Activation of cholinergic pathway induced vasodilation in rat aorta using aqueous and methanolic leaf extracts of Gynura procumbens.

Gynura procumbens (GP) is a herbal medicinal plant of South-East Asian origin, popularly recognised as 'Sambung nyawa'. The plant has been used traditionally to treat various diseases including hypertension. The anti-hypertensive activity of this plant has also been scientifically proven both in vivo and in vitro yet the investigation on its mechanisms of actions remains limited. Our previous study has demonstrated the vasodilatory action of both aqueous and methanol GP extracts possibly via activation of the cholinergic pathway and that kaempferol 3-O-rutinoside is the active ingredient responsible in mediating this effect. Hence, in this study we further confirm the involvement of the cholinergic pathway by using several pharmacological interventions, focusing on the downstream mechanism of this pathway. Our results showed that in the presence of endothelium, GP extracts induced vasodilation via activation of the muscarinic M3 receptors. However, in the absence of endothelium, GP mediated vasodilation possibly via stimulation of other muscarinic receptors and/or involvement of nicotinic receptors, a speculation that needs further investigations. GP-induced relaxation was markedly inhibited by nitric oxide (NO) blocker, L-NAME, suggesting that GP elicited ACh endothelium-dependent relaxation by producing NO in rat aortic rings. In conclusion, these data demonstrate that the vasodilatory effect of GP extracts appears to be mediated via cholinergic pathway.

Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract.

The objectives of this study were to evaluate, in vitro and in vivo, the contribution of muscarinic receptors to the effects of Ruscus extract. Ruscus extract was tested in competition binding experiments at recombinant human muscarinic receptors, heterologous expressed in Chinese Hamster Ovary (CHO) cells and in cellular assays measuring Ca2+ liberation and activator protein-1 (AP-1) reporter gene activation. The impact of muscarinic blockade on prolonged treatment outcome was evaluated using the hamster cheek pouch (HCP) microcirculation examining macromolecular permeability increase induced by histamine or ischemia/reperfusion (I/R), mean arteriolar and venular diameters, functional capillary density and I/R-induced leukocyte rolling and sticking. Ruscus extract exhibited affinities for muscarinic receptor subtypes at a range of 50-100µg/ml and behaved as partial agonist at human recombinant M1 and M3 receptors for Ca2+ liberation, confirmed in an AP-1 reporter gene assay. In the HCP model, topical application of atropine completely or partially blocked Ruscus extract-induced reductions of histamine- and I/R-induced increases of macromolecular permeability and leukocyte-endothelium interaction. Our results showed that Ruscus extract in vitro binds and activates different subtypes of muscarinic receptors and in vivo its anti-inflammatory effects are, at least partially, mediated via muscarinic receptors.

Insights into mechanisms underlying the gut and airways modulatory effects of Swertia chirata.

Swertia chirata is used in folk medicine for the treatment of constipation, colic, diarrhea, and asthma. This study was carried out in order to provide a pharmacological basis for its medicinal use in gastrointestinal and respiratory disorders. Crude extract of Swertia chirata (Sc.Cr) and its fractions were studied using rabbit isolated tissue preparations. In jejunum, Sc.Cr, which tested positive for alkaloids, flavonoids, saponins, tannins, and terpenes, caused stimulation at concentrations of 0.01-1.0 mg/mL, followed by a relaxant effect at higher concentrations. In the presence of atropine, the contractile effect was blocked and only relaxation occurred. Sc.Cr inhibited high K(+) (80 mM)-induced contractions at 0.01-10 mg/mL and shifted Ca(2+) concentration-response curves to the right, similar to that caused by verapamil. In trachea, Sc.Cr relaxed the carbachol (1 µM) and high K(+)-induced contractions, in a pattern similar to that of verapamil. Bioassay directed fractionation revealed the separation of spasmogenic and spasmolytic components in aqueous and organic fractions, respectively. The chloroform fraction exhibited a concentration-dependent (0.1-3.0 mg/mL) bronchodilator effect. These results indicate that Swertia chirata exhibits gut excitatory and inhibitory effects, mediated through cholinergic and Ca(2+) antagonist mechanisms, respectively, as well as bronchodilatation, via Ca(2+) channel blockade. Thus, this study provides a sound mechanistic background for the therapeutic application of Swertia chirata in gut motility disorders, such as constipation, colic, and diarrhea, and airways hyperactivity disease, such as asthma.

Characterization of a new muscarinic toxin from the venom of the Brazilian coral snake Micrurus lemniscatus in rat hippocampus.

