Comparison of functional antagonism between isoproterenol and M2 muscarinic receptors in guinea pig ileum and trachea.
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The ability of the M2 muscarinic receptor to mediate an inhibition of the relaxant effects of forskolin and isoproterenol was investigated in guinea pig ileum and trachea. In some experiments, trachea was first treated with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) mustard to inactivate M3 receptors. The contractile response to oxotremorine-M was measured subsequently in the presence of both histamine (10 microM) and isoproterenol (10 nM). Under these conditions, [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3b]-[1,4]benzodiazepine-6-one (AF-DX 116) antagonized the contractile response to oxotremorine-M in a manner consistent with an M3 mechanism. However, when the same experiment was repeated using forskolin (4 microM) instead of isoproterenol, the response to oxotremorine-M exhibited greater potency and was antagonized by AF-DX 116 in a manner consistent with an M2 mechanism. We also measured the effects of pertussis toxin treatment on the ability of isoproterenol to inhibit the contraction elicited by a single concentration of either histamine (0.3 microM) or oxotremorine-M (40 nM) in both the ileum and trachea. Pertussis toxin treatment had no significant effect on the potency of isoproterenol for inhibiting histamine-induced contractions in the ileum and trachea. In contrast, pertussis toxin treatment enhanced the relaxant potency of isoproterenol against oxotremorine-M-induced contractions in the ileum but not in the trachea. Also, pertussis toxin treatment enhanced the relaxant potency of forskolin against oxotremorine-M-induced contractions in the ileum and trachea. We investigated the relaxant potency of isoproterenol when very low, equi-effective (i.e., 20-34% of maximal response) concentrations of either histamine or oxotremorine-M were used to elicit contraction. Under these conditions, isoproterenol exhibited greater relaxant potency against histamine in the ileum but exhibited similar relaxant potencies against histamine and oxotremorine-M in the trachea. Following 4-DAMP mustard treatment, a low concentration of oxotremorine-M (10 nM) had no contractile effect in either the ileum or trachea. Nevertheless, in 4-DAMP mustard-treated tissue, oxotremorine-M (10 nM) reduced the relaxant potency of isoproterenol against histamine-induced contractions in the ileum, but not in the trachea. We conclude that in the trachea the M2 receptor mediates an inhibition of the relaxant effects of forskolin, but not isoproterenol, and the decreased relaxant potency of isoproterenol against contractions elicited by a muscarinic agonist relative to histamine is not due to activation of M2 receptors but rather to the greater contractile stimulus mediated by the M3 receptor compared with the H1 histamine receptor. (+info)
Selective activation of heterologously expressed G protein-gated K+ channels by M2 muscarinic receptors in rat sympathetic neurones.
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1. G protein-regulated inward rectifier K+ (GIRK) channels were over-expressed in dissociated rat superior cervical sympathetic (SCG) neurones by co-transfecting green fluorescent protein (GFP)-, GIRK1- and GIRK2-expressing plasmids using the biolistic technique. Membrane currents were subsequently recorded with whole-cell patch electrodes. 2. Co-transfected cells had larger Ba2+-sensitive inwardly rectifying currents and 13 mV more negative resting potentials (in 3 mM [K+]o) than non-transfected cells, or cells transfected with GIRK1 or GIRK2 alone. 3. Carbachol (CCh, 1-30 microM) increased the inwardly rectifying current in 70 % of GIRK1+ GIRK2-transfected cells by 261 +/- 53 % (n = 6, CCh 30 microM) at -120 mV, but had no effect in non-transfected cells or in cells transfected with GIRK1 or GIRK2 alone. Pertussis toxin prevented the effect of carbachol but had no effect on basal currents. 4. The effect of CCh was antagonized by 6 nM tripitramine but not by 100 nM pirenzepine, consistent with activation of endogenous M2 muscarinic acetylcholine receptors. 5. In contrast, inhibition of the voltage-activated Ca2+ current by CCh was antagonized by 100 nM pirenzepine but not by 6 nM tripitramine, indicating that it was mediated by M4 muscarinic acetylcholine receptors. 6. We conclude that endogenous M2 and M4 muscarinic receptors selectively couple to GIRK currents and Ca2+ currents respectively, with negligible cross-talk. (+info)
Muscarinic M3 receptor inactivation reveals a pertussis toxin-sensitive contractile response in the guinea pig colon: evidence for M2/M3 receptor interactions.
