Effects of 5-nitro-2-(3-phenylpropylamino) benzoic acid,anthracene-9-carboxylate,and zaprinast on endothelin-1-induced contractions of pregnant rat myometrium

OBJECTIVE: The effects of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), anthracene-9-carboxylate (9-AC) (chloride channel blockers) and zaprinast (an inhibitor of phosphodiesterase-5) on endothelin-1 (ET-1) induced contractions of pregnant rat myometrium were investigated in vitro. METHODS: Isolated myometrial strips were obtained from pregnant rats, and the strips were mounted in organ baths for recording of isometric tension (n=8). The effects of 10(-7) to 10(-4)M NPPB, 10(-7) to 10(-4)M 9-AC, and 10(-7) to 10(-4)M zaprinast on 10(-8)M ET-1-induced contractions of pregnant rat myometrial smooth muscle were recorded. RESULTS: NPPB and 9-AC increased the amplitude of ET-1-induced myometrial contractions, while decreasing the frequency, in a concentration-dependent manner. The amplitude of myometrial contractions were significantly increased by NPPB and 9-AC beginning from the concentration of 10(-6)M. The frequency of myometrial contractions were significantly decreased by NPPB and 9-AC beginning from the concentration of 10(-6)M. Zaprinast inhibited the amplitude and frequency of ET-1-induced myometrial contractions in a concentration-dependent manner. Zaprinast-induced decreases in amplitude and frequency of myometrial contractions reached statistical significance beginning from the concentrations of 10(-7)M and 10(-5)M, respectively. CONCLUSION: These data provide evidence that the ET-1-induced contractions of pregnant rat myometrial strips may be modulated by chloride channels. In addition, zaprinast effectively inhibited ET-1-induced contractions in pregnant rat myometrium.     

Comparison of the effects of nimesulide and 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanone (DFU) on contractions of isolated pregnant human myometrium

OBJECTIVE: To compare the effects of 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanone (DFU) and nimesulide, selective COX-2 inhibitors, on the amplitude and frequency of KCl-, oxytocin-, and PGF(2alpha)-stimulated contractions of isolated pregnant human myometrial strips. METHODS: Isolated myometrial strips were obtained from 20 pregnant women undergoing elective cesarean section. These strips were mounted in organ baths for recording of isometric tension. The effects of cumulative concentrations of nimesulide and DFU on KCl-, oxytocin-, and PGF(2alpha)-stimulated myometrial contractions were measured, and values for -log(10)EC(50) and mean maximal inhibition (E(max)) were compared. Nimesulide (10(-8) to 10(-4)M) and DFU (10(-8) to 10(-4)M) inhibited in a concentration-dependent manner the KCl-, oxytocin-, and PGF(2alpha)-stimulated contractions of myometrial strips, with a significant effect on the amplitude (10(-7) to 10(-4)M) and the frequency (10(-6) to 10(-4)M). RESULTS: The inhibitor effect of DFU was more potent than nimesulide on KCl-, oxytocin-, and PGF(2alpha)-stimulated myometrial contractions, however, the inhibitor effects of nimesulide and DFU was much greater on KCl-stimulated contractions than on oxytocin- and PGF(2alpha)-stimulated myometrial contractions (P < 0.05). There was no significant difference between E(max) values of nimesulide and DFU in all tissues (P > 0.05). Conclusion: DFU is a more potent inhibitor than nimesulide on KCl-, oxytocin-, and PGF(2alpha)-stimulated contractions of pregnant human myometrium. The inhibitor effects of nimesulide and DFU were predominantly on KCl-stimulated contractions.     

