The Effects of Female Sexual Hormones on the Expression of Aquaporin 5 in the Late-Pregnant Rat Uterus.

Thirteen mammalian aquaporin (AQP) water channels are known, and few of them play a role in the mammalian reproductive system. In our earlier study, the predominance of AQP5 in the late-pregnant rat uterus was proven. Our current aim was to investigate the effect of estrogen- and gestagen-related compounds on the expression of the AQP5 channel in the late-pregnant rat uterus. Furthermore, we examined the effect of hormonally-induced preterm delivery on the expression of AQP5 in the uterus. We treated pregnant Sprague-Dawley rats subcutaneously with 17ß-estradiol, clomiphene citrate, tamoxifen citrate, progesterone, levonorgestrel, and medroxyprogesterone acetate. Preterm delivery was induced by subcutaneous mifepristone and intravaginal prostaglandin E2. Reverse-transcriptase PCR and Western blot techniques were used for the detection of the changes in AQP5 mRNA and protein expressions. The amount of AQP5 significantly increased after progesterone and progesterone analogs treatment on 18 and 22 days of pregnancy. The 17ß-estradiol and estrogen receptor agonists did not influence the AQP5 mRNA level; however, estradiol induced a significant increase in the AQP5 protein level on the investigated days of gestation. Tamoxifen increased the AQP5 protein expression on day 18, while clomiphene citrate was ineffective. The hormonally-induced preterm birth significantly decreased the AQP5 level similarly to the day of delivery. We proved that AQP5 expression is influenced by both estrogen and progesterone in the late-pregnant rat uterus. The influence of progesterone on AQP5 expression is more predominant as compared with estrogen.

Oxytocin regulates the expression of aquaporin 5 in the late-pregnant rat uterus.

Aquaporins (AQPs) are integral membrane channels responsible for the transport of water across a cell membrane. Based on reports that AQPs are present and accumulate in the female reproductive tract late in pregnancy, our aim was to study the expression of AQP isoforms (AQP1, 2, 3, 5, 8, and 9) at the end of pregnancy in rat in order to determine if they play a role in parturition. Reverse-transcriptase PCR revealed that specific Aqp mRNAs were detectable in the myometrium of non-pregnant and late-pregnancy (Days 18, 20, 21, and 22 of pregnancy) rat uteri. The expression of Aqp5 mRNA and protein were most pronounced on Days 18-21, and were dramatically decreased on Day 22 of pregnancy. In contrast, a significant increase was found in the level of Aqp5 transcript in whole-blood samples on the last day of pregnancy. The effect of oxytocin on myometrial Aqp5 expression in an organ bath was also investigated. The level of Aqp5 mRNA significantly decreased 5 min after oxytocin (10(-8) M) administration, similarly to its profile on the day of delivery; this effect was sensitive to the oxytocin antagonist atosiban. The vasopressin analog desmopressin (3.7 × 10(-8) M), on the other hand, did not alter the expression of Aqp5, but did increased the amount of Aqp2 mRNA, an effect that was atosiban-resistant. These results lead us to propose that oxytocin selectively influences the expression of Aqp5 at the end of pregnancy, and may participate in events that lead to parturition in the rat. The sudden increase of AQP5 in the blood on the last day of pregnancy may serve as a marker that indicates the initiation of delivery.

Nocistatin inhibits pregnant rat uterine contractions in vitro: roles of calcitonin gene-related peptide and calcium-dependent potassium channel.

