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.

Xanthomicrol: a comprehensive review of its chemistry, distribution, biosynthesis and pharmacological activity.

Highly methoxylated flavones, which have known potential as cancer chemopreventive agents, accumulate on the leaf surfaces of some plant species and their physiological role is to protect the plant against harmful UV radiation. Xanthomicrol is one of the methoxylated flavones currently attracting most attention from researchers worldwide because of its promising pharmacological activities, including anti-spasmodic, anti-platelet and anti-cancer effects, among others. This review covers the chemistry and biological origin, distribution and pharmacological activity of xanthomicrol. Knowledge of the botanical distribution of this compound will not only encourage the use of plant sources for pharmacological purposes, but will also serve as a reference in the search for this valuable flavonoid in another genus or family. New approaches to xanthomicrol production are also described, including biotechnological attempts to develop xanthomicrol-producing plant cell factories.

M1 is a major subtype of muscarinic acetylcholine receptors on mouse colonic epithelial cells.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) are major regulators of gut epithelial functions. However, the precise subtype composition has not been clarified. METHODS: We characterized the pharmacological profile of mAChRs on mouse colonic crypts, employing [(3)H]-N-methyl scopolamine chloride as a radioligand and several subtype-selective chemicals, and the functional aspect by measuring short-circuit current (I sc) in Ussing chambers and by evaluating MAP kinase phosphorylation in mouse colonic mucosal sheets. RESULTS: The mAChRs were detected on the crypts (K d = 163.2 ± 32.3 pM, B max = 47.3 ± 2.6 fmol/mg of total cell protein). Muscarinic toxin 7 (MT-7, M1 subtype selective) gave a displacement curve with high affinity, but there was a part insensitive to MT-7 (18.8 ± 0.4 % of the total specific binding). The MT-7-insensitive component was displaced completely by darifenacin (M3 selective) with high affinity. ACh induced an increase in I sc, which was significantly enhanced by MT-7 but was completely inhibited by darifenacin or atropine. Colitis induction resulted in a significant decrease in the density of mAChRs, which occurred mainly in the MT-7-sensitive component (M1 subtype). Immunological experiments exhibited a reduction of M1 but not of M3 signal after colitis induction. Muscarinic stimulation induced an increase in MAP kinase phosphorylation, which was completely suppressed by MT-7 and was attenuated by inflammation, in mouse colonic epithelium. CONCLUSIONS: These results suggest that mAChRs in mouse colonic epithelial cells consist of two subtypes, M1 (80 %) and M3 (20 %). The major M1 subtype was likely to regulate epithelial chloride secretion negatively and was susceptible to inflammation and may be relevant to inflammatory gut dysfunction.

Gnaphaliin A and B relax smooth muscle of guinea-pig trachea and rat aorta via phosphodiesterase inhibition.

OBJECTIVES: To explore the relaxant mechanism of action of gnaphaliin A and gnaphaliin B in guinea-pig trachea and rat aorta, and to investigate the theoretical and experimental phosphodiesterase (PDE) inhibitory activity of these flavones. METHODS: The relaxant effect and the inhibition of calcium chloride induced contractions of both flavones were evaluated on guinea-pig trachea and rat aorta rings. The PDE inhibitory activity was evaluated using a cyclic nucleotide PDE colorimetric assay kit with cAMP and cGMP as substrates. The docking analysis was carried out with AutoDock4 software and X-ray structure of PDE type 5. The activity of both gnaphaliins was compared with the activity of sildenafil, rolipram, aminophylline, IBMX and enoximone. KEY FINDINGS: Gnaphaliin A and B were more actives as relaxants on rat aorta than guinea-pig trachea. They were less potent in the relaxation of guinea-pig trachea and rat aorta than sildenafil, but they were equal or more potent than the other PDE inhibitors tested. The relaxant effect of these flavones was potentiated by nitroprusside and forskolin, and blocked by 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one but not by 2',5'-dideoxyadenosine in guinea-pig trachea. L-NAME did not modify the relaxant effect of gnaphaliins. Gnaphaliins were more potent as PDE inhibitors when cGMP was used as substrate. Docking analysis revealed that gnaphaiins bind to the same binding site of sildenafil at PDE type 5. CONCLUSIONS: The results suggest that the main relaxant mechanism of action of gnaphaliin A and B is inhibition of PDEs with a preference to inhibit the degradation of cGMP. The docking study suggested that these flavones bind with high specificity to the same binding site of sildenafil at PDE type 5.

Hydroxylation of the diterpenes ent-kaur-16-en-19-oic and ent-beyer-15-en-19-oic acids by the fungus Aspergillus niger.

