Pharmacological and computational evaluation of fig for therapeutic potential in hyperactive gastrointestinal disorders.

BACKGROUND: Ficus palmata (Fig), are distributed in different parts of the world, and are used in traditional medicine to treat various ailments including inflammation, tumor, epilepsy, jaundice, influenza and bacillary dysentery. The present study aimed to evaluate the antidiarrheal, antisecretary, antispasmodic, antiulcer and anti motility properties of Ficus palmata. METHODS: In-vivo, in-vitro and in-silico techniques were used to investigate various gastrointestinal effects of Ficus palmata. Antidiarrheal, antisecretary, antispasmodic, antiulcer, anti motility and molecular docking were performed using castor oil induced diarrhea and fluid accumulation, isolated tissue preparations, ethanol-HCl induced ulcer assay, charcoal meal transit time and Auto Doc Vina. RESULTS: Ficus palmata crude extract (Fp.Cr) exhibited protection against castor oil-induced diarrhea in mice and dose-dependently inhibited intestinal fluid secretions. Fp.Cr caused relaxation of spontaneous and K+ (80 Mm)-induced contractions in isolated rabbit jejunum preparations. It showed protective effect against gastric ulcers induced by ethanol-hydrochloric acid in rats. Fp.Cr reduced distance travelled by charcoal meal in the gastrointestinal transit model in mice. The plant constituents: psoralenoside and bergapten showed high binding affinities (E-value ≥ - 6.5 Kcal/mol) against histaminergic H1, calmodulin and voltage gated L-type calcium channels, while showed moderate affinities (E-value ≥7 Kcal/mol) against dopaminergic D2, adrenergic α1, muscranic M3, mu-opioid, whereas revealed lower affinities (E-value ≥9.5 Kcal/mol) vs. muscranic M1, histaminergic H2 and H+/K+ ATPase pump. Germanicol acetate and psoralene exhibited weak affinities against aforementioned targets. CONCLUSION: This study reveals that Ficus palmata possesses anti-diarrheal, anti-secretory, anti-spasmodic, anti-motility and anti-ulcer activities. The various constituents reveal different binding affinities against target proteins, which mediate the gastrointestinal functions.

Development of a homogeneous high-throughput live-cell G-protein-coupled receptor binding assay.

The measurement of ligand receptor binding parameters for G-protein-coupled receptors is indispensable in the drug discovery process. Traditional ligand receptor binding assays require scale-up of cells and membrane preparations, which is an expensive and time-consuming process. In this report, the authors describe the development of a homogeneous live-cell binding assay for GPCRs using a fluorophore-labeled nonpeptide ligand. The model assay used Cy3B-labeled telenzepine and Chinese hamster ovary cells expressing M1 muscarinic acetylcholine receptors. This homogeneous live-cell fluorescence binding assay format is superior to the traditional binding methods because it measures binding of a ligand to intact receptors on living cells. The assay requires no washing or separation steps, thereby allowing a real-time kinetic readout for the determination of ligand association and dissociation from the intact receptors. The results also suggest that miniaturization is feasible without compromising the data quality.

Effects of chlorpyrifos oxon on M2 muscarinic receptor internalization in different cell types.

The muscarinic M2 receptor is a member of the G protein-coupled receptor (GPCR) superfamily. Agonist activation of GPCR leads to their phosphorylation, desensitization, internalization, and subsequent endocytic recycling or lysosomal degradation. Agonist-induced phosphorylation of M2 receptors is mediated by G-protein receptor kinase 2 (GRK2). The active metabolite of the organophosphorus insecticide chlorpyrifos, i.e., chlorpyrifos oxon (CPO), inhibited agonist-induced phosphorylation of human recombinant M2 receptors by GRK2 in vitro in a concentration-dependent manner. In both intact HEL 299 cells (human embryonic lung fibroblasts expressing M2 receptors) and CHO-M2 cells (stably expressing M2 receptors), the muscarinic agonist carbachol (100 microM) led to receptor internalization as determined by reduced specific binding to the membrane-impermeable radioligand [(3)H]-N-methylscopolamine (NMS). CPO alone (100 microM) exerted no significant effect on NMS binding in either HEL 299 or CHO-M2 cells. In HEL 299 cells, CPO did not influence carbachol-induced internalization, whereas in CHO-M2 cells CPO blocked internalization. In primary striatal neurons, M2 receptors appeared widely and diffusely distributed. Exposure to either carbachol or CPO led to apparent receptor internalization with an increased percent of cells exhibiting punctate domains of immunostaining, while combined exposure to both carbachol and CPO led to a significantly higher percent of cells exhibiting this appearance. The data suggest that CPO may differentially influence agonist-stimulated M2 receptor internalization in a cell-dependent manner.

