Mixed agonist-antagonist properties of clozapine at different human cloned muscarinic receptor subtypes expressed in Chinese hamster ovary cells. (1/212)

We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [3H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [35S] GTP gamma S binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug.  (+info)

Cholinergic blockade inhibits gastro-oesophageal reflux and transient lower oesophageal sphincter relaxation through a central mechanism. (2/212)

BACKGROUND: Atropine, an anticholinergic agent with central and peripheral actions, reduces gastro-oesophageal reflux (GOR) in normal subjects and patients with gastro-oesophageal reflux disease (GORD) by inhibiting the frequency of transient lower oesophageal sphincter relaxation (TLOSR). AIMS: To compare the effect of methscopolamine bromide (MSB), a peripherally acting anticholinergic agent, with atropine on the rate and mechanism of GOR in patients with GORD. METHODS: Oesophageal motility and pH were recorded for 120 minutes in 10 patients with GORD who were studied on three separate occasions. For the first two recording periods, either atropine (15 microg/kg bolus, 4 microg/kg/h infusion) or saline were infused intravenously. MSB (5 mg orally, four times daily) was given for three days prior to the third recording period. RESULTS: Atropine significantly reduced basal LOS pressure (12.6 (0.17) mm Hg to 7.9 (0.17) mm Hg), and the number of TLOSR (8.1 (0.56) to 2.8 (0. 55)) and reflux episodes (7.0 (0.63) to 2.0 (0.43)) (p<0.005 for all comparisons). MSB reduced basal LOS pressure (12.6 (0.17) to 8.7 (0. 15) mm Hg, p<0.005), but had no effect on the frequency of TLOSR (8. 1 (0.56) to 7.5 (0.59)) and reflux episodes (7.0 (0.63) to 4.9 (0. 60)) (p>0.05). CONCLUSION: In contrast to atropine, MSB has no effect on the rate of TLOSR or GOR in patients with GORD. Atropine induced inhibition of TLOSR and GOR is most likely mediated through a central cholinergic blockade.  (+info)

Pharmacological analysis of the novel mode of interaction between xanomeline and the M1 muscarinic acetylcholine receptor. (3/212)

Previous findings in our laboratory suggested that the M1 muscarinic acetylcholine receptor (mAChR) agonist xanomeline exhibits a novel mode of interaction that involves persistent binding to and activation of the M1 mAChR, subsequent to extensive washout, as well as a possible insurmountable element. In the present study, we examined this interaction in greater detail, using Chinese hamster ovary cells transfected with the genes for the M1 mAChR and neuronal nitric oxide synthase. Pretreatment of cells with xanomeline, followed by extensive washout, resulted in elevated basal levels of neuronal nitric oxide synthase activity that were suppressed by the antagonists atropine or pirenzepine in a concentration-dependent manner. Analysis of the data yielded estimates of Schild slope factors and pKB values for the antagonists that were consistent with a model of simple competition between these latter agents and the persistently bound form of xanomeline. The ability of the antagonists to produce parallel dextral shifts of the concentration-response curves to carbachol and xanomeline was also investigated. The interaction between xanomeline and pirenzepine appeared to be insurmountable, but this may have been due to an equilibrium artifact. In contrast, the interaction between xanomeline and atropine conformed to a model of competition, indicating that the mode of interaction of free xanomeline at the M1 mAChR is pharmacologically identical with that of the persistently bound form. Radioligand binding studies also showed that the presence of various concentrations of xanomeline had no significant effect on the calculated affinity of atropine or pirenzepine in inhibiting the binding of [3H]N-methylscopolamine. Overall, these findings suggest that the persistent attachment of xanomeline to the M1 mAChR does not prevent this agonist from interacting with the classic binding site in a competitive fashion.  (+info)

Pilocarpine modulates the cellular electrical properties of mammalian hearts by activating a cardiac M3 receptor and a K+ current. (4/212)