The endogenous neuropeptide nociceptin/orphanin FQ, translated from the prepronociceptin gene, exerts a contraction-inhibitory effect on the rat uterus. As nocistatin has been reported to cause functional antagonism of the pro-nociceptive effects of nociceptin, we set out to investigate its effects on the pregnant rat uterus and to elucidate its signalling pathway. The expression of prepronociceptin mRNA in the uterus and nocistatin levels in the uterus and the plasma were confirmed by RT-PCR and radioimmunoassay. The uterine levels of prepronociceptin mRNA and nocistatin were significantly increased by the last day of pregnancy, while the plasma nocistatin levels remained unchanged. In the isolated organ bath studies nocistatin inhibited the prostaglandin- and the KCl-evoked contractions in the uterus dose-dependently. This latter effect was decreased by preincubation with capsaicin. Incubation with calcitonin gene-related peptide after capsaicin treatment caused an elevation in the contraction-inhibitory effect of nocistatin. The effect of nocistatin was also decreased by the Ca(2+)-dependent K(+) channel inhibitor paxilline, against spontaneous uterine contractions. Nociceptin potentiated the action of nocistatin. Naloxone decreased the effect of nocistatin administered either alone or in combination with nociceptin. In Ca(2+)-poor environment, this effect of naloxone was suspended. Enzyme immunoassay for the uterine intracellular cAMP levels partially confirmed the results of in vitro contractility studies. We conclude that nocistatin, generated locally in the uterus, exerts an inhibitory effect, the mechanism being mediated in part by Ca(2+)-dependent K(+) channels, the elevation of cAMP levels and sensory neuropeptides.

Increase of the uterus-relaxant effect of nifedipine by the Abcg2 efflux protein inhibitor KO134 in the rat in vivo.

BACKGROUND/AIM: High Abcg2 (ATP-Binding Cassette Transporter Subfamily G, Member-2) levels have been found in reproductive tissues, such as the placenta and uterus. The substrate specificity of Abcg2 is very wide, including uterus-relaxant agents (e.g. nifedipine and prazosine). Through the use of a potent inhibitor (KO134), intracellular accumulation of the substrate can be increased. Nifedipine, commonly used in acute tocolytic therapy, exerts a greater tocolytic effect and has fewer side-effects than ß2-adrenergic receptor agonists. The aims of the present study were to investigate the expression of Abcg2 in the rat uterus during gestation and the uterus-relaxant effect of nifedipine in the presence of the Abcg2 inhibitor KO134. MATERIALS AND METHODS: Real-time Polymerase Chain Reaction (PCR) and western blot analyses were performed to detect the levels of Abcg2 during gestation in the rat. The uterus-relaxant effect of nifedipine in vivo was investigated by the intra-uterine pressure measuring method, described by Csapo. RESULTS: Low levels of Abcg2 were found in non-pregnant animals and early-pregnancy (days 6, 8 and 10), but on day 15 of gestation, a sharp increase in Abcg2 levels was observed, which reached its maximum on day 18 and later decreased until the end of gestation. The post-partum levels were similar to those in non-pregnant rats. The in vivo contractility studies revealed that nifedipine had a strong uterus-relaxant effect on spontaneous contractions, and that this effect was significantly and dose-dependently increased by the Abcg2 blocker KO134. CONCLUSION: The administration of efflux pump inhibitors in combination with tocolytic agents may be of novel therapeutic relevance in the management of pre-term labour.

Direct antiproliferative effect of nonsteroidal 17ß-hydroxysteroid dehydrogenase type 1 inhibitors in vitro.

Inhibition of the local formation of estrogens seems to be an attractive strategy for pharmacological intervention in hormone-dependent disorders. The direct antiproliferative properties of ten nonsteroidal 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) inhibitors were investigated on human cancer cell lines of gynecological origin. The mechanism of the antiproliferative action was approximated by cell cycle analysis, fluorescent microscopy, BrdU assay, determination of caspase-3 activity and quantification of the expression of cell cycle regulators at mRNA level. Treatment of HeLa cells with some of the compounds resulted in a concentration-dependent inhibition of the G1-S transition and an increase in the apoptotic population. The most effective agents increased the expression of tumor suppressors p21 and p53, while CDK2 and Rb were down-regulated. The reported anticancer actions of the tested compounds are independent of the 17ß-HSD1-inhibiting capacity. These results indicate that it is possible to combine direct antiproliferative activity and 17ß-HSD1 inhibition resulting in novel agents with dual mode of action.