Distension-induced gastric accommodation in functional dyspepsia: effect of autonomic manipulation.

Functional dyspepsia (FD) is associated with impaired gastric accommodation and autonomic dysregulation. The aim of this study was to investigate the effects of autonomic manipulation on distension-induced gastric accommodation in subjects with and without FD, using a newly developed gastric barostat paradigm. Twelve healthy subjects (HS) and 18 subjects with FD had four barostat examinations each: no intervention, intravenous atropine (1 mg), vagal stimulation (mental relaxation with deep breathing) and acute stress stimulation (serial subtraction task). Intrabag pressure increased from 1 to 15 mmHg in 5 min (ramp phase), and was maintained at 15 mmHg for 5 min (tonic phase). Volume responses were analysed using predefined parameters. There were no significant group differences in accommodation variables between HS and subjects with FD. The FD group could be subdivided into two distinct subgroups: subgroup 1 (n = 7, 38%) with low maximum volume and accommodation rate, and subgroup 2 with normal accommodation (n = 11). In subgroup 1, but not in subgroup 2 atropine increased maximum volume and accommodation rate substantially. Neither mental stress nor mental relaxation changed any of the accommodation variables. In a subgroup of subjects with FD, impairment of distension-induced gastric accommodation can be improved by cholinergic blockade, but not by acute physiological autonomic manipulation.

Urodynamics and bladder muscarinic receptors in rats with cerebral infarction and bladder outlet obstruction.

We characterized muscarinic receptor binding and urodynamic parameters in rats with cerebral infarction and chronic bladder outlet obstruction as models of detrusor overactivity. Bladder weight showed little significant difference between the cerebral-infarcted and sham rats, but the bladder weight was about three times greater in the bladder outlet-obstructed rats. Bladder capacity and voided volume were significantly lower (36.7 and 55.1%, respectively) in the cerebral-infarcted than in the sham rats. Involuntary contractions before micturition were seen in the bladder outlet-obstructed rats but not in sham rats. The bladder outlet-obstructed rats showed significant increases (2.65 and 2.57 times, respectively) in bladder capacity and voided volume, compared with those in sham rats. Bmax values for specific [N-methyl-3H]scopolamine ([3H]NMS) binding in the bladder were significantly (34%) increased in the cerebral-infarcted rats compared with sham rats, whereas Kd was unaffected by infarction. On the other hand, there was little significant change in Kd and Bmax for specific [3H]NMS binding in the bladder-obstructed rats compared with sham rats. In conclusion, the present study shows that cerebral infarction but not bladder outlet obstruction in rats causes up-regulation of bladder muscarinic receptors, and that such regulation of bladder muscarinic receptors may be at least partly associated with the symptoms of detrusor overactivity subsequent to cerebral infarction.

High performance liquid chromatographic determination of 3-methylflavone-8-carboxylic acid, the main active metabolite of flavoxate hydrochloride in human urine.

High performance liquid chromatographic (HPLC) method was presented for the determination of 3-methylflavone-8-carboxylic acid as the main active metabolite of flavoxate hydrochloride (FX) in human urine. The proposed method was based on using CN column with mobile phase consisting of acetonitrile-12 mM ammonium acetate (40:60, v/v) and adjusted to apparent pH 4.0 with flow rate of 1.5 ml min(-1). Quantitation was achieved with UV detection at 220 nm. The proposed method was utilized to the determination of dissolution rate for tablets containing flavoxate hydrochloride. The urinary excretion pattern has been calculated using the proposed method.

Treatment of the overactive bladder syndrome with muscarinic receptor antagonists: a matter of metabolites?

Antagonists of muscarinic acetylcholine receptors, such as darifenacin, oxybutynin, propiverine, solifenacin, tolterodine, and trospium, are the mainstay of the treatment of the overactive bladder syndrome. Fesoterodine is a newer drug awaiting regulatory approval. We briefly review the pharmacological activity of their metabolites and discuss how active metabolites may contribute to their efficacy and tolerability in vivo. Except for trospium, and perhaps solifenacin, all of the above drugs form active metabolites, and their presence and activity need to be taken into consideration when elucidating relationships between pharmacokinetics and pharmacodynamics of these drugs. Moreover, the ratios between parent compounds and metabolites may differ depending on genotype of the metabolizing enzymes, concomitant medication, and/or drug formulation. Differential generation of active metabolites of darifenacin or tolterodine are unlikely to influence the overall clinical profile of these drugs in a major way because the active metabolites exhibit a similar pharmacological profile as the parent compound. In contrast, metabolites of oxybutynin and propiverine may behave quantitatively or even qualitatively differently from their parent compounds and this may have an impact on the overall clinical profile of these drugs. We conclude that more comprehensive studies of drug metabolites are required for an improved understanding of their clinical effects.

