Non-genomic uterorelaxant actions of corticosteroid hormones in rats: An in vitro and in vivo study.

AIMS: We aimed to identify the short-term effects of a glucocorticoid (GC) and a mineralocorticoid (MC) on non-pregnant and late pregnant rat uterine contractions to estimate their tocolytic potential. METHODS: The in vitro contractility studies were performed with uterine tissues from non-pregnant and 22-day pregnant SPRD rats. The cumulative dose-response of fludrocortisone (FLU) and dexamethasone (DEX) was measured alone or in the presence of steroid receptor antagonist mifepristone (MIF) or spironolactone (SPR). [35S]GTPγS and cAMP immunoassays were carried out to detect the activated G-proteins and cAMP, respectively. The in vivo uterine action of single doses of FLU and DEX was measured by smooth muscle electromyography. The results were statistically analyzed with an unpaired t-test. RESULTS: FLU and DEX relaxed both pregnant (33 and 34%) and non-pregnant (37 and 34%) uteri in vitro. MIF inhibited the relaxing effect of DEX, especially in the pregnant uterus, but reduced the effect of FLD only in non-pregnant tissues. GTPγS studies showed a MIF-sensitive elevation in activated G-proteins both in pregnant and non-pregnant uteri by DEX, whereas FLU induced activation only in non-pregnant samples. DEX relaxed pregnant and non-pregnant uteri in vivo in a MIF-sensitive way. SIGNIFICANCE: DEX can inhibit contractions in the late pregnant uterus in a non-genomic manner, while FLU seems to be ineffective. Its action is mediated by a G-protein-coupled receptor that can be blocked by mifepristone. Further investigations are necessary to determine the required dose and duration of GCs in the therapy of premature birth.

High Fat High Sucrose Diet Modifies Uterine Contractility and Cervical Resistance in Pregnant Rats: The Roles of Sex Hormones, Adipokines and Cytokines.

BACKGROUND: In obesity, the adipose tissue becomes a very significant endocrine organ producing different factors called adipokines, such as leptin, adiponectin and kisspeptin; however, no data are available about their actions on uterine contraction in obese pregnant rats. Our aim was to study the impact of obesity on pregnant uterine contraction in a rat model. METHODS: Obesity was induced by the consumption of a high fat high sucrose diet (HFHSD) for 9 weeks, including pregnancy. Glucose tolerance, sex hormone, cytokine and adipokine levels were measured. Uterine contractions and cervical resistance, as well as their responses to adipokines, were tested along with the expressions of their uterine receptors. RESULTS: HFHSD increased body weight, and altered glucose tolerance and fat composition. The uterine leptin and kisspeptin pathway affect increased. The levels of proinflammatory cytokines were reduced, while the plasma level of progesterone was increased, resulting in weaker uterine contractions, and improving the uterine relaxing effects of adipokines. HFHSD reduced cervical resistance, but the core effect of adipokines is difficult to determine. CONCLUSIONS: Obesity in pregnant rats reduces uterine contractility and cytokine-induced inflammatory processes, and therefore obese pregnant rat methods are partially applicable for modelling human processes.

Synthesis of 1-(para-methoxyphenyl)tetrazolyl-Substituted 1,2,3,4-Tetrahydroisoquinolines and Their Transformations Involving Activated Alkynes.

1-(p-Methoxyphenyl)tetrazolyl-substituted 6,7-dimethoxy(6,7-methylenedioxy)-1,2,3,4-tetrahydroisoquinolines formed tetrazolyl-substituted azocines in high yields by using activated alkynes. Unsubstituted at 6,7,8-aromatic fragment 1-tetrazolylisoquinoline interacted in several pathways forming tetrazolyl-substituted azocines, 1-tetrazolyl-1-R-vinylisoquinolines and 3-azaspiro[5.5]undeca-1,7,9-triene.

A new approach to alkaloid-like systems: synthesis and crystal structure of 1-(2-acetyl-11-meth-oxy-5,6-di-hydro-[1,3]dioxolo[4,5-g]pyrrolo-[2,1-a]isoquinolin-1-yl)propan-2-one.

The title compound, C19H19NO5, (I), is the product of a domino reaction between cotarnine chloride and acetyl-acetylene catalysed by copper(I) iodide. The mol-ecule of (I) comprises a fused tetra-cyclic system containing two terminal five-membered rings (pyrrole and 1,3-dioxole) and two central six-membered rings (di-hydro-pyridine and benzene). The five-membered 1,3-dioxole ring has an envelope conformation and the central six-membered di-hydro-pyridine ring adopts a twist-boat conformation. The acyl substituent is almost coplanar with the pyrrole ring, whereas the meth-oxy substituent is twisted by 27.93 (16)° relative to the benzene ring. The 2-oxopropan-1-yl substituent is roughly perpendicular to the pyrrole ring. In the crystal, mol-ecules are stacked along the a-axis direction; the stacks are linked by weak C-H⋯O hydrogen bonds into puckered layers lying parallel to (001).