1. Pilocarpine, a muscarinic acetylcholine receptor (mAChR) agonist, is widely used for treatment of xerostomia and glaucoma. It can also cause many other cellular responses by activating different subtypes of mAChRs in different tissues. However, the potential role of pilocarpine in modulating cardiac function remained unstudied. 2. We found that pilocarpine produced concentration-dependent (0.1-10 microM) decrease in sinus rhythm and action potential duration, and hyperpolarization of membrane potential in guinea-pig hearts. The effects were nearly completely reversed by 1 microM atropine or 2 nM 4DAMP methiodide (an M3-selective antagonist). 3. Patch-clamp recordings in dispersed myocytes from guinea-pig and canine atria revealed that pilocarpine induces a novel K+ current with delayed rectifying properties. The current was suppressed by low concentrations of M3-selective antagonists 4DAMP methiodide (2-10 nM), 4DAMP mustard (4-20 nM, an ackylating agent) and p-F-HHSiD (20-200 nM). Antagonists towards other subtypes (M1, M2 or M4) all failed to alter the current. 4. The affinity of pilocarpine (KD) at mAChRs derived from displacement binding of [3H]-NMS in the homogenates from dog atria was 2.2 microM (65% of the total binding) and that of 4DAMP methiodide was 2.8 nM (70% of total binding), consistent with the concentration of pilocarpine needed for the current induction and for the modulation of the cardiac electrical activity and the concentration of 4DAMP to block pilocarpine effects. 5. Our data indicate, for the first time, that pilocarpine modulates the cellular electrical properties of the hearts, likely by activating a K+ current mediated by M3 receptors.  (+info)

Characterization of prejunctional and postjunctional muscarinic receptors of the ascending reflex contraction in rat ileum. (5/212)

The ascending reflex contraction of the small intestine involves predominantly cholinergic neurotransmission. The orally projecting neural excitatory pathway of the myenteric reflex was studied in an in vitro model of rat ileal segments. The contractile response elicited by aboral field stimulation was significantly inhibited by a range of muscarinic receptor antagonists. Methoctramine and tripitramine (both M(2) selective, pIC(50) = 9.3 and 8.8, respectively), darifenacin and hexahydrosiladifenidol (both M(3) selective, pIC(50) = 7.3 and 7.7, respectively), and pirenzepine (M(1) selective, pIC(50) = 7.0). In radioligand binding experiments on synaptosomal and smooth muscle plasma membrane fractions, we examined whether prejunctional or postjunctional muscarinic receptors exist that could potentially contribute to the reflex contraction. In the synaptosomal fraction, the muscarinic ligand [(3)H]N-methylscopolamine labeled a homogeneous population of receptors (Hill coefficient = 1) with a K(d) value of 0.31 +/- 0.09 nM and a B(max) value of 185 +/- 16.6 fmol/mg protein. The ratio of potency of subtype-selective muscarinic receptor antagonists in competition studies was tripitramine (pK(i) = 8.7 +/- 0.3) > 1/6 x methoctramine (pK(i) = 7.9 +/- 0.02) > 1/25 x darifenacin (pK(i) = 7. 3 +/- 0.2) > 1/316 x hexahydrosiladifenidol (pK(i) = 6.2 +/- 0.1) > 1/2511 x pirenzepine (pK(i) = 5.3 +/- 0.1). In the smooth muscle plasma membrane fraction, the K(d) value was 0.29 +/- 0.05 nM and the B(max) value was 770 +/- 29 fmol/mg. The competition studies revealed a similar ratio of potency of the respective antagonists. These data suggest that muscarinic M(2) receptors, located at prejunctional and postjunctional sites, are predominantly involved in the ascending reflex contraction.  (+info)

Muscarinic cholinergic receptors activate both inhibitory and stimulatory growth mechanisms in NIH3T3 cells. (6/212)

Activation of G(q) protein-coupled receptors can either stimulate or inhibit cell growth. Previously, these opposite effects were explained by differences in the cell models. Here we show that activation of m3 muscarinic acetylcholine receptors ectopically expressed in NIH3T3 cells can cause stimulation and inhibition of growth in the same cell. A clonal cell line was selected from cells that formed foci agonist dependently (3T3/m3 cells). In quiescent 3T3/m3 cells, carbachol stimulated DNA synthesis. In contrast, when 3T3/m3 cells were growing, either due to the presence of serum or after transformation with oncogenic v-src, carbachol inhibited growth. This inhibition was not due to reduction of extracellular signal-regulated kinase activity because carbachol induced extracellular signal-regulated kinase phosphorylation in both quiescent and growing 3T3/m3 cells. Investigating the cell cycle mechanisms involved in growth inhibition, we found that carbachol treatment decreased cyclin D1 levels, increased p21(cip1) expression, and led to hypophosphorylation of the retinoblastoma gene product (Rb). Proteasome inhibitors blocked the carbachol-induced degradation of cyclin D1. Effects on p21(cip1) were blocked by a protein kinase C inhibitor. Thus, m3 muscarinic acetylcholine receptors couple to both growth-stimulatory and -inhibitory signaling pathways in NIH3T3 cells, and the observed effects of receptor activation depend on the context of cellular growth.  (+info)