Effects of endothelin-1 and calcium channel blockers on contractions in human myometrium. A study on myometrial strips from normal and diabetic pregnant women

OBJECTIVE: We studied the effect of endothelin (ET)-1 on spontaneous contractions and the effects of nimodipine and isradipine on ET-1-induced contractions in myometrial tissue from normal and diabetic pregnant women. STUDY DESIGN: We isolated myometrial strips from seven normal pregnant and seven gestational diabetic women undergoing elective cesarean section at term. Three sets of experimental studies were performed with three myometrial strips obtained from every woman and mounted in organ baths for recording of isometric tension. In the first set, the effect of increasing concentrations of ET-1 (10(-11)-10(-8) M) on spontaneous contractions was recorded. In the second and third sets, effects of increasing concentration of nimodipine (10(-6)-3.10(-5) M) and isradipine (10(-5)-3.10(-4) M), respectively, on contractions following pretreatment with 10(-8) M ET-1 were recorded. RESULTS: ET-1, beginning from 10(-9) M and 10(-10) M, significantly increased the amplitude of contractions in normal and diabetic strips, respectively. ET-1, beginning from 10(-9) M, also increased the frequency of contractions in normal and diabetic strips. The amplitude of contractions was significantly higher in diabetic strips as compared with normal strips at 10(-9) and 10(-8) M. There was no significant difference in the frequency of contractions between normal and diabetic strips. ET-1 at 10(-8) M also increased the basal tone of all normal and diabetic strips. Nimodipine, beginning from 10(-6) M and 3.10(-6) M, and isradipine, beginning from 10(-5) M and 3.10(-5) M, significantly decreased the amplitude of contractions in normal and diabetic strips, respectively. Nimodipine at 10(-5) M and 3.10(-5) M and isradipine at 3.10(-4) M significantly decreased the frequency of contractions in normal strips. Nimodipine, except at 3.10(-5) M, and isradipine did not significantly decrease the frequency of contractions in diabetic strips. CONCLUSION: Gestational diabetes increases ET-1-induced contractile response in human myometrium. The contractile effect of ET-1 in normal and diabetic myometrium is mediated partly by dihydropyridine-sensitive calcium channels since it is significantly reduced by nimodipine and isradipine. The promising data from this study warrant clinical studies on the definitive place of nimodipine and isradipine in the treatment of preterm labor, especially in a diabetic woman, to avoid metabolic complications of beta-mimetic tocolytics.     

Effects of recombinant human relaxin on pregnant rat uterine artery and myometrium in vitro

OBJECTIVE: The purpose of this study was to evaluate the effects of recombinant human relaxin on the uterine artery and myometrial contractility in pregnant rats. STUDY DESIGN: Uterine artery and myometrial rings from mid and term pregnant rats were used. Relaxin effect was studied on phenylephrine-induced contraction in the presence or absence of nitric oxide synthase inhibitor, N omega-nitro-l-arginine methyl ester, soluble guanylate cyclase inhibitor, 1H-oxadiazolo-quinoxaline-1-one, or adenylate cyclase inhibitor, SQ-22,536. The myometrial inhibitory effect of relaxin was studied on spontaneous and oxytocin- or protein kinase C activator-induced contractions. RESULTS: Uterine artery relaxation by relaxin was greater at mid pregnancy compared with term. Relaxin effect was decreased by SQ-22,536, 1H-oxadiazolo-quinoxaline-1-one and N omega-nitro-l-arginine methyl ester at mid pregnancy. Relaxin inhibited spontaneous contractions at mid pregnancy but not at term. Relaxin had no effect on oxytocin- or indolactam-V-induced contractions. CONCLUSION: Relaxin effect is mediated by nitric oxide, soluble guanylate cyclase, and adenylate cyclase in mid pregnant uterine artery. Relaxin inhibits spontaneous uterine activity at mid pregnancy. Relaxin effect decreased at term gestation in both tissues.     

Magnesium prevents seizure-induced reduction in excitatory amino acid receptor (kainate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) binding in pregnant rat brain

