfusion on the ultrastructure and activity of mitochondria isolated from the rat heart

Summary Mitochondria isolated from heart tissue after a 1-min perfusion with Hanks medium were found to have significantly lower rates of State-3 respiration and respiratory control ratios compared to mitochondria isolated from non-perfused hearts. Examination of the mitochondrial preparations by electron microscopy revealed that a large proportion of the mitochondria isolated from perfused heart tissue were swollen and broken compared to mitochondria from non-perfused hearts.     

Effect of oleic, lauric and myristic acids on phenylephrine-induced contractions of isolated rat vas deferens

D-004, a lipid extract of Roystonea regia fruits that contains oleic, lauric and myristic acids as major components inhibits alpha1-adrenoreceptors-mediated contractile responses in isolated rat vas deferens and prostate trips; no study has demonstrated a similar effect for oleic, lauric or myristic acids individually. Therefore, the effects of D-004 (250 microg/mL), oleic (100 microg/mL), lauric (50 microg/mL) or myristic (25 microg/mL) acids and their combined effects on phenylephrine (PHE: 10(-7)-10(-4) mol/L) induced contractions has been studied. No treatment changed the basal tone of the preparations, but all inhibited PHE-induced contractions. D-004 produced the highest inhibition, followed by lauric acid, which was more effective than myristic and oleic acids against PHE-induced contractions of control group. D-004 and the mixture of the three acids produced similar inhibitions.     

Use of a novel and highly selective oxytocin receptor antagonist to characterize uterine contractions in the rat

Spontaneous and induced uterine contractions in the rat were found to be inhibited by a novel and selective oxytocin receptor antagonist GSK221149A (3R,6R)-3-Indan-2-yl-1-[(1R)-1-(2-methyl-1,3-oxazol-4-yl)-2-morpholin-4-yl-2-oxoethyl]-6-[(1S)-1-methylpropyl]-2,5-piperazinedione. GSK221149A displayed nanomolar affinity (K(i) = 0.65 nM) for human recombinant oxytocin receptors with >1,400-fold selectivity over human V1a, V1b, and V2 receptors. GSK221149A had similar affinity (K(i) = 4.1 nM) and selectivity for native oxytocin receptors from rat and produced a functional, competitive block of oxytocin-induced contractions in isolated rat myometrial strips with a pA(2) value of 8.18. Intravenous administration of GSK221149A produced a dose-dependent decrease in oxytocin-induced uterine contractions in anesthetized rats with an ID(50) = 0.27 +/- 0.60 mg/kg (corresponding plasma concentrations were 88 ng/ml). Oral administration of GSK221149A (5 mg/kg) was effective in inhibiting oxytocin-induced uterine contractions after single and multiple (4-day) dosing. Spontaneous uterine contractions in late-term pregnant rats (19-21 days gestation) were significantly reduced by intravenous administration of 0.3 mg/kg of GSK221149A. These results provide further evidence that selective oxytocin receptor antagonism may offer an effective treatment for preterm labor.     

Effect of gossypol on the ultrastructure of rat spermatozoa

Summary Gossypol was found to induce sterility in male rats when administered orally. A reduction in the number of spermatozoa in the epididymis from the gossypol-treated rats was observed when compared to the control animals. An examination of the spermatozoa from the treated rats showed the following ultrastructural modifications: disorganization of the mitochondrial sheath and missing cell membrane from the middle piece, broken cell membrane and missing members of both outer fibers and inner microtubules of the principal piece, and broken cell membrane of the sperm head. Serial mating experiments proved that gossypol-treated males were indeed sterile. The results suggest that gossypol at low concentrations is able to affect the motility of spermatozoa, thus contributing to its contraceptive action.     

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.