Uterus-relaxing effect of ß2-agonists in combination with phosphodiesterase inhibitors: studies on pregnant rat in vivo and on pregnant human myometrium in vitro.

AIMS: Our aims were to examine the effects of a simultaneous stimulation of ß(2) -adrenergic receptors and inhibition of uterine phosphodiesterases (PDE), in the pregnant rat uterus in vivo and on human uterine tissue in vitro. We also set out to measure cAMP levels and detect the expressions of the isoenzymes PDE4B and PDE4D in human uterine tissue samples. MATERIAL AND METHODS: Preterm birth was induced in Sprague-Dawley rats with bacterial lipopolysaccharide. The uterine effects of terbutaline alone or in combination with rolipram were tested in vivo. Human myometrial strips from cesarean sections at full-term pregnancy and at preterm labor were stimulated with oxytocin, and the inhibitory effects of theophylline, rolipram and terbutaline were studied. The myometrial accumulation of cAMP in the presence of rolipram and terbutaline was determined by enzyme immunoassay. The expressions of PDE4B and PDE4D proteins were detected by Western blotting. RESULTS: The selective PDE4 inhibitor rolipram was more effective than the non-selective PDE inhibitor theophylline in inhibiting the oxytocin-induced contractions in the human uterus. The uterus-relaxing effects of low doses of terbutaline were markedly potentiated by rolipram, both in rats and in human tissues. The changes in uterine cAMP levels correlated with these results. At preterm labor, PDE4B was the predominant form of PDE4 expressed; at full term, PDE4D was expressed more strongly. CONCLUSIONS: A combination of selective PDE4 inhibitors and ß(2) -agonists should be considered for the treatment of preterm contractions.

Antiproliferative effect of normal and 13-epi-D-homoestrone and their 3-methyl ethers on human reproductive cancer cell lines.

The possibility of the therapeutic use of estrogens emerged following the recognition that certain estradiol analogs, and particularly metabolites (e.g. the A-ring metabolite 2-hydroxyestrone, etc.) inhibit the differentiation of diverse tumor cell lines. Until recently, despite the investigation of numerous synthetic d-ring-substituted estrone derivatives, no analysis had been published on the effects of D-ring expansion of estrone on its tumor-suppressing activity. The aim of the present study was to characterize the antiproliferative effects of normal and 13-epi-D-homoestrone and their 3-methyl ethers (1-4) on human reproductive cancer cell lines. The antitumor activities of the two epimer pairs on HeLa, MCF-7 and Ishikawa cells were determined. Normal D-homoestrone exerted the greatest cytostatic effect on HeLa cells (IC(50)=5.5 µM) and was subjected to further investigations to elucidate its mechanism of action on apoptosis induction. Morphological changes detected by Hoechst 33258-propidium iodide double staining, the cell cycle arrest at phase G2/M and the subsequent increase in the proportion of the subG1 fraction determined by flow cytometric analysis and the significant increase in the activity of caspase-3 confirmed the induction of apoptosis in HeLa cells treated with D-homoestrone. D-Homoestrone was also tested on a non-cancerous human lung fibroblast cell line (MRC-5) to determine its selective toxicity. The concentration in which it inhibited cell proliferation by 50% was at least six times higher for the fibroblast cells than for cervical cancer cells. No significant in vivo estrogenic activity was observed as concerns the uterus weight of gonadectomized rats after a 7-day treatment with normal D-homoestrone. These results led to the conclusion that normal D-homoestrone is a novel antitumor compound with a similar activity on HeLa cells as that of the reference agent cisplatin, but its selectivity toward non-cancerous cells is significantly higher than that of cisplatin. It may be considered to be a basic lead molecule for the preclinical development of potential anticancer agents.

