Detection of stress and the effects of central nervous system depressants by gastrointestinal smooth muscle electromyography in wakeful rats.

AIMS: The altered gut-brain interaction can be in the background of functional gastrointestinal (GI) disorders. In the GI tract, the slow-wave myoelectric signals can be detected by electromyography (EMG). The aims of our study were to follow up the stress induced alteration in the GI tract by smooth muscle EMG in wakeful rats. MAIN METHODS: The GI tract myoelectric activity of male rats was measured by an electrode pair under the abdominal skin, the responses were detected and analyzed by a software using fast Fourier transformation. Animals were immobilized and treated with either diazepam or haloperidol. The plasma corticosterone level was determined by ELISA kit, the levels of drugs were measured by HPLC, while the direct GI effects of the compounds were tested in an organ bath. KEY FINDINGS: Significant correlation (r2 = 0.52) was found between the immobilization induced increase in the EMG spectra of the GI tract segments and the increase in corticosterone plasma levels. The stress-reducing effects of diazepam and haloperidol were also detectable by smooth muscle EMG in the GI tract. No direct smooth muscle actions of the drugs were found in organ bath studies. SIGNIFICANCE: The smooth muscle EMG instrument can measure the level of acute stress and is applicable for the investigation of central nervous system affecting drugs through the GI tract in awake rats. This is the first tool to measure the stress response via the GI tract reactions. The technique may open a new perspective in the diagnosis and therapy of psychosomatic disorders.

Hydrogen peroxide via thromboxane A2 receptors mediates myogenic response of small skeletal muscle veins in rats.

UNLABELLED: We aimed to test two hypotheses: (1) isolated small veins develop substantial myogenic tone in response to elevation of intraluminal pressure, (2) H2O2 contributes to the mediation of myogenic response via activation of arachidonic acid (AA) cascade and release constrictor prostaglandins. METHODS: Small veins were isolated from gracilis muscle of male rats, then cannulated. Changes of diameter to increases in intraluminal pressure, H2O2 and arachidonic acid in the presence and absence of various inhibitors were measured by videomicroscope and microangiometer. At the end of experiments the passive diameter were obtained in Ca2+ -free PSS solution. RESULTS: Isolated rat gracilis muscle small veins developed a substantial myogenic tone in response to increases in intraluminal pressure (1-12 mmHg). Calculated maximum myogenic tone was 70 ± 5% at 10 mmHg. Presence of catalase or indomethacin or SQ 29,548 reduced significantly the pressure-induced myogenic response. Also, H2O2 (10-9-10-5 M) and arachidonic acid (10-7-10-4 M) elicited concentration dependent constrictions, which were inhibited by the presence of indomethacin or SQ 29,548. CONCLUSION: We propose that both myogenic response and pressure-induced release of H2O2 play important roles in regulating the vasomotor function of venules both in physiological and pathological conditions.

Metabolic effects of mulberry leaves: exploring potential benefits in type 2 diabetes and hyperuricemia.

The leaves of Morus alba L. have a long history in Traditional Chinese Medicine and also became valued by the ethnopharmacology of many other cultures. The worldwide known antidiabetic use of the drug has been suggested to arise from a complex combination effect of various constituents. Moreover, the drug is also a potential antihyperuricemic agent. Considering that type 2 diabetes and hyperuricemia are vice-versa in each other's important risk factors, the use of mulberry originated phytotherapeutics might provide an excellent option for the prevention and/or treatment of both conditions. Here we report a series of relevant in vitro and in vivo studies on the bioactivity of an extract of mulberry leaves and its fractions obtained by a stepwise gradient on silica gel. In vivo antihyperglycemic and antihyperuricemic activity, plasma antioxidant status, as well as in vitro glucose consumption by adipocytes in the presence or absence of insulin, xanthine oxidase inhibition, free radical scavenging activity, and inhibition of lipid peroxidation were tested. Known bioactive constituents of M. alba (chlorogenic acid, rutin, isoquercitrin, and loliolide) were identified and quantified from the HPLC-DAD fingerprint chromatograms. Iminosugar contents were investigated by MS/MS, 1-deoxynojirimycin was quantified, and amounts of 2-O-alpha-D-galactopyranosyl-1-deoxynojirimicin and fagomine were additionally estimated.

Systematic counselling by general practitioners for promoting physical activity in elderly patients: a feasibility study.