OBJECTIVE: Our purpose was to evaluate the effect of seizures on kainate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor binding in maternal rat brain and whether maternal peripheral administration of magnesium sulfate can decrease this effect. STUDY DESIGN: Rats were implanted with a bipolar electrode into the hippocampus. One week of recovery was allowed before breeding. Pregnant rats were randomly assigned to 1 of 4 groups, as follows: group 1, sodium chloride and no seizures (n = 5); group 2, magnesium sulfate and no seizures (n = 4); group 3, sodium chloride and seizures (n = 8); and group 4, magnesium sulfate and seizures (n = 9). Doses of sodium chloride or magnesium sulfate were administered every 20 minutes for 4 hours to all rats on gestational days 9, 11, 13, 15, 17, and 19, followed by seizure induction (groups 3 and 4). On gestational day 20, rats were perfused, brains were dissected, and cryostat sections were taken, labeled in vitro, and placed on Hyperfilm for 4 weeks. The ligands used included kainate receptor agonist and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor agonist and antagonist. Optical density measurements of binding in 15 brain regions on each section were evaluated by 1- and 2-way analysis of variance. RESULTS: Seizure activity was associated with decreased alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor binding of its agonist in pregnant rat brains (seizure effect, 25.9 +/- 3.2 and 92.6 +/- 3.4 fmol/mg tissue in hindbrain and forebrain, respectively; no seizure effect, 44.5 +/- 4.7 and 110. 7 +/- 5.0 fmol/mg tissue in hindbrain and forebrain, respectively; P <.01). Magnesium administration was associated with increased binding of tritiated alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (magnesium effect, 44.9 +/- 4.2 and 110.4 +/- 4.5 fmol/mg tissue in hindbrain and forebrain, respectively; sodium chloride effect, 25.5 +/- 3.7 and 92.9 +/- 4.0 fmol/mg tissue in hindbrain and forebrain, respectively; P <.01). The same trend was seen with the kainate receptor in the hippocampus and hypothalamus, with a significant interaction effect between seizure and magnesium (P <.05). CONCLUSIONS: The mechanism for maternal rat brain injury resulting from seizure activity may be, at least in part, associated with alteration in the function of excitatory amino acid receptors. Administration of magnesium sulfate can counteract this effect and may reduce resultant maternal brain damage.     

A study of (Ca2+-Mg2+)-, and actomyosin-ATPase activities in pregnant myometrium and the functional modification by calmodulin

In order to clarify the biochemical bases of myometrial contractions, (Ca2+-Mg2+)-ATPase activity, actomyosin (ACT)-ATPase activity and ACT superprecipitation were studied during pregnancy. The results indicate that; (Ca2+-Mg2+)-ATPase activity in nonpregnant ovariectomized rats was markedly increased after estradiol treatment (15 micrograms/day for 3 days) from 12.0 +/- 20.1 to 132.5 +/- 22.8 nmolePi/mg/min (p less than 0.01). The activity on days 19, 20, 21 and 22 of pregnancy was 48.0 +/- 5.0, 48.2 +/- 7.5, 112.3 +/- 18.5 and 27.3 +/- 8.8 nmolePi/mg/min, respectively, showing marked prepartum increase and rapid decrease after delivery. In human myometrium, basal (Ca2+-Mg2+)-ATPase activity showed no significant change but at term the activity was decreased (79.4 +/- 9.8 to 60.7 +/- 7.4 nmolePi/mg/min, p less than 0.05) in the presence of calmodulin (CMD). Human myometrial ACT-ATPase activity, on the other hand, was stimulated with CMD (1st trimester, term without labor and with labor: 295 to 1,134, 550 to 1,243 and 897 to 4,735 pmolePi/mg/min, p less than 0.05). Myometrial CMD concentrations, however, showed no change during pregnancy. ACT prepared from rabbit uterus showed an enhanced interaction of contractile proteins in the presence of CMD. These data indicate that CMD stimulates (Ca2+-Mg2+)-ATPase activity in early pregnancy but inhibits at term and increases ACT-ATPase activity. Since the myometrial CMD concentration remains unchanged during pregnancy, there may exist a function which alters CMD action on ATPase activity as pregnancy advances.     

5-(2-bromoethyl)-2'-deoxyuridine: a selective inhibitor of herpes simplex viruses in vitro

A new pyrimidine analog, 5-(2-bromoethyl)-2'-deoxyuridine (BEUdR), was tested in vitro for antiviral activity on Herpes simplex virus types 1 and 2. As reference compounds, ACG, BVUdR and PAA were used. Compared to ACG and BVUdR, BEUdR resulted less potent on both HSV-1 and HSV-2. However, a 50% inhibition of the multiplication of uninfected cells could be obtained only at very high BEUdR concentration (ID50 = 8500 microM). This makes BEUdR the least toxic analog known and gives it a selective index comparable to, if not better, than of ACG and BVUdR.     