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The role of M2 and M3 receptors in the contractile and phosphoinositide responses elicited to oxotremorine-M was investigated in the guinea pig colon. Under standard conditions, both the contractile and phosphoinositide responses were insensitive to pertussis toxin and irreversibly antagonized by alkylation of M3 receptors with N-(2-chloroethyl)-4-piperidinyl diphenylacetate. After treatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate, the remaining contractile response was sensitive to pertussis toxin and weakly antagonized by the M2- and M4-selective antagonist AF-DX 116. In contrast, the residual phosphoinositide response was unaffected by pertussis toxin. The pertussis toxin sensitivity of the remaining contractile response suggests that the M2 receptor is mediating the contraction, whereas its weak antagonism by AF-DX 116 suggests that an alternate muscarinic subtype mediates the response. To explain this enigma, we investigated a mathematical model for receptor action based on an interaction between two receptor subtypes (M2 and M3). This model predicts that a response mediated by both the M2 and M3 receptor can be pertussis toxin sensitive yet exhibit an antagonistic profile indicative of an M3 response. (+info)
A cost-effectiveness clinical decision analysis model for schizophrenia.
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A model was developed to estimate the medical costs and effectiveness outcomes of three antipsychotic treatments (olanzapine, haloperidol, and risperidone) for patients with schizophrenia. A decision analytic Markov model was used to determine the cost-effectiveness of treatments and outcomes that patients treated for schizophrenia may experience over a 5-year period. Model parameter estimates were based on clinical trial data, published medical literature, and, when needed, clinician judgment. Direct medical costs were incorporated into the model, and outcomes were expressed by using three effectiveness indicators: the Brief Psychiatric Rating Scale, quality-adjusted life years, and lack of relapse. Over a 5-year period, patients on olanzapine had an additional 6.8 months in a disability-free health state based on Brief Psychiatric Rating Scale scores and more than 2 additional months in a disability-free health state based on quality-adjusted life years, and they experienced 13% fewer relapses compared with patients on haloperidol. The estimated 5-year medical cost associated with olanzapine therapy was $1,539 less than that for haloperidol therapy. Compared with risperidone therapy, olanzapine therapy cost $1,875 less over a 5-year period. Patients on olanzapine had approximately 1.6 weeks more time in a disability-free health state (based on Brief Psychiatric Rating Scale scores) and 2% fewer relapses compared with patients on risperidone. Sensitivity analyses indicated the model was sensitive to changes in drug costs and shortened hospital stay. Compared with both haloperidol and risperidone therapy, olanzapine therapy was less expensive and provided superior effectiveness outcomes even with conservative values for key parameters such as relapse and discontinuation rates. (+info)
Atypical antipsychotics and formulary decisions.
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Although drug costs are a small fraction of the total direct costs of treating schizophrenia, managed care has focused on drug acquisition costs as an area of concern. There is pressure to demonstrate by outcome measures that the increased cost of the newer atypical antipsychotics versus traditional neuroleptics is justified. Decision makers want to be convinced that newer, more expensive treatment translates to value. Evidence accumulated to date suggests that the atypical agents are cost-effective. Studies show patients taking atypical antipsychotics have an improved quality of life, are more easily rehabilitated and reintegrated into the community, return to full- or part-time work more often, and prefer the newer agents to conventional antipsychotics. These benefits have been shown in studies of olanzapine versus haloperidol. Just as important, patients taking atypical antipsychotics show decreased medical care resource utilization, which results in cost savings. (+info)
Molecular probes for muscarinic receptors: functionalized congeners of selective muscarinic antagonists.