ß(2)-Adrenergic activity of 6-methoxykaempferol-3-O-glucoside on rat uterus: in vitro and in silico studies.

6-Methoxykaempferol-3-O-glucoside (6-MKG) was isolated from a Sudanese herb (El-hazha). The pharmacological effects of 6-MKG were tested on isolated non-pregnant or late-pregnant rat uteri in vitro, whilst docking studies were carried out modelling of the binding of 6-MKG to the rat ß(2)-adrenoceptor in silico. In vitro studies revealed that 6-MKG was able to relax both the non-pregnant and the late-pregnant uterine contractility with 50% of the E(max) of terbutaline, whilst the EC(50) for 6-MKG was at least half than that of terbutaline. The ß(2)-adrenoceptors antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol(ICI118,551) competitively antagonised the relaxing effect of 6-MKG. Radioligand binding and cAMP studies confirmed the ß(2)-adrenoceptors agonistic property of the compound. In in silico docking studies, 6-MKG bound to rat ß(2)-adrenoceptors with low ∆G(bind) value (-11.53±0.06 kcal/mol) and it interacted with four residues of the active site (Asp(113), Asn(312), Cys(191)and Tyr(316)). It is concluded that 6-MKG exerts weak ß(2)-adrenoceptor agonistic activity and can be considered a natural compound with potential therapeutic significance in the field of premature pregnant uterine contractions and asthmatic problems.

Improving the relaxing effect of terbutaline with phosphodiesterase inhibitors: studies on pregnant rat uteri in vitro.

AIMS: Previous results by our group showed that the in vitro uterus-relaxing potency of ß(2)-adrenergic receptor (ß(2)-AR) agonists and uterine cAMP accumulation are enhanced in case of visceral inflammation. Our aim was to study the effects of the non-selective phosphodiesterase (PDE) inhibitor theophylline and the selective PDE4 inhibitor rolipram on the uteri of intact late-pregnant female rats (on days 20 and 22 of pregnancy) and of pregnant rats treated with lipopolysaccharide (LPS) to evoke preterm labor (on day 20). MAIN METHODS: The effects of theophylline and rolipram alone and of rolipram with terbutaline were investigated in isolated organ system. Contractions were evoked with KCl. The forskolin- and terbutaline-stimulated cAMP accumulations were determined by enzyme immunoassay, with or without rolipram. KEY FINDINGS: The maximum uterus-relaxing effects of theophylline and rolipram decreased significantly (p<0.05) with the progression of pregnancy in intact rats. The most pronounced effect of rolipram was detected in rats challenged with LPS on day 20. Rolipram increased the in vitro effect of terbutaline both in intact and in LPS-treated rats. In the presence of rolipram, the forskolin- and terbutaline-stimulated cAMP accumulations were higher in LPS-treated than in intact rats. SIGNIFICANCE: The previous findings led us to conclude that the combined administration of PDE4 inhibitors with ß(2)-agonists is of therapeutic value for the inhibition for uterine contractions, especially in the case of genital inflammation, which often triggers preterm birth. Combination therapy in general is associated with lesser side-effects, as a consequence of lower effective doses of each drug.

Potentiation of the uterus-relaxing effects of ß-adrenergic agonists with nifedipine: studies on rats and the human myometrium.

OBJECTIVE: We investigated how progesterone and salmeterol modify the effect of nifedipine in an in vivo preterm birth model in rats, and how terbutaline and nifedipine modify the contractions of the isolated human myometrium. DESIGN: Experimental animal and human myometrial studies. SAMPLE: Twenty-four female Sprague-Dawley rats and 13 human uterine tissues sampled from cesarean section. METHODS: Preterm birth was induced in Sprague-Dawley rats with a combination of mifepristone and prostaglandin-E(2). The animals were treated with nifedipine or its combination with salmeterol and progesterone. Additionally, isolated human myometrial strips from cesarean sections were stimulated with oxytocin, and the inhibitory effects of nifedipine and terbutaline were studied. RESULTS: Nifedipine delayed the preterm delivery in the rats, but its effect was tripled by the addition of ß(2)-mimetics, or abolished after progesterone pretreatment. Synergism was observed in the relaxing effects of nifedipine and terbutaline on the isolated human myometrium. CONCLUSION: The action of nifedipine in delaying labor is impeded by progesterone. A combination of nifedipine and ß(2)-agonists should be considered for the treatment or prevention of preterm birth.