Conservative management of cervical intraepithelial neoplasia (CIN(2-3)) in pregnant women

The management of cervical intraepithelial neoplasia (CIN(2-3)) diagnosed during pregnancy was the subject of this study. Two hundred and eight pregnant women with an abnormal cytology were assessed in our unit over a 10-year period. The age of the patients ranged from 20 to 45 (mean 28) years. Seventy-eight of these women were histologically proven to have CIN(2-3). All patients were followed up every 8-10 weeks by cytology and colposcopy during pregnancy and reassessed 8-12 weeks postpartum. The disease persisted in 30 cases (38.4%), whereas in the remaining 48 cases it regressed to CIN(1). No case of invasive disease developed during the follow-up period in these pregnant patients. Conservative management of CIN(2-3) during pregnancy is acceptable, but close follow-up and colposcopic expertise are necessary.     

Activity of matrix metalloproteinase-2 and -9 and contents of their tissue inhibitors in uterine leiomyoma and corresponding myometrium

Background and aim. Matrix metalloproteinase-2 and -9 (MMP-2 and -9) are proteolytic enzymes degrading extracellular matrix proteins, mainly collagen type IV. Recent reports show that these proteases may be implicated in the growth of uterine leiomyoma. The aim of the present study was to evaluate the activity of MMP-2 and MMP-9, the contents of their tissue inhibitors (TIMP-1 and TIMP-2) and the immunolocalization of collagen type IV in uterine leiomyoma and corresponding myometrium.

Materials and methods. Material for the study comprised specimens of uterine leiomyomas and corresponding myometrium derived from 20 hysterectomized women. The activity of MMP-2 and MMP-9 in tissue extracts was evaluated by semi-quantitative zymography. TIMPs were measured by enzyme-linked inmmunosorbent assay. Protein immunohistochemistry was applied for detection of collagen type IV.

Results. Activity and activation ratio of MMP-2 were significantly higher in leiomyomas than myometrium. The activity of MMP-9 was weak and did not differ between the investigated tissues. Contents of TIPM-1 and TIPM-2 were similar in both tissues. In both leiomyomas and myometrium, collagen type IV was localized in the extracellular matrix embedding bundles of smooth muscle cells, but was absent in areas of extracellular matrix accumulation within leiomyomas and in larger septa separating muscle fibers in normal myometrium.

Conclusion. MMP-2 may be implicated in pathogenesis of leiomyoma.     

Cyclooxygenase (COX)-2 and granulosa cell apoptosis in vitro

Purpose : C-myc was studied in cyclooxygenase (COX)-2 associated granulosa cell apoptosis. Methods : Granulosa cells (N = 5 cases) were incubated for 24 h in either 1 or 50 M COX-2 inhibitor, 1 or 50 M COX-1/COX-2 inhibitor, negative or positive controls. Single primer polymerase chain reaction of c-myc exon 1 were performed. Bisbenzimide-stained control single-stranded (ssDNA) were hybridized to SYBR Gold-stained ssDNA and fluorescent images analyzed. Results : C-myc was disrupted by the high-dose COX-2 inhibitor. Cell viability decreased with COX-1 and COX-2 inhibition. However, cell viability was similar for the positive control and at low-dose COX-2 inhibition. Conclusions : Inhibition of both COX-1 and COX-2 initiated apoptosis without disrupting c-myc suggesting a protective effect on c-myc. The low dosage of the COX-2 inhibitor did not disrupt c-myc and cell viability. C-myc sensitization was not part of apoptosis.     

Activity of matrix metalloproteinase-2 and -9 and contents of their tissue inhibitors in uterine leiomyoma and corresponding myometrium.