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The muscarinic agonist oxotremorine and the tricyclic muscarinic antagonists pirenzepine and telenzepine have been derivatized using a functionalized congener approach for the purpose of synthesizing high affinity ligand probes that are suitable for conjugation with prosthetic groups, for receptor cross-linking, fluorescent and radioactive detection, etc. A novel fluorescent conjugate of TAC (telenzepine amine congener), an n-decylamino derivative of the m1-selective antagonist, with the fluorescent trisulfonated pyrene dye Cascade Blue may be useful for assaying the receptor as an alternative to radiotracers. In a rat m3 receptor mutant containing a single amino acid substitution in the sixth transmembrane domain (Asn507 to Ala) the parent telenzepine lost 636-fold in affinity, while TAC lost only 27-fold. Thus, the decylamino group of TAC stabilizes the bound state and thus enhances potency by acting as a distal anchor in the receptor binding site. We have built a computer-assisted molecular model of the transmembrane regions of muscarinic receptors based on homology with the G-protein coupled receptor rhodopsin, for which a low resolution structure is known. We have coordinated the antagonist pharmacophore (tricyclic and piperazine moieties) with residues of the third and seventh helices of the rat m3 receptor. Although the decylamino chain of TAC is likely to be highly flexible and may adopt many conformations, we located one possible site for a salt bridge formation with the positively charged -NH3+ group, i.e. Asp113 in helix II. (+info)
Roles of threonine 192 and asparagine 382 in agonist and antagonist interactions with M1 muscarinic receptors.
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Conserved amino acids, such as Thr in transmembrane domains (TM) V and Asn in TM VI of muscarinic receptors, may be important in agonist binding and/or receptor activation. In order to determine the functional roles of Thr192 and Asn382 in human M1 receptors in ligand binding and receptor activation processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala. HM1 wild-type (WT) and mutant receptors [HM1(Thr192Ala) and HM1(Asn382Ala)] were stably expressed in A9 L cells. The Kd values for 3H-(R)-QNB and Ki values for other classical muscarinic antagonists were similar at HM1(WT) and HM1(Thr192Ala) mutant receptors, yet higher at HM1(Asn382Ala) mutant receptors. Carbachol exhibited lower potency and efficacy in stimulating PI hydrolysis via HM1(Thr192Ala) mutant receptors, and intermediate agonist activity at the HM1(Asn382Ala) mutant receptors. The Asn382 residue in TM VI but not the Thr192 residue in TM V of the human M1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved differentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less important than Thr192 in agonist activation of M1 receptors. Molecular modelling studies indicate that substitution of Thr192 or Asn382 results in the loss of hydrogen-bond interactions and changes in the agonist binding mode associated with an increase in hydrophobic interactions between ligand and receptor. (+info)
Inverse agonist activity of pirenzepine at M2 muscarinic acetylcholine receptors.
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1. The intrinsic properties of muscarinic ligands were studied through their binding properties and their abilities to modulate the GTPase activity of G proteins coupled to muscarinic M2 receptors in pig atrial sarcolemma. 2. Competition binding experiments were performed with [3H]-oxotremorine-M to assess the affinity of receptors coupled to G proteins (R*), with [3H]-N-methylscopolamine ([3H]-NMS) to estimate the affinities of coupled and uncoupled receptors (R*+R) and with [3H]-NMS in the presence of GppNHp to assess the affinity of uncoupled receptors (R). 3. The ranking of Ki values for the agonist carbachol was R*<R*+R>R (174, 155, 115 nM), suggesting inverse agonism. 4. The Vmax of the basal high affinity GTPase activity of pig atrial sarcolemma was increased by mastoparan and decreased by GPAnt-2 indicating the relevance of this activity to G proteins coupled to receptors (R*). The K(M) value (0.26-0.33 microM) was not modified by mastoparan or GPAnt-2. 5. Carbachol increased the Vmax of GTP hydrolysis (EC50 8.1+/-0.3 microM), whereas atropine and AF-DX 116, up to 1 mM, did not modify it. Pirenzepine decreased the Vmax of GTP hydrolysis (EC50 77.5+/-10.3 microM). This effect was enhanced when KCI was substituted for NaCl (EC50 11.0+/-0.8 microM) and was antagonized by atropine and AF-DX 116 (IC50 0.91+/-0.71 and 197+/-85 nM). 6. Pirenzepine is proposed as an inverse agonist and atropine and AF-DX 116 as neutral antagonists at the muscarinic M2 receptor. (+info)