Propiverine and metabolites: differences in binding to muscarinic receptors and in functional models of detrusor contraction.

Propiverine is a commonly used antimuscarinic drug used as therapy for symptoms of an overactive bladder. Propiverine is extensively biotransformed into several metabolites that could contribute to its spasmolytic action. In fact, three propiverine metabolites (M-5, M-6 and M-14) have been shown to affect various detrusor functions, including contractile responses and L-type calcium-currents, in humans, pigs and mice, albeit with different potency. The aim of our study was to provide experimental evidence for the relationship between the binding of propiverine and its metabolites to human muscarinic receptor subtypes (hM(1)-hM(5)) expressed in chinese hamster ovary cells, and to examine the effects of these compounds on muscarinic receptor-mediated detrusor function. Propiverine, M-5, M-6 and M-14 bound to hM(1)-hM(5) receptors with the same order of affinity for all five subtypes: M-6 > propiverine > M-14 > M-5. In HEK-293 cells expressing hM(3), carbachol-induced release of intracellular Ca(2+) ([Ca(2+)](i)) was suppressed by propiverine and its metabolites; the respective concentration-response curves for carbachol-induced Ca(2+)-responses were shifted to the right. At higher concentrations, propiverine and M-14, but not M-5 and M-6, directly elevated [Ca(2+)](i). These results were confirmed for propiverine in human detrusor smooth muscle cells (hDSMC). Propiverine and the three metabolites decreased detrusor contractions evoked by electric field stimulation in a concentration-dependent manner, the order of potency being the same as the order of binding affinity. We conclude that, in comparison with the parent compound, loss of the aliphatic side chain in propiverine metabolites is associated with higher binding affinity to hM(1)-hM(5) receptors and higher functional potency. Change from a tertiary to a secondary amine (M-14) results in lower binding affinity and reduced potency. Oxidation of the nitrogen (M-5) further lowers binding affinity as well as functional potency.

Muscarinic acetylcholine receptor: thermodynamic analysis of the interaction of agonists and antagonists.

The thermodynamic parameters of the interaction of agonists and antagonists with heart and brain muscarinic receptors were determined. The binding of quinuclidinyl [3H]benzilate and the inhibition of quinuclidinyl benzilate (QNB) binding by agonists and antagonists were examined at temperatures between 2 degrees C and 27 degrees C. The density of specific binding sites and the relative proportions of high- and low-affinity binding components of drugs were unaffected by the temperature changes. The binding of atropine was entropy driven in brain and heart membranes. In contrast, net values of these thermodynamic parameters for QNB binding and for the high-affinity binding component of pirenzepine to brain membranes were decreased with the enhancement of the temperature. The low-affinity binding component of the agonists carbachol, oxotremorine and pilocarpine was enthalpy driven. Their high-affinity binding component was entropy driven at 2 degrees C and became enthalpy driven when the incubation temperature was increased. The guanine nucleotide Gpp[NH]p partly prevented the temperature-dependent decrease of net entropy and enthalpy values. Considering that the net changes of thermodynamic parameters are relevant of the interactions between the ligand, the receptor protein and the adjoining membranous molecules, a three-state conformational model is proposed for the muscarinic receptor protein. The receptor selectivity is reappreciated owing to these three states of the receptor protein and the different components of the muscarinic receptor complexes.

Reversibility of diabetes- and diuresis-induced alterations in rat bladder dome muscarinic receptors.

Previous studies from our laboratory demonstrated that 8 weeks of streptozocin (STZ)-induced diabetes and sucrose-fed diuresis resulted in increases in the density of muscarinic receptors in rat bladder dome and that early insulin treatment (started 3 days after the onset of diabetes) prevented the diabetes-induced upregulation (J Pharmacol Exp Ther 248:81-88, 1989; Diabetes 40: 1150-1156, 1991; J Urol 147:760-763, 1992). To determine whether diabetes- and diuresis-induced alterations in muscarinic receptors in rat bladder dome are reversible, we administered insulin (beginning 8 weeks after the onset of diabetes) or removed sucrose from drinking water of diuretic rats (beginning 8 weeks after the onset of diuresis). Five groups of rats were maintained for 16 weeks: 1) STZ-induced diabetic rats (65 mg/kg intravenously); 2) insulin-treated diabetic rats (5-8 U/day insulin subcutaneously beginning 8 weeks after the onset of diabetes); 3) sucrose-fed diuretic rats (5% sucrose in drinking water throughout 16 weeks); 4) sucrose-removed rats (sucrose withdrawn from drinking water after 8 weeks of the sucrose-induced diuretic state); and 5) age-matched control rats. Radioligand receptor binding experiments with [3H]quinuclidinyl benzilate showed an increase in the density of muscarinic receptors in bladder dome of diabetic and sucrose-fed rats compared with age-matched control rats. Removing the 5% sucrose from the drinking water of diuretic rats reversed the increased water intake and urine output, decreased the bladder hypertrophy that accompanied the diuretic state, and corrected the upregulation of the muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of ligand binding to G protein-coupled receptors by arachidonic acid.