BACKGROUND AND AIM: Matrix metalloproteinase-2 and -9 (MMP-2 and -9) are proteolytic enzymes degrading extracellular matrix proteins, mainly collagen type IV. Recent reports show that these proteases may be implicated in the growth of uterine leiomyoma. The aim of the present study was to evaluate the activity of MMP-2 and MMP-9, the contents of their tissue inhibitors (TIMP-1 and TIMP-2) and the immunolocalization of collagen type IV in uterine leiomyoma and corresponding myometrium. MATERIALS AND METHODS: Material for the study comprised specimens of uterine leiomyomas and corresponding myometrium derived from 20 hysterectomized women. The activity of MMP-2 and MMP-9 in tissue extracts was evaluated by semi-quantitative zymography. TIMPs were measured by enzyme-linked inmmunosorbent assay. Protein immunohistochemistry was applied for detection of collagen type IV. RESULTS: Activity and activation ratio of MMP-2 were significantly higher in leiomyomas than myometrium. The activity of MMP-9 was weak and did not differ between the investigated tissues. Contents of TIPM-1 and TIPM-2 were similar in both tissues. In both leiomyomas and myometrium, collagen type IV was localized in the extracellular matrix embedding bundles of smooth muscle cells, but was absent in areas of extracellular matrix accumulation within leiomyomas and in larger septa separating muscle fibers in normal myometrium. CONCLUSION: MMP-2 may be implicated in pathogenesis of leiomyoma.     

Matrix metalloproteinase (MMP)-2 and MMP-9 in seminal plasma

Purpose To evaluate the latent and active forms of MMP-2 and MMP-9 in human semen samples and to investigate their association with semen parameters. Methods Basic semen analysis was performed in 82 semen samples. Seminal plasma was analyzed with gelatin zymography. Results Both latent and active forms of MMP-2 and MMP-9 were detected in human seminal plasma. The latent forms were the predominant ones. MMP-2 and MMP-9, either in latent or active forms, were not correlated with semen parameters. ProMMP-9 levels were higher in semen samples with abnormally low concentration (≤ 19 × 10⁶/ml) compared with semen samples with concentration ≥ 50 × 10⁶/ml. Conclusion MMP-2 and MMP-9 are both present in human semen. The latent forms of both MMPs are the predominant ones. ProMMP-9 is elevated in samples of low sperm concentration. Both latent and active forms of MMP-2 and MMP-9 are present in human seminal plasma; ProMMP-9 is associated with low concentration and poor sperm morphology.     

Activity of matrix metalloproteinases -2 and -9 (MMP-2 and MMP-9) and content of their tissue inhibitors in endometrial cancer--a preliminary study

OBJECTIVES: Matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) are enzymes degrading collagen type IV and other components of the basement membrane. Their activity is suppressed by tissue inhibitors of metalloproteinases--TIMP-1 and TIMP-2. Substantial evidence indicates that MMP2 and MMP-9 play an important role in the spread of malignant tumours. The aim of the study was to evaluate the activity of MMP-2 and MMP-9 and contents of their inhibitors: TIMP-1 and TIMP-2 in endometrial cancer and normal endometrium. MATERIAL AND METHODS: Material for the study comprised 28 samples of endometrial cancers and 15 samples of normal endmetrium. A two-step method for extraction of MMPs was applied. The activity of MMP-2 and MMP-9 was measured with semi-quantitative zymography. TIMP-1 and TIMP-2 contents were measured with ELISA method. RESULTS: Mean activity and activation ratio of MMP-9 was significantly higher in endometrial cancers compared with normal myometrium, whereas mean activity and activation ratio of MMP-2 did not differ significantly between investigated groups. Mean content of TIMP-1 and TIMP-2 did not differ between cancer and control tissues. No unequivocal association between activity of investigated MMPs or contents of their inhibitors and clinicopathological features of endometrial cancers was observed. CONCLUSIONS: Results of the study suggest that MMP-9 may play an important role in the progression of endometrial cancer, whereas MMP-2 does not seem to be involved in this process. Action of MMP-9 may be further enhanced by relative deficiency of TIMP-1.