Khat - a controversial plant.

Khat (Catha edulis) is a shrub or tree whose leaves have been chewed for centuries by people who live in the Eastern part of Africa and the Arabian Peninsula. It has recently turned up in North America and Europe, particularly among emigrants and refugees from countries such as Somalia, Ethiopia and Yemen. Khat contains a number of chemicals, among which are two controlled substances, cathinone (Schedule I) and cathine (Schedule IV). Both chemicals are stimulant drugs with effects similar to amphetamine. Chewing the leaves makes people feel more alert and talkative, and suppresses appetite. Chewing khat leaves releases cathinone, a stimulant that produces the feeling of euphoria. When cathinone is broken down in the body, it produces chemicals including cathine and norephedrine, which have a similar structure to amphetamine and adrenaline (epinephrine). Regular khat use is associated with a rise in arterial blood pressure and pulse rate, corresponding with levels of cathinone in the plasma. Moreover, regular khat chewers have gingivitis and loose teeth, but there appears to be no convincing unusual incidence of oral cancer. Among khat users in Yemen there is, however, a higher incidence of esophageal cancer compared with gastric cancer. Long term use or abuse can cause insomnia, anorexia, gastric disorders, depression, liver damage and cardiac complications, including myocardial infarction. Manic and delusional behavior, violence, suicidal depression, hallucinations, paranoia and khat-induced psychosis have also been reported. On the basis of the scientific data it seems clear that khat use has negative consequences on the economic development of a country and on the health of the society.

Modification of the effect of nifedipine in the pregnant rat myometrium: the influence of progesterone and terbutaline.

AIMS: The aims of the study were to investigate the effects of nifedipine on potassium chloride (KCl)-evoked rat uterine contractions on different days of pregnancy in vitro, and the alterations in the effects of nifedipine on combination with terbutaline or progesterone. MAIN METHODS: In rat myometrial rings taken on different days of pregnancy, rhythmic contractions were evoked with KCl in an isolated organ bath. KEY FINDINGS: The relaxing effect of nifedipine was most expressed in the 25 mM KCl-induced uterine contractions, reaching the maximum on the last day of pregnancy (day 22). This effect was decreased by progesterone pretreatment in vivo. Synergism was observed in the uterus-relaxing effect of nifedipine+terbutaline, though the extent of potentiation depended on the sequence of administration of the two compounds. When terbutaline was added first in a single dose, the maximal inhibitory effect of nifedipine was lower. This decrease in the inhibition was suspended by a Ca(2+)-poor buffer, indicating the role of Ca(2+) channel activating effect of terbutaline. SIGNIFICANCE: It is concluded that the uterus-relaxing effect of nifedipine is weakened by progesterone and may be enhanced by low concentrations of beta-mimetics. However, the administration of terbutaline cannot precede the administration of nifedipine.

Progesterone decreases the relaxing effect of the beta3-adrenergic receptor agonist BRL 37344 in the pregnant rat myometrium.