RESEARCH QUESTIONS: To investigate how the daily physical activities of elderly patients can be enhanced by systematic counselling conducted by general practitioners (GPs). METHODS: In this feasibility study with pre-post design, 29 people (14 females, mean age 72.2 years, SD = 6.1) were enrolled during routine visits by two general practitioners. A baseline assessment of current physical activity based on the stages according to the Transtheoretical Model was followed by a counselling session. The target behaviour was defined by performance of 30 minutes of daily moderate-intensity activities that increase the breathing rate, on five days per week. At the 2-month follow-up, subjects were assessed for improvement in stage of physical activity since baseline. After the end of the intervention, participating GPs and patients were asked questions focusing on the feasibility, acceptance and usefulness of counselling. RESULTS: Interview results showed that the two GPs considered the counselling protocol easy to handle and useful for promoting physical activity. Counselling sessions were especially encouraging for the not sufficiently active people. Most of them would like to have additional counselling session. At baseline, 9 of 29 people were sufficiently active. After 2 months, this proportion was 21 of 29. The mean of the number of minutes of physical activity during the previous 4 weeks increased from 247 to 436 minutes (weekly). CONCLUSIONS: The programme was judged positively by the general practitioners and the participating elderly patients. Systematic counselling by general practitioners led to an increase in the physical activity behaviour. Therefore, a more rigorous randomised controlled trial with adequate followup is recommended.

Kappa-receptor selective binding of opioid ligands with a heterocyclic bicyclo[3.3.1]nonan-9-one structure.

Previous pharmacological results have suggested that members of the heterocyclic bicyclo[3.3.1]nonan-9-one-like compounds are potent kappa-opioid receptor specific agonists. One lead molecule of this series. called compound 1 (dimethyl 7-methyl-2,4-di-2-pyridyl-3.7-diazabicyclo[3.3.1]nonan-9-one-1,5-dicarboxylate) exhibited high affinity for [3H]ethylketocyclazocine and [3H]U-69.593 binding sites in guinea pig cerebellar membranes which known to be a good source for kappa1 receptors. It was shown by molecular modelling that heterocyclic bicyclo[3.3.1]nonan-9-ones fit very well with the structure of ketazocine, a prototypic kappa-selective benzomorphan compound; when compared to the arylacetamide structure of U-69.593, a specific kappa1-receptor agonist, a similar geometry was found with a slightly different distribution of the charges. It is postulated, that the essential structural skeleton involved in the opioid activity is an aryl-propyl-amine element distributed along the N7-C6-C5-C4-aryl bonds.

Noncompetitive nature of oxytocin antagonists with general structure Mpa(1)Xxx(2)Sar(7)Arg(8).

Eight oxytocin (OT) antagonists with general structure Mpa(1)Sar(7)Arg(8), substituted at position 2 with conformationally constrained and bulky amino acids, were synthesized and pharmacologically tested. Binding affinities and selectivities of compounds for OT, and vasopressin receptor subtypes were investigated. In vitro effects of antagonists were evaluated via inhibition of OT-induced contractions of isolated guinea-pig uterus. The abilities of OT antagonists to inhibit spontaneous contractility in 24 h postpartum rat uterus were investigated. These peptides exhibited pseudoirreversible pharmacological properties, and comprise a novel group of OT antagonists for potential clinical use. Their noncompetitive pharmacological nature can be of therapeutic benefit through a sustained effect on myometrium.

Use of antisense oligonucleotides to verify the role of the alpha(1A)-adrenergic receptor in the contractility of the rat uterus post partum.

The adrenergic system plays a major role in the regulation of the contractility of the uterus during pregnancy. This study investigated the role of the alpha(1A)-adrenergic receptor (AR) in this regulation. The use of partial phosphorothioate antisense oligodeoxynucleotides (AONs) permitted the sequence-selective inhibition of AR gene expression. AONs were injected together with a cationic liposomal carrier agent into the post partum rat uterus. Incubation for 12 or 24 h with the most effective AON (480-AON) caused a 58.7 or 53.0% inhibition, respectively, of the expression of the alpha(1A)-AR density, whereas incubation for 36 or 48 h resulted in only a 38.8 or 26.7% inhibition, respectively. The decrease of the alpha(1A)-AR density by 480-AON was demonstrated by Western blot analysis and a radioreceptor binding assay on rat uterus preparations 24 h after delivery. The changes in the contractility of the uterus after AON treatment were measured on isolated rat uterine tissue by electric field stimulation. The significant decrease in the ability of the uterus to contract was indicated by the area under the curve method. The electric field studies revealed that the specific alpha(1A)-blockers 5-methylurapidil and WB 4101 inhibited the rhythmic contraction by about 74 and 70% in the control uteri and by 25 and 20% in 480-AON-treated uteri, respectively. The curves for the beta-mimetic (terbutaline) and alpha(1D)-antagonist (BMY7370) inhibitors were unchanged after 480-AON treatment of the uteri. These results suggest the importance of the alpha(1A)-AR in the tocolytic effect exerted by the alpha(1)-antagonist, although high concentrations of antagonists can not exclude the role of alpha(1D)-ARs, too. Additionally, these prove that the knockdown transformation by AONs offers a useful animal model for the investigation of receptors controlling the function of uterine tissue.