AIMS: We have isolated a new muscarinic protein (MT-Mlα) from the venom of the Brazilian coral snake Micrurus lemniscatus. MAIN METHODS: This small protein, which had a molecular mass of 7,048Da, shared high sequence homology with three-finger proteins that act on cholinergic receptors. The first 12 amino acid residues of the N-terminal sequence were determined to be: Leu-Ile-Cys-Phe-Ile-Cys-Phe-Ser-Pro-Thr-Ala-His. KEY FINDINGS: The MT-Mlα was able to displace the [(3)H]QNB binding in the hippocampus of rats. The binding curve in competition experiments with MT-Mlα was indicative of two types of [(3)H]QNB-binding site with pK(i) values of 9.08±0.67 and 6.17±0.19, n=4, suggesting that various muscarinic acetylcholine receptor (mAChR) subtypes may be the target proteins of MT-Mlα. The MT-Mlα and the M(1) antagonist pirenzepine caused a dose-dependent block on total [(3)H]inositol phosphate accumulation induced by carbachol. The IC(50) values for MT-Mlα and pirenzepine were, respectively, 33.1 and 2.26 nM. Taken together, these studies indicate that the MT-Mlα has antagonist effect on mAChRs in rat hippocampus. SIGNIFICANCE: The results of the present study show, for the first time, that mAChRs function is drastically affected by MT-Mlα since it not only has affinity for mAChRs but also has the ability to inhibit mAChRs.

A 100K well screen for a muscarinic receptor using the Epic label-free system--a reflection on the benefits of the label-free approach to screening seven-transmembrane receptors.

Seven-transmembrane receptors (7TMRs) are a family of proteins of great interest as therapeutic targets because of their abundance on the cell surface, diverse effects in modulating cell behavior and success as a key class of drugs. We have evaluated the Epic label-free system for the purpose of identifying antagonists of the muscarinic M3 receptor. We compared the data generated from the label-free technology with data for the same compounds in a calcium flux assay. We have shown that this technology can be used for high throughput screening (HTS) of 7TMRs and as an orthogonal approach to enable rapid evaluation of HTS outputs. A number of compounds have been identified which were not found in a functional HTS measuring the output from a single pathway, which may offer new approaches to inhibiting responses through this receptor.

Methylisogermabullone isolated from radish roots stimulates small bowel motility via activation of acetylcholinergic receptors.

We have previously reported that extract of radish roots exhibits an increase in gastrointestinal motility through the activation of muscarinic acetylcholine (ACh) receptors. Based on the stimulatory activity-guided fractionation on rat ileal segments, this study isolated methylisogermabullone (MIGB, C23H31O5NS, MW 433) from methanol extracts of radish roots. MIGB caused a significant increase of the isolated rat ileal contraction in a concentration-dependent manner (23-693 microM), and the pattern of MIGB-induced ileal contraction was different in the time course to that produced by ACh. The EC50 value of MIGB, to produce 50% maximum ileal contraction, was estimated to be 45.5 microM. MIGB (230 microM)-induced ileal contractions were enhanced by pretreatment of segments with ACh (0.1 microM). Ileal contractions produced by MIGB (230 microM) or ACh (0.1 microM) at submaximal concentration were partially inhibited by pretreatment of hexamethonium (0.1 mM), a ganglionic blocker, whereas they were almost completely abolished by atropine (10 microM). Oral administration of MIGB to mice stimulated the small intestinal transit of charcoal in a dose-dependent manner (10-100 mg kg(-1)), and MIGB (100 mg kg(-1))-induced stimulation of small intestinal transit was significantly attenuated by co-administration of atropine (50 mg kg(-1)). Taken together, these results demonstrate that MIGB isolated from radish roots stimulates the small bowel motility through the activation of ACh receptors. These findings suggest that MIGB may become a potential regulatory agent for therapeutic intervention in dysfunction of gastrointestinal motility.

Pharmacological profile of enantiomerically pure chiral muscarinic agonists: desoxymuscarines.

The enantiomers desoxymuscarine 6 were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M2 (heart force and rate) and M3 (ileum and bladder) receptor subtypes together with the enantiomers of the parent compound muscarine 1. The eutomers (+)-1 and (+)-6 and distomers (-)-1 and (-)-6 were also assayed in vivo on pithed rat. Affinity, relative efficacy and enantio-selectivity were also determined for the compounds under study at M2 (heart force and rate) and M3 (ileum and bladder), in order to investigate muscarinic receptor heterogeneity. The results of this study have been discussed in comparison with the data previously reported for the structurally related fluoromuscarine (+)-4 and difluoromuscarines (+)-5 and (-)-5.

Partial characterization of endogenous modulators of muscarinic acetylcholine receptors in human frontal cortex.

A soluble fraction from human frontal cortex with molecular weight less than 10 kD was tested for the presence of endogenous substances capable of modulating the [3H]-QNB binding to crude P1 + P2 fractions from the same region. The soluble fraction was able to decrease [3H]-QNB binding in a dose-response manner with an IC50 of about 30 micrograms/ml. The effect appeared to be noncompetitive in nature, since Bmax but not Kd was significantly affected; however, in some specimens a biphasic profile, with an initial inhibition of 88-90% of [3H]-QNB binding and 50-60% ulterior binding recuperation was also found. The modulator appeared to have a molecular weight less than 10,000 Daltons and was heat and trypsin resistant. These results point out the existence of an endogenous factor, which could be heterogeneous in regard to its molecular nature or to its action sites.