Although the published results regarding the function of the beta(3)-adrenergic receptors (beta(3)-ARs) in the regulation of smooth muscle activity are very promising, the question of the mechanism of beta(3)-ARs' action in the pregnant myometrium cannot be fully answered by human investigations. To assess whether it possesses an essential role in the regulation of uterine contractility in pregnant rats, as in humans, we performed functional, western blotting and molecular biology experiments on the late-pregnant rat myometrium. The influence of progesterone on the function of the beta(3)-ARs was also investigated. We demonstrated the presence and the functional activity of the beta(3)-ARs in the late-pregnant rat myometrium. The maximum dose-dependent uterus-relaxing effect of the selective beta(3)-agonist BRL 37344 was recorded at the end of pregnancy in rats, similarly as in humans. The extent of its relaxing action was regarded as moderate. The expression of beta(3)-AR protein and mRNA remained unchanged during the investigated period. The administration of progesterone had no effect on the beta(3)-AR mRNA and protein expression or the maximum relaxation effect of BRL 37344, but shifted the dose-response curve to the right and decreased the synthesis of the second messenger, cAMP. It can be concluded that the beta(3)-ARs play an additional role in the regulation of the contractile activity of the pregnant rat uterus. The inhibitory effect of progesterone on the functional activity of the beta(3)-ARs may have important consequences in the case of human application if this effect is also demonstrated in pregnant human myometrial tissue.

The roles of alpha2-adrenoceptor subtypes in the control of cervical resistance in the late-pregnant rat.

The roles of the alpha(2)-adrenoceptor subtypes in the regulation of cervical resistance have previously not been investigated. The aim of the present study was to identify these receptors in the late-pregnant cervix and determine their functions in vitro in the rat. The expressions of the alpha(2)-adrenoceptor subtypes were determined by means of RT-PCR and Western blotting techniques. The changes in cervical resistance due to subtype-selective antagonists were investigated in stretching tests. The cyclic AMP immunoassay technique was used to detect the level of cyclic AMP following stimulation of the alpha(2)-adrenoceptors with or without pertussis toxin. On pregnancy days 18, 20, 21 and 22, the RT-PCR and Western blotting studies revealed the expressions of all three alpha(2)-adrenoceptor subtype mRNAs and proteins. On days 18 and 20, noradrenaline increased and decreased the resistance, respectively. Its effect was blocked by each of the antagonists used, except ARC 239 on both days. On day 21, noradrenaline again increased the resistance, this effect being maintained only in the presence of spiroxatrine. Noradrenaline was ineffective on day 22. These results were supported by the changes in cyclic AMP levels. Pertussis toxin pretreatment eliminated the changes in the cyclic AMP level on days 18 and 21. We presume that the alpha(2A)- and alpha(2C)-adrenoceptors play predominant roles in the regulation of cervical resistance on days 18-21. Depending on the day of pregnancy, stimulation of these alpha(2)-adrenoceptors could even result in opposite effects. This fluctuation can be explained by the changes in the G(i)/G(s)-coupling of the alpha(2A)- and alpha(2C)-adrenoceptors.

Antitumor activity of alkaloids derived from Amaryllidaceae species.

The aim of the present study was to investigate the anticancer properties of five alkaloids isolated from Amaryllidaceae, including the inhibitory effect on P-glycoprotein (P-gp) and the apoptosis-inducing capacity. The tested alkaloids were evaluated for their multidrug resistance (MDR)-reversing activity on human MDR1-gene-transfected L5178 mouse lymphoma cells, using the rhodamine-123 (Rh-123) assay. Trisphaeridine and pretazettine increased the intracellular Rh-123 concentration 30- and 50-fold, respectively, as compared to the non-treated cells, and 2-O-acetyllycorine and trisphaeridine were demonstrated by means of the checkerboard method to enhance the antiproliferative activity of doxorubicin on L5178 MDR mouse lymphoma cells. The MTT assay revealed that pretazettine, trisphaeridine and 2-O-acetyllycorine displayed excellent antiproliferative effects on both the human and the mouse cell lines. The apoptosis-inducing activities of selected agents (2-O-acetyllycorine and trisphaeridine) were measured via acridine orange and ethidium bromide dual staining and flow cytometry of the subG1 population.