Arachidonic acid (AA), released in response to muscarinic acetylcholine receptor (mAChR) stimulation, previously has been reported to function as a reversible feedback inhibitor of the mAChR. To determine if the effects of AA on binding to the mAChR are subtype specific and whether AA inhibits ligand binding to other G protein-coupled receptors (GPCRs), the effects of AA on ligand binding to the mAChR subtypes (M1, M2, M3, M4, and M5) and to the micro-opioid receptor, beta2-adrenergic receptor (beta2-AR), 5-hydroxytryptamine receptor (5-HTR), and nicotinic receptors were examined. AA was found to inhibit ligand binding to all mAChR subtypes, to the beta2-AR, the 5-HTR, and to the micro-opioid receptor. However, AA does not inhibit ligand binding to the nicotinic receptor, even at high concentrations of AA. Thus, AA inhibits several types of GPCRs, with 50% inhibition occurring at 3-25 MuM, whereas the nicotinic receptor, a non-GPCR, remains unaffected. Further research is needed to determine the mechanism by which AA inhibits GPCR function.

A high n-6 polyunsaturated fatty acid diet reduces muscarinic M2/M4 receptor binding in the rat brain.

The aim of this study was to examine the influence of different fat diets on muscarinic acetylcholine receptor binding. Nineteen male Sprague-Dawley rats were divided into four groups and fed a diet of either high saturated fat, n-6 polyunsaturated fatty acid (PUFA), n-3 PUFA or low fat (control) for 8 weeks. Using quantitative autoradiography, [(3)H]pirenzepine binding to muscarinic M1/M4 receptors and [(3)H]AF-DX384 binding to M2/M4 receptors were measured throughout the brain in all four groups. The main findings were that compared to the low fat control group, M2/M4 receptor binding was significantly reduced in the dorsolateral, dorsomedial and ventromedial parts of the caudate putamen (61-64%, p < 0.05), anterior cingulate cortex (59%, p < 0.01), dentate gyrus and CA1-3 fields of the hippocampus (32-43%, p < 0.01) of rats on a high n-6 PUFA diet; however, no differences in M1/M4 receptor binding densities between the four groups were observed. These results suggest that a diet high in n-6 PUFA, but not of n-3 PUFAs or saturated fat, may selectively alter M2/M4 receptor-mediated signal transduction in the rat brain.

Serum levels of anticholinergic drugs in treatment of acute extrapyramidal side effects.

A simple, sensitive, and specific radioreceptor assay has been developed for the measurement of anticholinergic drugs in human serum. The assay is based on the competitive inhibition by free anticholinergic drugs in a 0.2-mL sample of serum with the specific binding of the potent muscarinic antagonists, tritiated quinuclidinyl benzilate, to solubilized brain muscarinic receptors. Anticholinergic activity could be detected regardless of drug structure and was quantified against atropine standards. Although the serum levels of anticholinergic drugs varied considerably in 35 patients receiving both neuroleptic and anticholinergic drugs, there was a highly significant inverse correlation between the presence of acute extrapyramidal side effects due to neuroleptics and the serum levels of anticholinergics.

Altered ontogenesis of muscarinic cholinergic receptor in mouse brain: effect of L-thyroxine and betamethasone.

The effects of L-T4 and betamethasone treatment of newborn mice on the development of the cholinergic muscarinic receptor in certain brain regions was studied using the potent labeled muscarinic antagonist [3H]4-N-methyl-piperidyl benzilate. Treatment with both L-T4 and betamethasone caused an accelerated accumulation of muscarinic receptors in the cortex 16 days post partum, with a subsequent reduction in level at 30 days. In the cerebellum and caudate putamen, only betamethasone caused a similar early accumulation of muscarinic receptors, while the later effect, namely a reduction in the level at 30 days, was seen with both hormones in these two regions as well as in the hippocampus. The results can explain some behavioral effects observed in other studies after treatment with these hormones.

Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging.