Herbal laxatives and antiemetics in pregnancy.

Constipation appears in the 2nd and 3rd trimester of pregnancy, while nausea is the strongest in the 1st trimester. This review summarizes the applicability of herbal laxatives and antiemetics in pregnancy. A human study has shown that flax oil as laxative is safe from 2nd trimester. Human data is not available about the rhubarb, but animal studies reveal that its emodin content induces fetal abnormalities. Fenugreek induces teratogenic malformation both in human and animals. Senna seed is proved as a safe laxative in pregnancy. The antiemetic ginger is safe during 1st trimester, but it reduces the gestational period when applied from the 2nd trimester. Cannabis induces fetal neurological disorders while fennel can shorten the gestational age. There is herbal alternative for laxative therapy (senna) for the whole length of pregnancy, but nausea and vomiting might be reduced by herbal medicine (ginger) safely in the 1st trimester, only.

Kynurenic acid and its analogue can alter the opioid receptor G-protein signaling after acute treatment via NMDA receptor in rat cortex and striatum.

Previously, we have shown that the N-methyl d-aspartate (NMDA)-receptor antagonist kynurenic acid (KYNA) and its analogue KYNA1 do not bind directly to mu, kappa and delta opioid receptors in vitro. On the other hand, chronic administration of KYNA and KYNA1 resulted in region (cortex vs striatum) and opioid receptor-type specific alterations in G-protein activation of mouse brain homogenates. Here we describe for the first time the acute effect of KYNA and KYNA1 on opioid receptor function with the possible involvement of the NMDA receptor. The acute 30minute in vivo KYNA1 and KYNA treatments altered opioid receptor G-protein signaling or ligand potency depending on the opioid receptor type and brain region (rat cortex vs striatum) using [35S]GTPγS binding assays. Pretreatment with the NMDA receptor antagonist MK-801 impaired or reversed the effects of KYNA1 and KYNA. These results suggest an NMDA receptor mediated effect. After acute 30minute treatment HPLC measurements revealed a similar KYNA1 and a higher KYNA plasma concentration compared to cerebrospinal fluid concentrations. Finally, KYNA, KYNA1 and MK-801 showed comparable results in opioid receptor G-protein activity and ligand potency with acute in vivo treatments when they were administered in vitro for 30min on isolated cortex and striatum slices. We previously demonstrated that KYNA1 and KYNA acutely altered opioid receptor function in vivo and in vitro through the NMDA receptor depending on the opioid receptor type and brain region. This study may lead to a new, indirect approach to influence opioid receptor signaling.

Obesity in pregnancy: a novel concept on the roles of adipokines in uterine contractility.

Obesity is a global health problem even among pregnant women. Obesity alters quality of labor, such as preterm labor, prolonged labor, and higher oxytocin requirements in pregnant women. The most important factors to play a role in the altered gestational period and serve as drug targets to treat the consequences are female sexual hormones, calcium channels, adrenergic system, oxytocin, and prostaglandins. However, we have limited information about the impact of obesity on the pregnant uterine contractility and gestation time. Adipose tissue, which is the largest endocrine and paracrine organ, especially in obesity, is responsible for the production of adipokines and various cytokines and chemokines, and there are no reliable data available describing the relation between body mass index, glucose intolerance, and adipokines during pregnancy. Recent data suggest that the dysregulation of leptin, adiponectin, and kisspeptin during pregnancy contributes to gestational diabetes mellitus and pre-eclampsia. A preclinical method for obese pregnancy should be developed to clarify the action of adipokines and assess their impact in obesity. The deeper understanding of the adipokines-induced processes in obese pregnancy may be a step closer to the prevention and therapy of preterm delivery or prolonged pregnancy. Gestational weight gain is one of the factors that could influence the prenatal development, birth weight, and adiposity of newborn.

Identification of myoelectric signals of pregnant rat uterus: new method to detect myometrial contraction.