The diterpenes ent-kaur-16-en-19-oic acid (1) and ent-beyer-15-en-19-oic acid (2) are the major constituents of a spasmolytic diterpenic mixture obtained from the roots of Viguiera hypargyrea, a Mexican medicinal plant. Microbial transformation of 1 and 2 was performed with Aspergillus niger. Two metabolites, ent-7alpha,11beta-dihydroxy-kaur-16-en-19-oic acid (4) and ent-1beta,7alpha-dihydroxy-kaur-16-en-19-oic acid (5), were isolated from the incubation of 1, and one metabolite, ent-1beta,7alpha-dihydroxy-beyer-15-en-19-oic acid (6), was isolated in high yield (40%) from 2. The structures were elucidated on the basis of spectroscopic analyses and confirmed by X-ray crystallographic studies. Compounds 1-4 and 6 and methyl ester derivatives 4a and 6a were evaluated for their ability to inhibit the electrically induced contraction of guinea-pig ileum. Compounds 1, 3, 4, 4a and 5 were significantly active. These results showed that dihydroxylation of 1 at 7beta, 11alpha-, and 1alpha, 7beta-positions resulted in a loss of potency.

Muscarinic receptor changes in the gerbil thalamus during aging.

Here we studied muscarinic receptors in the gerbil thalamus at 8 different ages - from 6 to 36 months - using receptor and functional autoradiography. The pharmacological profile inhibiting [(3)H]N-methyl scopolamine ([(3)H]NMS) binding with 50 and 200 nM pirenzepine, 30 nM pFHHSiD and 100 nM AF-DX 116 revealed the predominance of the M(2) muscarinic subtype in the thalamic nuclei studied, mainly in the anteroventral, anteromedial and paraventricular thalamic nuclei. These data correlated with the highest [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTP gamma S) binding induced in these nuclei by the muscarinic agonist oxotremorine in functional autoradiographic assays. Significant aging-dependent increases in the functional response in these three nuclei were observed, but only the anteroventral and anteromedial thalamic nuclei showed aging-dependent increases in [(3)H]NMS binding. Since these nuclei exert relevant functions, in which cholinergic pathways are involved and acetylcholine release is reported to decrease during aging, we suggest that the anteroventral and anteromedial thalamic nuclei would play critical roles in the cholinergic transmission that require compensatory mechanisms during the aging process and that are not observed in other thalamic nuclei.

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.

Multimodal sensory testing of the rectum and rectosigmoid: development and reproducibility of a new method.

Evaluation of rectal and rectosigmoid sensation is important in basic, clinical and pharmacological studies. New methods to evoke and assess multimodal (electrical, thermal and mechanical) experimental pain of the upper gut activate distinct pathways and mimics clinical pain. The aims of the current study were to characterize the sensory response and reproducibility to multimodal stimulation of rectum and the rectosigmoid. A multimodal rectal probe was developed. Mucosal electrostimulation was delivered at the recto-sigmoid junction. In Rectum, impedance planimetry was used for measurement of cross-sectional area (CSA) during distension. Circulation of water within the bag at either 4 or 60 degrees C was applied for thermal stimulation. The method was tested in 12 healthy volunteers (six men mean age 32 years) on two subsequent days. Mechanical and sensory responses and referred pain areas were assessed. Stimulation with electrical, thermal and mechanical modalities resulted in different sensory perceptions. The relationship between stimulus intensity and sensory response was linear for all modalities. Sensory response to different modalities did not differ between investigation days (all P-values > 0.1). Approximately 75% of subjects felt referred pain in distinct skin locations. Between-days reproducibility was good for all modalities [intra-class correlation (ICC) > or = 0.6]. At sensory threshold, CSA showed best reproducibility (ICC > or = 0.9). At pain detection threshold stretch ratio, CSA and electrostimulation showed best reproducibility (ICC = 1.0; 0.9; 0.9). The present model was easily implemented, robust and showed good reproducibility. It can be used to study pathophysiology or pharmacological interventions in healthy controls and in patients with diseases involving the distal hindgut.

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.

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.

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.

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.

Isoliquiritigenin, one of the antispasmodic principles of Glycyrrhiza ularensis roots, acts in the lower part of intestine.