Characterization of late-pregnant rat uterine contraction via the contractility ratio in vitro significance of alpha1-adrenoceptors.

The aim of this study was to characterize the ability of late-pregnant (days 15-22) rat uterine tissue rings to contract in response to electric field stimulation in vitro. For this purpose, maximum rhythmic contractions were elicited by optimum choice of the period time and the pulse width, the two main parameters of electric field stimulation. In parallel, the plasma 17beta-estradiol and progesterone levels were determined. It was found that the contractility ratio, i.e. the quotient of the optimum pulse width and the period time, is a good parameter with which to express the contractility. The larger the contractility ratio, the better the ability to contract. Evaluation of the area under the curve did not furnish information relating to the contractility in this method. A very close correlation was observed between the contractility ratio and the quotient of the 17beta-estradiol and progesterone levels on different days, demonstrating that the in vitro ability characterized by the contractility ratio is in keeping with the physiological regularity. There was also a very close correlation between the contractility ratio and the quotient of the alpha1- and beta-adrenergic receptors, suggesting the main role of the numbers of alpha1-receptor in pregnant uterine contractility. It is believed that this is the first in vitro model to give a numerical measure concerning the ontogeny of uterine contractility in late pregnancy.

[Pharmacological models to investigate the role of the adrenergic system on post-partum rat uterus]. / Farmakológiai modell az adrenerg rendszer szerepének vizsgálatára post partum patkány uteruson.

The adrenergic system plays a major role in the regulation of the pregnant uterine contractility. Our aim was to develop an experimental animal model to study the role of the alpha 1A-adrenergic receptor (AR) in uterine motor activity by antisense oligonucleotides (AONs). AONs were injected with DOTAP and pluronic gel into the uterine lumen of post-partum rats 2-3 hours after delivery. The decrease of the alpha 1A-AR density by AON was demonstrated by RT-PCR method, Western blot analysis and radioligand binding assay on rat uterus preparations 24 h after delivery. The changes in the contractility of the uterus were measured on isolated rat uterine tissue by electric field stimulation (EFS). The EFS investigation demonstrated that the effect of the specific alpha 1A-blocker 5-methylurapidil and WB4101 was significantly decreased in the AON-treated rat uterus as compared to the control group but the effect of the beta-mimetic terbutalin and alpha 1D-antagonist BMY7378 was unchanged. Our result suggest that the alpha 1A-ARs play a very important role in the regulation of uterine contractility, and may serve as the basis for a subsequent new group of tocolytics (uterus selective alpha 1-antagonists), which may lead to more selective therapy than currently used beta-mimetics.

Tritiated kappa receptor antagonist norbinal torphimine: synthesis and in vitro binding in three different tissues.

Recently a new antagonist with high selectivity for the kappa receptors (norbinaltorphimine) was developed and tested in various systems. This compound was radiolabelled with tritium resulting in high specific radioactivity (47.2 Ci/mmol). [3H]Norbinaltorphimine was characterized by in vitro radioligand binding assays. The radioligand binds to kappa-opioid receptors with a high potency and selectivity in guinea pig, frog and rat brain membranes. Our results suggest the kappa1 specificity of the radioligand.

Mu-opioid receptor specific antagonist cyprodime: characterization by in vitro radioligand and [35S]GTPgammaS binding assays.