Inhibition of GTPase activity of Gi proteins and decreased agonist affinity at M2 muscarinic acetylcholine receptors by spermine and methoctramine. (7/212)

1. The effects of spermine and methoctramine, a selective M2 muscarinic receptor antagonist, were studied on the high-affinity GTPase activity of G proteins, and on ligand binding to M2 muscarinic receptors in pig heart sarcolemma. 2. The spontaneous GTP hydrolysis by pig heart sarcolemma and its stimulation by mastoparan or carbachol were prevented by pertussis toxin and inhibited by methoctramine (IC50s: 21, 13 and 0.005 microM, respectively), and spermine (IC50s: 967, 278 and 11 microM). Spermine and methoctramine also inhibited spontaneous GTP hydrolysis by rat peritoneal mast cell membranes which do not respond to carbachol. 3. The neutral muscarinic antagonists, AF-DX 116 and atropine, did not modify the inhibitory effect of high concentrations of methoctramine, indicating that this effect was not related to the antagonist binding site of muscarinic receptors. We suggest that methoctramine behaves as a receptor antagonist at nanomolar concentrations and interacts with G proteins at micromolar concentrations. 4. Spermine did not modify the binding of the tritiated muscarinic antagonist [3H]-NMS, but decreased the binding of the agonist [3H]-Oxo-M. Spermine elicited a rightward shift of the carbachol/[3H]-NMS binding isotherm with a decrease in the proportion of sites with high-affinity for carbachol, suggesting that polyamines uncouple Gi proteins from receptors. 5. The inhibition of GTPase activity by polyamines, preventing the re-association of alpha and betagamma subunits of Gi proteins, might sustain the regulatory effect of Gi subunits on downstream effectors. The level of intracellular polyamines might be important for the control of the transduction of extracellular signals through Gi protein-coupled receptors.  (+info)

Subtype-selective inhibition of [methyl-3H]-N-methylscopolamine binding to muscarinic receptors by alpha-truxillic acid esters. (8/212)

Seven esters of alpha-truxillic acid have been synthesized: bis-3-piperidylpropyl ester and its quaternary bis-N-ethyl derivative, bis-N-diethylaminopropyl ester and its quaternary bis-N-methyl derivative, and bis-4-piperidylbutyl ester and its quaternary bis-N-methyl and bis-N-ethyl derivatives. All esters inhibited the specific binding of muscarinic receptor antagonist [methyl-3H]-N-methylscopolamine ([3H]-NMS) to muscarinic receptors in membranes of CHO cell lines stably expressing the human gene for the M1, M2, M3 or M4 subtype of muscarinic receptors. All esters displayed the highest potency at the M2 and the lowest potency at the M3 receptor subtype. In experiments performed on the M2 muscarinic receptor subtype, the affinity between the receptors and the esters was greatly increased when the concentration of ions was diminished. The highest affinities were found for the tertiary bis-3-piperidylpropyl and bis-4-piperidylbutyl aminoesters (equilibrium dissociation constants of 52 and 179 pM, respectively, in the low ionic strength medium). All investigated esters slowed down the dissociation of [3H]-NMS from the M2 muscarinic receptor subtype. [3H]-NMS dissociation from the M1, M3 and M4 muscarinic receptor subtypes was investigated in experiments with the bis-4-piperidylbutyl aminoester and also found to be decelerated. It is concluded that the esters of alpha-truxillic acid act as M2-selective allosteric modulators of muscarinic receptors and that, by their potency, the tertiary bis-3-piperidylpropyl and bis-4-piperidylbutyl aminoesters surpass the other known allosteric modulators of these receptors.  (+info)

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