The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [3H]-antagonist as well as of the labeled natural neurotransmitter, [3H]-acetylcholine [( 3H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [3H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [3H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [3H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [3H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms.

Expression and pharmacological characterization of the human M1 muscarinic receptor in Saccharomyces cerevisiae.

The yeast S. cerevisiae has been examined as a heterologous host for the expression of mammalian neurotransmitter receptors which couple to guanine nucleotide regulatory (G) proteins. A cloned gene encoding the M1 subtype of human muscarinic receptor (HM1) was transformed into S. cerevisiae on a high copy plasmid under the control of the promoter for the yeast alcohol dehydrogenase (ADH) gene. Northern blotting demonstrated the presence of HM1 transcripts in transformants, and crude membranes prepared from these cells showed saturable binding of the muscarinic antagonist [3H]N-methyl scopolamine with a Kd of 179 pM and Bmax of 20 fmol/mg protein. Competition binding studies revealed pharmacological properties for these sites which were comparable to those reported for the M1 site in mammalian tissues. Yeast expressing HM1 did not exhibit high affinity agonist binding or cell-cycle arrest in the presence of muscarinic agonists, indicating that the mammalian receptor did not couple to the endogenous yeast G protein.

Effects of cycloheximide and tunicamycin on cell surface expression of pancreatic muscarinic acetylcholine receptors.

The importance of glycosylation in cell surface expression of muscarinic receptors in cultured guinea pig pancreatic acini was investigated. Recovery of the muscarinic receptor population after carbachol-induced down regulation was blocked by cycloheximide but not by tunicamycin, although tunicamycin reduced [3H]mannose incorporation into acinar macromolecules by up to 90%. Tunicamycin treatment also failed to alter carbachol stimulation of amylase secretion from cultured acini. These results indicate that glycosylation of the glandular subtype of muscarinic receptor in the pancreatic acinar cell is not necessary for its insertion in the plasma membrane or for its functional activity.

Expression of the breast cancer associated gene pS2 and the pancreatic spasmolytic polypeptide gene (hSP) in diffuse type of stomach carcinoma.

Expression of the pancreatic spasmolytic peptide (hSP) gene and pS2 (a gene isolated from oestrogen-induced breast carcinoma cells) were analysed in 36 samples of human stomach carcinoma. 17 tumours were investigated at the RNA level (by northern blots) as well as at the gene product level (by immunochemistry). Since pS2 had been shown to be expressed in normal stomach mucosa its activity in carcinoma samples was expected. Surprisingly, strong pS2 immunoreactivity was noted in the diffuse carcinoma type, whereas the intestinal type displayed weak reactivity. The tumour samples showing strong immunostaining expressed the regular 0.6 kb pS2 RNA band and weak staining was paralleled by aberrant transcripts. Additionally, only in tumour samples with regular pS2 transcription was the typical 0.7 kb hSP RNA band seen; samples with aberrant pS2 bands did not express hSP at all. This is the first demonstration of hSP gene activity in a human tumour.

Clinical pharmacology and toxicology of ipratropium bromide.

Anticholinergic drugs inhibit a variety of intrapulmonary events related to airflow obstruction. When administered as an inhaled aerosol, approximately 90 percent of ipratropium bromide (as with beta-adrenergic aerosols) can be assumed to be swallowed. Peak pharmacologic effects occur prior to any detectable plasma drug concentrations. Ipratropium does not exhibit the well-known toxic effects of atropine, and doses many times those required for maximum therapeutic benefit do not produce any effects on the eye, urinary bladder, heart rate, or mucociliary function. Ipratropium seems to act primarily on large- and intermediate-size airways; beta-adrenergic agents, on the other hand, appear to act primarily on the smaller airways. The drug is a promising addition to the therapeutic armamentarium, and may be especially useful in certain groups of patients whose condition is less responsive to other agents.

Tricyclic compounds as selective antimuscarinics. 2. Structure-activity relationships of M1-selective antimuscarinics related to pirenzepine.

In order to gain some insight into those structural features that control M1 selectivity, a selected set of pirenzepine analogues has been studied in which both the tricyclic ring system and the basic side chain have been varied. Binding studies were conducted in rat tissue homogenates from cerebral cortex (M1) and gastric fundus (M2). The ratio of IC50 values of the test compounds in the two different tissues was taken as a measure of M1 receptor selectivity. Several derivatives, especially those with flexible side chains, i.e. high degree of freedom of rotation around single bonds, proved to be nonselective. Among semirigid compounds only those containing 6-membered ring systems (11, 13, 14, and 15) showed significant M1 selectivity. Principles of structure-activity and structure-selectivity are discussed.