The use of compounds with high selectivity for each opioid receptor (mu, delta and kappa) is crucial for understanding the mechanisms of opioid actions. Until recently non-peptide mu-opioid receptor selective antagonists were not available. However, N-cyclopropylmethyl-4,14-dimethoxy-morphinan-6-one (cyprodime) has shown a very high selectivity for mu-opioid receptor in in vivo bioassays. This compound also exhibited a higher affinity for mu-opioid receptor than for delta- and kappa-opioid receptors in binding assays in brain membranes, although the degree of selectivity was lower than in in vitro bioassays. Cyprodime has recently been radiolabelled with tritium resulting in high specific radioactivity (36.1 Ci/mmol). We found in in vitro binding experiments that this radioligand bound with high affinity (K(d) 3. 8+/-0.18 nM) to membranes of rat brain affording a B(max) of 87. 1+/-4.83 fmol/mg. Competition studies using mu, delta and kappa tritiated specific ligands confirmed the selective labelling of cyprodime to a mu-opioid receptor population. The mu-opioid receptor selective agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO) was readily displaced by cyprodime (K(i) values in the low nanomolar range) while the competition for delta- ([D-Pen(2), D-Pen(5)]enkephalin (DPDPE)) and kappa- (5alpha,7alpha, 8beta-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl]-benzene-acetamide (U69,593)) opioid receptor selective compounds was several orders of magnitude less. We also found that cyprodime inhibits morphine-stimulated [35S]GTPgammaS binding. The EC(50) value of morphine increased about 500-fold in the presence of 10 microM cyprodime. These findings clearly indicate that cyprodime is a useful selective antagonist for mu-opioid receptor characterization.

Correlation between alpha1/beta-adrenoceptor ratio and spontaneous uterine motor activity in the post-partum rat.

The spontaneous uterine motor activity of the post-partum rat was investigated in parallel with the in-vitro determination of the density of the alpha1 and beta-adrenergic receptors of the myometrium. The in-vivo experiments were performed by an improved method, using a Millar catheter fitted with a latex microballoon. The spontaneous contractility of the post-partum rat uterus was found to be highest 24 h after delivery, indicating that this time is the most suitable for pharmacological examinations of tocolytic agents. A very close correlation was found between the results of the in-vivo experiments and the alpha1/beta-adrenergic receptor ratio assessed by an in-vitro receptor assay, thus indicating that the state of the adrenergic receptor system fundamentally determines the contractility of the uterus. This conclusion is supported by the fact that the pharmacological sensitivity of the rat uterus to prazosin and fenoterol changed as a function of the post-partum time in accordance with the alpha1/beta-adrenoceptor ratio. These results and the relevant data available reveal a crucially important role of an alpha1-adrenoceptor-mediated process, implicating alpha1-blockers as theoretically potent agents for inhibition of premature uterine contractions.

Evidence of non-synaptic regulation of postpartum uterine contractility in the rat.

Myometrial tissue rings from postpartum rats (24 h after delivery) were studied in vitro by electric field stimulation, and the alpha1/beta2-adrenoceptor ratio was determined by a radioligand binding technique. Pregnancy-denervated uterine rings were stimulated by long-duration pulses (100 ms). The contractions were inhibited by beta2-agonists (terbutaline and fenoterol) and alpha-antagonists (phentolamine, urapidil and yohimbine) in a concentration-dependent manner. Their effects were not altered by the adrenergic neuron-blocking agent bretylium. The alpha-antagonists (except phentolamine) elicited the same maximal inhibition as the beta2-agonists. Receptor assays revealed that the alpha1/beta2 ratio was about 2 in the measured uteri. It was concluded that the inhibitory effects of alpha-antagonists and beta2-agonists are mediated via non-synaptic adrenoceptors of the denervated postpartum rat uterus. The same inhibitory activity could be explained by the greater amount of alpha-receptors. It is believed that this is the first functional proof of the existence of non-synaptic alpha1-adrenoceptors in smooth muscle.

New nepenthone and thevinone derivatives.

The diastereoselective reaction of thevinone (2a) and nepenthone (2c) and their dihydro derivatives (2b and d) with Grignard reagents afforded new N-substituted (20S)- and (20R)-phenyl-6,14-ethenomorphinan derivatives (6a-y). The Grignard reaction of the N-substituted-N-demethyl derivatives 4a-f and 4m-r with methylmagnesium iodide resulted in the (20R)-phenyl tertiary alcohols 5a-f and 5m-r, respectively, but the conversion of 4g-1 and that of the N-substituted-dihydrothevinone derivatives with phenylmagnesium bromide afforded the (20S)-phenyl derivatives 5g-l and 5s-y, respectively. The N-cyclopropylmethyl-, N-beta-phenylethyl-, and N-propyl derivatives were prepared by the 3-O-demethylation of compounds 5. For the synthesis of the N-allyl-, N-dimethylallyl-, and N-propargyl compounds 2a-d were reacted with the corresponding Grignard reagent, and treatment of the products with cyanogen bromide gave the cyanamides 8a-d. These latter compounds were transformed into 10a, b,d, whose alkylation led to the target derivatives 6d-f, j-l, p-r, and w-y. The biochemical investigation of these substances showed that the affinities to the delta-opioid receptors were high, but the selectivity was low. In two cases (6c and 11d) a mu-opioid receptor specificity was observed.