Glycyrrhizae radix is used to treat abdominal pain as a component of shakuyakukanzoto (shaoyao-gancao-tang), a traditional Chinese medicine formulation. Previously, we have reported the isolation of glycycoumarin as a potent antispasmodic with an IC50 value of 2.93+/-0.94 microM for carbamylcholine (CCh)-induced contraction of mouse jejunum from an aqueous extract of Glycyrrhizae radix (licorice), and clarified that its mechanism of action involves inhibition of phosphodiesterase 3. The purpose of the present study was to examine an antispasmodic principle of licorice other than glycycoumarin. Isoliquiritigenin was isolated from an aqueous extract of licorice as a potent relaxant, which inhibited the contraction induced by various types of stimulants, such as CCh, KCl, and BaCl2 with IC50 values of 4.96+/-1.97 microM, 4.03+/-1.34 microM and 3.70+/-0.58 microM, respectively, which are close to those of papaverine. However, the amount of isoliquiritigenin in the aqueous extract of licorice was very small. When the aqueous licorice extract was treated with naringinase, the amounts of glycosides such as isoliquiritin, which were abundant but had much less potent relaxant activity, were decreased while isoliquiritigenin was increased. At the time, the relaxant activity of the treated sample was increased significantly, shifting the IC50 from 358+/-104 to 150+/-38 microg/ml for CCh-induced contraction. Isoliquiritigenin also showed the most potent inhibition of mouse rectal contraction induced by CCh with an IC50 value of 1.70+/-0.07 microM. These results suggest that isoliquiritigenin acts as a potent relaxant in the lower part of the intestine by transformation from its glycosides.

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.

M1 muscarinic receptors contribute to, whereas M4 receptors inhibit, dopamine D1 receptor-induced [3H]-cyclic AMP accumulation in rat striatal slices.

In rat striatal slices labelled with [(3)H]-adenine and in the presence of 1 mM 3-isobutyl-1-methylxantine (IBMX), cyclic [(3)H]-AMP ([(3)H]-cAMP) accumulation induced by the dopamine D(1) receptor agonist SKF-81297 (1 microM; 177 +/- 13% of basal) was inhibited by the general muscarinic agonist carbachol (maximum inhibition 72 +/- 3%, IC(50) 0.30 +/- 0.06 microM). The muscarinic toxin 7 (MT-7), a selective antagonist at muscarinic M(1) receptors, reduced the effect of SKF-81297 by 40+/-7% (IC(50) 251+/- 57 pM) and enhanced the inhibitory action of a submaximal (1 microM) concentration of carbachol (69 +/- 4% vs. 40 +/- 7% inhibition, IC(50) 386 +/- 105 pM). The toxin MT-1, agonist at M(1) receptors, stimulated [(3)H]-cAMP accumulation in a modest but significant manner (137 +/- 11% of basal at 400 nM), an action additive to that of D(1) receptor activation and blocked by MT-7 (10 nM). The effects of MT-7 on D(1) receptor-induced [(3)H]-cAMP accumulation and the carbachol inhibition were mimicked by the PKC inhibitors Ro-318220 (200 nM) and Gö-6976 (200 nM). Taken together our results indicate that in addition to the inhibitory role of M(4) receptors, in rat striatum acetylcholine stimulates cAMP formation through the activation of M(1 )receptors and PKC stimulation.

Differential effects of cocaine-induced seizures and lethality on M(1)-like muscarinic and dopaminergic D (1)- and D (2)-like binding receptors in mice brain.

This work was designed to study the changes produced by cocaine-induced seizures and lethality on dopaminergic D(1)- and D(2)-like receptors, muscarinic M(1)-like binding sites, as well as acetylcholinesterase activity in mice prefrontal cortex (PFC) and striatum (ST). Binding assays were performed in brain homogenates from the PFC and ST and ligands used were [(3)H]-N-methylscopolamine, [(3)H]-NMS (in the presence of carbachol), [(3)H]-SCH 23390 and [(3)H]-spiroperidol (in presence of mianserin), for muscarinic (M(1)-like), D(1)- and D(2)-like receptors, respectively. Brain acetylcholinesterase (AChE) activity was also determined in these brain areas. Cocaine-induced SE decreased [(3)H]-SCH 23390 binding in both ST and PFC areas. A decrease in [(3)H]-NMS binding and an increase in [(3)H]-spiroperidol binding in PFC was also observed. Cocaine-induced lethality increased [(3)H]-spiroperidol binding in both areas and decreased [(3)H]-NMS binding only in PFC, while no difference was seen in [(3)H]-SCH 23390 binding. Neither SE, nor lethality altered [(3)H]-NMS binding in ST. AChE activity increased after SE in ST while after death the increase occurred in both PFC and ST. In conclusion, cocaine-induced SE and lethality produces differential changes in brain cholinergic and dopaminergic receptors, depending on the brain area studied suggesting an extensive and complex involvement of these with cocaine toxicity in central nervous system.

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.