AIM: To develop an electromyography method for pregnant rat uterus in vivo and to separate myometrial signals from the gastrointestinal tract signals. METHODS: Pregnant Sprague-Dawley rats (n=8) were anaesthetized and their stomach, small intestine, and large intestine were removed from the abdomen. A pair of thread electrodes was inserted into the uterus, while a pair of disk electrodes was placed subcutaneously above the myometrium. Additionally, a strain gauge sensor was fixed on the surface of the myometrium and cecum for the parallel detection of mechanical contractions in rats (n=18) with intact gastrointestinal tract. The filtered electric signals were amplified and recorded by an online computer system and analyzed by fast Fourier transformation. The frequency of the electric activity was characterized by cycle per minute (cpm), the magnitude of the activity was described as power spectrum density maximum (PsDmax). RESULTS: The frequency of the pregnant uterine activity was 1-3 cpm, which falls within the same range as that of cecum. Measuring by both electrodes, oxytocin (1 µg/kg) increased and terbutaline (50 µg/kg) decreased the PsDmax by 25%-50% (P<0.001) and 25%-40% (P<0.01), respectively. We found a strong positive correlation between the alterations of PsDmax values and the strain gauge sensor-detected mechanical contractions (area under curve). The GI specific compounds (neostigmine, atropine) mainly affected the cecal activity, while myometrium specific drugs (oxytocin, terbutaline) influenced the myometrial signals only. Conclusion Our method proved to be able to detect the myoelectric activity that reflects the mechanical contraction. The overlapping myometrial and cecal signals are not separable, but they can be distinguished based on the much higher activity and different pharmacological reactivity of the pregnant uterus. Thus, the early signs of contractions can be detected and labor may be predicted in a fast and sensitive way.

Exploring the first Rimonabant analog-opioid peptide hybrid compound, as bivalent ligand for CB1 and opioid receptors.

Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavior. Due to the co-localization of µ-opioid (MOR) and CB1 receptors in various regions of the central nervous system (CNS) and their ability to form heterodimers, bivalent ligands targeting to both these systems may be good candidates to investigate the existence of possible cross-talking or synergistic effects, also at sub-effective doses. In this work, we selected from a small series of new Rimonabant analogs one CB1R reverse agonist to be conjugated to the opioid fragment Tyr-D-Ala-Gly-Phe-NH2. The bivalent compound (9) has been used for in vitro binding assays, for in vivo antinociception models and in vitro hypothalamic perfusion test, to evaluate the neurotransmitters release.

The effects of progesterone on the alpha2-adrenergic receptor subtypes in late-pregnant uterine contractions in vitro.

BACKGROUND: The adrenergic system and progesterone play major roles in the control of the uterine function. Our aims were to clarify the changes in function and expression of the α2-adrenergic receptor (AR) subtypes after progesterone pretreatment in late pregnancy. METHODS: Sprague Dawley rats from pregnancy day 15 were treated with progesterone for 7 days. The myometrial expressions of the α2-AR subtypes were determined by RT-PCR and Western blot analysis. In vitro contractions were stimulated with (-)-noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (α2A), ARC 239 (α2B/C) and spiroxatrine (α2A). The accumulation of myometrial cAMP was also measured. The activated G-protein level was investigated via GTPγS binding assays. RESULTS: Progesterone pretreatment decreased the contractile effect of (-)-noradrenaline through the α2-ARs. The most significant reduction was found through the α2B-ARs. The mRNA of all of the α2-AR subtypes was increased. Progesterone pretreatment increased the myometrial cAMP level in the presence of BRL 44408 (p < 0.001), spiroxatrine (p < 0.001) or the spiroxatrine + BRL 44408 combination (p < 0.05). Progesterone pretreatment increased the G-protein-activating effect of (-)-noradrenaline in the presence of the spiroxatrine + BRL 44408 combination. CONCLUSIONS: The expression of the α2-AR subtypes is progesterone-sensitive. It decreases the contractile response of (-)-noradrenaline through the α2B-AR subtype, blocks the function of α2A-AR subtype and alters the G protein coupling of these receptors, promoting a Gs-dependent pathway. A combination of α2C-AR agonists and α2B-AR antagonists with progesterone could be considered for the treatment or prevention of preterm birth.

Evaluation of the analgesic effect of 4-anilidopiperidine scaffold containing ureas and carbamates.

Fentanyl is a powerful opiate analgesic typically used for the treatment of severe and chronic pain, but its prescription is strongly limited by the well-documented side-effects. Different approaches have been applied to develop strong analgesic drugs with reduced pharmacologic side-effects. One of the most promising is the design of multitarget drugs. In this paper we report the synthesis, characterization and biological evaluation of twelve new 4-anilidopiperidine (fentanyl analogues). In vivo hot-Plate test, shows a moderate antinociceptive activity for compounds OMDM585 and OMDM586, despite the weak binding affinity on both µ and δ-opioid receptors. A strong inverse agonist activity in the GTP-binding assay was revealed suggesting the involvement of alternative systems in the brain. Fatty acid amide hydrolase inhibition was evaluated, together with binding assays of cannabinoid receptors. We can conclude that compounds OMDM585 and 586 are capable to elicit antinociception due to their multitarget activity on different systems involved in pain modulation.

