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)
Modulation of phosphatidylinositol turnover on central nicotinic receptors.
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AIM: To study the modulatory effects of phosphatidylinositol (PI) turnover on nicotinic receptors in CNS, and to study the relationship between brain nicotinic receptors and PI turnover. METHODS: Effects of inositol phosphatase inhibitor lithium chloride (LiCl) and muscarinic receptor agonist oxotremorine (Oxo) on nicotine-induced convulsions were investigated in mice. RESULTS: The effects of nicotine for producing convulsions were modified by LiCl 2.5-10 mmol.kg-1, revealing the convulsive effects of nicotine > 0.8 mg.kg-1 were increased by acute pretreatment with LiCl rather than oxotremorine. Mice were given LiCl 5.0 mmol.kg-1 once a day for 7 d, the ED50 value of nicotine for producing convulsions was increased from 0.58 to 0.97 mg.kg-1, suggesting that the sensitivity of central nicotinic receptors for mediating convulsions was decreased by chronic treatment with LiCl. CONCLUSION: The functions of central nicotinic receptors were modulated by PI turnover. (+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)
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)
Cholinergic and GABAergic regulation of nitric oxide synthesis in the guinea pig ileum.
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Nitric oxide (NO) synthesis was examined in intact longitudinal muscle-myenteric plexus preparations of the guinea pig ileum by determining the formation of [3H]citrulline during incubation with [3H]arginine. Spontaneous [3H]citrulline production after 30 min was 80-90 dpm/mg, which constituted approximately 1% of the tissue radioactivity. Electrical stimulation (10 Hz) led to a threefold increase in [3H]citrulline formation. Removal of calcium from the medium or addition of NG-nitro-L-arginine strongly inhibited both spontaneous and electrically induced production of [3H]citrulline. TTX reduced the electrically induced but not spontaneous [3H]citrulline formation. The electrically induced formation of [3H]citrulline was diminished by (+)-tubocurarine and mecamylamine and enhanced by scopolamine, which suggests that endogenous ACh inhibits, via muscarinic receptors, and stimulates, via nicotinic receptors, the NO synthesis in the myenteric plexus. The GABAA receptor agonist muscimol and GABA also reduced the electrically evoked formation of [3H]citrulline, whereas baclofen was without effect. Bicuculline antagonized the inhibitory effect of GABA. It is concluded that nitrergic myenteric neurons are equipped with GABAA receptors, which mediate inhibition of NO synthesis. (+info)
Determination of [35S]guanosine-5'-O-(3-thio)triphosphate binding mediated by cholinergic muscarinic receptors in membranes from Chinese hamster ovary cells and rat striatum using an anti-G protein scintillation proximity assay.
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An assay for measuring agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate (GTPgamma35S) binding to heterotrimeric GTP binding proteins was developed for use in 96-well format using commercially available anti-G protein antibodies captured by anti-IgG-coated scintillation proximity assay beads. Use of an anti-Galphaq/11 antibody to measure GTPgamma35S binding mediated by M1, M3, and M5 receptors stably expressed in Chinese hamster ovary (CHO) cells resulted in a marked increase in agonist-stimulated/basal binding ratio compared with whole membrane binding. Pertussis toxin (PTX) treatment of CHO M1 cells before membrane preparation resulted in a marked reduction in agonist-stimulated GTPgamma35S binding to whole membranes. Direct coupling of M1 receptors in CHO cells to inhibitory G proteins was demonstrated using an anti-Galphai(1-3) antibody, and this binding was inhibited by 76% following PTX treatment. However, PTX had no effect on M1-mediated binding determined using anti-Galphaq/11. CHO M2 receptors mediated robust agonist-stimulated GTPgamma35S binding measured with anti-Galphai(1-3), but coupled only weakly to Galphaq/11. Using membranes from rat striatum, GTPgamma35S binding stimulated by oxotremorine M was demonstrated using anti-Galphaq/11, anti-Galphai(1-3), and anti-Galphao antibodies. Agonist-stimulated binding to striatal membranes showed a marked antibody-dependent GDP requirement with robust signals obtained using 0.1 microM GDP for anti-Galphaq/11 compared with 50 microM GDP for anti-Galphai(1-3) and anti-Galphao. The potencies observed for pirenzepine and AFDX 116 blockade of agonist-stimulated GTPgamma35S binding to striatal membranes determined with anti-Galphaq/11 and anti-Galphao suggested mediation of these responses primarily by M1 and M4 receptors, respectively. Antibody capture GTPgamma35S binding using scintillation proximity assay technology provides a convenient, productive alternative to immunoprecipitation for exploration of receptor-G protein interaction in cells and tissues. (+info)
The effect of miotics on the intraocular pressure of conscious owl monkeys.
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The intraocular pressure of conscious, unsedated owl monkeys (Aotus trivirgatus) was measured with an applanation tonometer. Untreated eyes of the conscious animals were found to have higher values than those reported for owl monkeys anesthetized with pentobarbitone. Locally applied pilocarpine, carbachol, and oxotremorine gave concentration-related reduction in pressure, oxotremorine being the most potent and having longer duration of effect than the other compounds. Slight reductions were also observed with aceclidine and R. S. 86. These results are discussed in relation to the effects of miotics in man. (+info)
Lack of effect of McN-A-343 on membrane current and contraction in guinea pig ventricular myocytes.
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We asked whether agonist occupancy of M(1) muscarinic receptor (mAChR) causes increased L-type Ca(2+) [I(Ca(L))] and contractions in ventricular myocytes. Voltage-clamp pulses evoked I(Ca(L)) in guinea pig ventricular myocytes superfused with Tyrode's solution (22-24 degrees C). The mAChR agonists carbachol (Cch, nonselective), McN-A-343 (McN, M(1)-selective), and oxotremorine (Oxo, M(2)-selective) were tested at 0.1 mM. None of these agonists affected basal I(Ca(L)). McN did not change isoproterenol-stimulated I(Ca(L)) in 13 of 15 cells. The slight decrease in two cells was not muscarinic because atropine (1 microM) did not antagonize it. Carbachol or Oxo decreased isoproterenol-stimulated I(Ca(L)) by 87 +/- 6.7 (n = 8 cells) and 49 +/- 9.0% (n = 4 cells), respectively. Atropine blocked inhibition by Cch or Oxo. External stimulation evoked contractions of single myocytes (35 degrees C). McN increased contraction in 1 of 22 cells stimulated at 0.2 Hz and in 0 of 16 cells stimulated at 1.0 Hz. Carbachol significantly increased contraction in 10 of 15 cells at 0.2 Hz and in 8 of 10 cells at 1.0 Hz stimulus frequency. Summarily, the M(1)-selective agonist McN had a negligible role to regulate I(Ca(L)). The antiadrenergic effect of mAChR agonists is attributable to M(2) receptor occupancy. That Cch, but not McN, increased cell shortening excludes participation of M(1) mAChR in the stimulant effect of Cch on guinea pig ventricular myocyte contractions. (+info)