Effects of experimentally induced diabetes mellitus on pharmacologically and electrically elicited myometrial contractility.

1. Diabetes is one of the most frequent complications of gestation, affecting approximately 7% of pregnancies. However, little is known about its effects on electrically and pharmacologically stimulated myometrial contractility. The aim of the present study was to investigate the consequences of streptozotocin (STZ)-induced diabetes on: (i) electrical field stimulation (EFS)-evoked contraction of isolated uterine rings as a function of gestational age; and (ii) the uterotonic and tocolytic actions of α- and ß-adrenoceptor stimulation, respectively. The effects of oxytocin in late pregnancy were also investigated. 2. During pregnancy, EFS-evoked contractions of isolated uterine rings from intact rats declined, whereas isolated uterine rings from diabetic rats exhibited continuously low sensitivity to EFS. 3. In non-pregnant rats, diabetes resulted in increased noradrenaline-mediated contractility and a decreased relaxation response to terbutaline. At the mRNA level, diabetes enhanced the expression of α1B-adrenoceptors in non-pregnant rats from 14.65 to 18.39 µg/mL (P < 0.05), whereas the expression of α1D-adrenoceptors decreased (from 42.87 to 35.67 µg/mL; P < 0.05). During pregnancy, the responses to these sympathomimetics did not differ between diabetic and intact rats. 4. In late pregnancy (on Days 15 and 21), oxytocin caused greater maximum contractility of uterine rings from diabetic rats without affecting the EC(50). In addition, on Day 15 of pregnancy, the expression of oxytocin receptors in the myometrium of diabetic rats was higher than that in intact rats. 5. The results of the present study indicate that experimental diabetes facilitates gestation-induced denervation and increases myometrial sensitivity to oxytocin in late pregnancy. If similar mechanisms operate in humans, this could contribute to a tendency to premature uterine contractions in diabetes-complicated pregnancies.

Efficient approach to androstene-fused arylpyrazolines as potent antiproliferative agents. Experimental and theoretical studies of substituent effects on BF(3)-catalyzed intramolecular [3 + 2] cycloadditions of olefinic phenylhydrazones.

Highly diastereoselective Lewis acid induced intramolecular 1,3-dipolar cycloadditions of alkenyl phenylhydrazones (containing various substituents on the aromatic ring) obtained from a d-secopregnene aldehyde were carried out under fairly mild conditions to furnish androst-5-ene-fused arylpyrazolines in good to excellent yields. The ability of phenylhydrazones to undergo cyclization was found to be affected significantly by the electronic features of the substituents on the aromatic moiety. The rates of the ring-closure reactions were observed to be increased by electron-donating and decreased by electron-withdrawing groups. The experimental findings on the BF(3)-catalyzed transformations were supported by calculations of the proposed mechanism at the BLYP/6-31G(d) level of theory, indicating a noteworthy dependence, mainly of the initial complexation step, and hence of the whole process, on the character of the substituent. The cycloaddition was estimated to occur via a zwitterionic intermediate rather than involving a pure concerted mechanism. The antiproliferative activities of the structurally related pyrazoline derivatives were tested in vitro on three malignant human cell lines (HeLa, MCF7, and A431): the microculture tetrazolium assay revealed that several compounds exerted marked cell growth-inhibitory effects. The highest cytotoxic activities, displayed by the p-methoxyphenylpyrazoline derivative 7d (IC(50) values: 2.01, 2.16, and 1.41 microM on HeLa, MCF7, and A341 cells, respectively), were better than those of cisplatin (IC(50) values: 12.43, 9.63, and 2.84 microM, respectively).

Antiproliferative activity of Hungarian Asteraceae species against human cancer cell lines. Part II.