The effects of estrogen on the α2-adrenergic receptor subtypes in rat uterine function in late pregnancy in vitro.

AIM: To assess the effect of 17ß-estradiol pretreatment on the function and expression of α2- adrenergic receptors (ARs) subtypes in late pregnancy in rats. METHODS: Sprague-Dawley rats (n=37) were treated with 17ß-estradiol for 4 days starting from the 18th day of pregnancy. The myometrial expression of the α2-AR subtypes was determined by real time polymerase chain reaction and Western blot analysis. In vitro contractions were stimulated with (-)-noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (α2A), ARC 239 (α2B/C), and spiroxatrine (α2A). The cyclic adenosine monophosphate (cAMP) accumulation was also measured. The activated G-protein level was investigated by guanosine 5'-O-[gamma-thio]triphosphate (GTPγS) binding assay. RESULTS: 17ß-estradiol pretreatment decreased the contractile effect of (-)-noradrenaline via the α2-ARs, and abolished the contractile effect via the α2B-ARs. All the α2-AR subtypes' mRNA was significantly decreased. 17ß-estradiol pretreatment significantly increased the myometrial cAMP level in the presence of BRL 44408 (P=0.001), ARC 239 (P=0.007), and spiroxatrine (P=0.045), but did not modify it in the presence of spiroxatrine + BRL 44408 combination (P=0.073). It also inhibited the G-protein-activating effect of (-)-noradrenaline by 25% in the presence of BRL 44408 + spiroxatrine combination. CONCLUSIONS: The expression of the α2-AR subtypes is sensitive to 17ß-estradiol, which decreases the contractile response of (-)-noradrenaline via the α2B-AR subtype, and might cause changes in G-protein signaling pathway. Estrogen dysregulation may be responsible for preterm labor or uterine inertia via the α2-ARs.

The effects of female sexual hormones on the expression and function of α1A- and α1D-adrenoceptor subtypes in the late-pregnant rat myometrium.

The aim of the study was to investigate the roles of α1-adrenoceptor subtypes in the last-day pregnant rat uterus in vitro by the administration of subtype-specific antagonists (the α1A-adrenoceptor antagonist WB 4101 and the α1D-adrenoceptor antagonist BMY 7378) after 17ß-estradiol or progesterone pretreatment. In isolated organ bath studies, contractions were elicited with (-)-noradrenaline (10(-8)-10(-5)M) in the presence of propranolol (10(-5)M) and yohimbine (10(-6)M) in order to avoid ß-, and α2-adrenergic action. The myometrial expressions of the α1-adrenoceptor subtypes were determined by means of the real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques. The activated G protein levels were investigated through radiolabelled GTP binding assays. Both 17ß-estradiol and progesterone pretreatment changed the myometrial contracting effect of (-)-noradrenaline. In the presence of WB 4101, progesterone pretreatment decreased the (-)-noradrenaline-induced myometrial contraction. In the presence of BMY 7378, both the 17ß-estradiol and the progesterone pretreatment reduced the effect of (-)-noradrenaline. The mRNA and protein expressions of the α1A-adrenoceptors were decreased after 17ß-estradiol pretreatment. (-)-Noradrenaline increased the [(35)S]GTPγS binding of the α1-adrenoceptors, which was most markedly elevated by progesterone. Pertussis toxin inhibited the [(35)S]GTPγS binding-stimulating effect of (-)-noradrenaline, indicating the role of Gi proteins in the signal mechanisms. 17ß-estradiol pretreatment blocks the expression of the α1A-adrenoceptors, whereas it does not influence the expression of the α1D-adrenoceptors. Progesterone pretreatment does not have any effect on the myometrial mRNA and protein expressions of the α1-adrenoceptors, but it alters the G protein coupling of these receptors, promoting a Gi-dependent pathway.

Low dosage of rimonabant leads to anxiolytic-like behavior via inhibiting expression levels and G-protein activity of kappa opioid receptors in a cannabinoid receptor independent manner.