The antiproliferative activities of aqueous and organic extracts prepared from 26 Hungarian species of the tribes Cynereae and Lactuceae (Asteraceae) were tested in vitro against HeLa (cervix epithelial adenocarcinoma), A431 (skin epidermoid carcinoma) and MCF7 (breast epithelial adenocarcinoma) cells by using the MTT assay. Of the tested 200 extracts of different plant parts obtained with n-hexane, chloroform, 50% methanol and water, 16 extracts displayed noteworthy cell growth inhibitory activity (>50% inhibition at a concentration of 10 microg/mL). The IC50 values of these extracts were determined, and their direct cytotoxic effects were measured. High differences between the antiproliferative and cytotoxic activities, demonstrating a real cell proliferation inhibitory activity rather than direct killing effects, were found for some Centaurea, Cirsium, Cichorium, Lactuca, Onopordum and Scorsonera extracts.

The roles of the alpha1-adrenergic receptor subtypes in rat embryonic implantation.

OBJECTIVE: To focus on the possible roles of alpha(1)-adrenergic receptors (alpha(1)-ARs) in rat embryonic implantation. DESIGN: Laboratory study. SETTING: Animal and pharmacology laboratory at Department of Pharmacodynamics and Biopharmacy, University of Szeged, Hungary. ANIMAL(S): Pregnant and nonpregnant Sprague-Dawley rats. INTERVENTION(S): Uterus tissues were collected during the peri-implantation period. MAIN OUTCOME MEASURE(S): We used a reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting to demonstrate the expressions of mRNAs and the protein expressions of the alpha(1)-AR subtypes in the early-pregnant uterus. Electric field stimulation was applied to test the pharmacologic reactivity of the alpha(1A)-AR, and the physiologic role of this receptor was tested in a knock-down transformed animal model using an antisense oligonucleotide that elicits sequence-selective inhibition of the alpha(1A)-AR gene expression. RESULT(S): The presence of all alpha(1)-AR subtypes (alpha(1A), alpha(1B), and alpha(1D)) was proved, with a predominance of alpha(1A)-AR. The maximal expression of the alpha(1A)-AR was attained on the day of implantation. The selective alpha(1A) antagonist 5-methylurapidil inhibited the contraction in a dose-dependent manner. The number of implantation sites was decreased ( approximately 75%) in the alpha(1A)-AR knock-down transformed rats. CONCLUSION(S): We assume that the alpha(1A)-AR predominance plays a crucial role in embryonic implantation in the rat.

Antiproliferative effect of flavonoids and sesquiterpenoids from Achillea millefolium s.l. on cultured human tumour cell lines.

The antiproliferative activities of n-hexane, chloroform, aqueous-methanol and aqueous extracts of the aerial parts of the Achillea millefolium aggregate on three human tumour cell lines were investigated by means of MTT assays. The chloroform-soluble extract exerted high tumour cell proliferation inhibitory activities on HeLa and MCF-7 cells, and a moderate effect on A431 cells; accordingly, it was subjected to detailed bioactivity-guided fractionation. As a result of the multistep chromatographic purifications (VLC, CPC, PLC, gel filtration), five flavonoids (apigenin, luteolin, centaureidin, casticin and artemetin) and five sesquiterpenoids (paulitin, isopaulitin, psilostachyin C, desacetylmatricarin and sintenin) were isolated and identified by spectroscopic methods. The antiproliferative assay demonstrated that centaureidin is the most effective constituent of the aerial parts of yarrow: high cell growth inhibitory activities were observed especially on HeLa (IC(50) 0.0819 microm) and MCF-7 (IC(50) 0.1250 microm) cells. Casticin and paulitin were also highly effective against all three tumour cell lines (IC(50) 1.286-4.76 microm), while apigenin, luteolin and isopaulitin proved to be moderately active (IC(50) 6.95-32.88 microm). Artemetin, psilostachyin C, desacetylmatricarin and sintenin did not display antiproliferative effects against these cell lines. This is the first report on the occurrence of seco-pseudoguaianolides (paulitin, isopaulitin and psilostachyin C) in the Achillea genus.