WHAT IS KNOWN: There is an increasing number of studies demonstrating the direct effect of the cannabinoid receptor 1 (CB1) antagonist/inverse agonist rimonabant on the opioid system. The kappa opioid receptors (KORs) are well known to mediate depression- and anxiety-like behavior. Clinical studies on chronic rimonabant administration have revealed that rimonabant leads to a very similar pathophysiology, suggesting a potential impact of rimonabant on KORs. OBJECTIVES: Our objectives were to examine the putative effects of rimonabant on KOR ligand binding, G-protein activity, protein expression and how all these contribute to the development of depression- and anxiety-like behavior. RESULTS: In Chinese hamster ovary (CHO) cell membranes transfected with rat KOR (CHO-rKOR) rimonabant inhibited KOR agonist [3H]U69593 binding in the micromolar range in competition binding experiments and specifically reduced KOR basal activity at lower micromolar concentrations in [35S]GTPγS binding assays. Rimonabant significantly inhibited dynorphin (1-11)-induced [35S]GTPγS binding in micromolar range in CHO-rKOR cells, CB1 knockout (CB1 K.O.) and CB1/CB2 double knockout mouse forebrain membranes. A single dose of i.p. 0.1 mg/kg rimonabant significantly reduced dynorphin (1-11)-induced KOR G-protein activity and KOR protein expression levels 24 h following the administration in both wild type and CB1 K.O. mice forebrain. Furthermore, in elevated plus maze mice showed an anxiolytic-like effect upon rimonabant injection that could be reversed by 1 mg/kg KOR antagonist norbinaltorphimine. The anxiolytic-like effects were further confirmed with the light­dark box test. CONCLUSION: Rimonabant reduced KOR ligand binding, receptor mediated G-protein activity and protein expression level, which overall leads to altered anxiety-like behavior.

Design, Synthesis and Biological Evaluation of Two Opioid Agonist and Cav 2.2 Blocker Multitarget Ligands.

N-type voltage-dependent Ca(2+) channels (CaV 2.2) are located at nerve endings in the central and peripheral nervous systems and are strongly associated with the pathological processes of cerebral ischaemia and neuropathic pain. CaV 2.2 blockers such as the ω-conotoxin MVIIA (Prialt) are analgesic and have opioid-sparing effects. With the aim to develop new multitarget analgesic compounds, we designed the first ω-conotoxin/opioid peptidomimetics based on the enkephalin-like sequence Tyr-D-Ala-Gly-Phe (for the opioid portion) and two fragments derived from the loop-2 pharmacophore of ω-conotoxin MVIIA. Antinociceptive activity evaluated in vitro and in vivo revealed differential affinity for CaV 2.2 and opioid receptors and no significant synergistic activity.

Inhibition of opioid receptor mediated G-protein activity after chronic administration of kynurenic acid and its derivative without direct binding to opioid receptors.

There is an increasing number of evidence showing analgesic properties of the kynurenic acid (KYNA), and also some studies demonstrate that kynurenine might interact with the opioid system. Therefore in this study, for the first time we investigated the direct binding affinity of KYNA and its structural analog KYNA-1 towards mu, kappa and delta opioid receptor in competition binding experiments applying opioid receptor specific radioligands. The binding affinity measurements were performed in Chinese hamster ovary cell lines overexpressing the corresponding opioid receptor (mu and kappa opioid receptor were rat, delta opioid receptor were mouse sequence). Additionally we also examined the chronic effect of these compounds on mu, kappa and delta opioid receptor and also nociceptin peptide receptor mediated G-protein activity in [(35)S]GTPγS binding assays performed in mouse cortex and striatum membranes. Our results showed that KYNA and KYNA-1 had no affinity towards any of the three classic opioid receptors. On the other hand the compounds significantly decreased opioid and nociceptin receptor mediated G-protein activity or in some cases enhanced the potency of the activating ligand. Moreover, the alterations were receptor and brain region specific. Accordingly, we conclude that KYNA and KYNA-1 do not interact directly with the opioid receptors, but more likely alter the receptor functions intracellularly.

Novel cyclic biphalin analogue with improved antinociceptive properties.

Two novel opioid analogues have been designed by substituting the native d-Ala residues in position 2,2' of biphalin with two residues of d-penicillamine or l-penicillamine and by forming a disulfide bond between the thiol groups. The so-obtained compound 9 containing d-penicillamines showed excellent µ/δ mixed receptor affinities (K i (δ) = 5.2 nM; K i (µ) = 1.9 nM), together with an efficacious capacity to trigger the second messenger and a very good in vivo antinociceptive activity, whereas product 10 was scarcely active. An explanation of the two different pharmacological behaviors of products 9 and 10 was found by studying their conformational properties.