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Presynaptic inhibitory muscarinic autoreceptors modulating 3H-acetylcholine release in human isolated detrusor muscle
1194
Abstracts
Vol. 60, Nos. 13/14, 1997
64 PRESYNAPTIC INHIBITORY MUSCARlNlC AUTORECEPTORS MODULATING 3H-ACETYLCHOLINE RELEASE IN HUMAN ISOLATED DETRUSOR MUSCLE GD’Agostino, ‘M.Tagliani, lS.M.Candura, E.Chiossa, *G.P.Franceschetti, C.Gregotti and # MTonini. Institute of Pharmacology, University of Pavia, l S.fvlaugeri Foundation IRCCS, Pavia, r)ivision of Urology, Hospital of Voghera (Pavia) and department of Internal Medicine and Therapeutics, Division of Pharmacology and Toxicology, University of Pavia, I -27100 Pavia, Italy The presence of presynaptic muscarinic mechanism(s) modulating the release of newly-synthesized tritiated acetylcholine @-I-ACh) from cholinergic nerves was investigated in human isolated detrusor muscle ships. Specimens from the anterior pad of the urinary bladder dome were obtained from patients undergoing total cystectomy due to bladder base malignancy. Muscular strips (20 mm long, 4 mm wide), prepared by removing serosal and mucosal layers and mounted isometrically in a modified Krebs solution (37 C), were preincubated with sH-choline and stimulated at 10 Hz to improve their neuronal sH-ACh content. Electrical field stimulation (EFS) induced contractile responses and tritium-outflow. These effects were prevented by TTX. Most of the evoked outflow was recognized as sH-ACh by HPLC analysis. When two EFSs (Sl and S2) were carried out at 5 Hz (000 pulses, 0.1 ms, 00 V) 30 min apart, an S2/St ratio of 0.84 was calculated. Muscarone (10-1000 nM), a potent muscarinic agonist, caused a concentration-dependent inhibition of the outflow of sH-ACh (pECy, 7.30) with maximal inhibition by about 50%. Physostigmine (100 nM) markedly reduced (by 50%) the evoked release whereas atropine (100 nM) increased it by 25%. Maximal (50%) facilitation of release was observed for atropine when six intermittent EFSs were applied at IO Hz (100 pulses, 1 ms, 12 V). Atropine and the subtype-selective antagonists CDAMP (M3), methoctramine (Md M4) and tripitramine (M2) increased the evoked release in a concentration-dependent fashion. The calculated pEC50 potencies were 9.07, 8.00, 7.97, and 8.73, respectively. These findings provide direct evidence that in the human urinary bladder acetylcholine release is under the local inhibitory control of muscarinic autoreceptors. The comparison between the observed rank order of pre- and postjunctional potency of muscarinic antagonists and their affinities at muscarinic receptor subtypes suggests that these autoreceptors may be regarded as Mz or a mixture of M2 and M4 receptors.
65 SPECIFIC AGONISTS FOR TWO SUBTYPES OF PRESYNAPTIC MUSCARINIC WHICH REGULATE ACETYLCHOLINE RELEASE IN GUINEA PIG A UERBACH B.V.R. Sastry and R.F. Ochillo; Vanderbilt
University
Medical Center, Nashville,
RECEPTORS PLEXUS
TN 37232-2125 U.S.A.
Several furan analogs of muscarine (M) were synthesized and evaluated pharmacologically to characterize the muscarinic receptors (MR) which regulate acetylcholine (ACh) release (1,2). Two furan analogs, 5methoxyfurmethide (5MOFT) and 5hydroxymethylfurmethide (5HMFT) indicated two subtypes of presynaptic MRs which regulate ACh release. 5-MOFT produced a contractile response of guinea pig longitudinal ileal muscle (EC,, 28mM), which disappeared upon depletion of tissue ACh with hemicholinium-3. 5-HMFT (4 x 1O‘aM) blocked nicotine-induced release of ACh and subsequent contraction of the muscle. The effects of both 5-MOFT and HMFT were blocked by atropine. These agonists did not exhibit significant effects on tissues containing M2 or M3 receptors. Both agonists were weak to induce phosphoinositol hydrolysis or attenuate CAMP accumulation in cultured human astrocytoma and neuroblastoma glioma cells, respectively (3,4). These results indicate that Auerbach plexus contains two novel muscarinic receptors (Mi, MS), one excitatory and the other inhibitory, which maintain homeostasis of ACh release. (Supported by The Smokeless Tobacco Research Council, Inc.; The Council for Tobacco Research, U.S.A., Inc.; and The Study Center for Anesthesia Toxicology of Vanderbilt University.) 1.) 2.) 3.) 4.)
Chaturvedi, A.K., Rowell, P.P. and Sastry, B.V.R., Pharmacol. Res. Comm. 13:829-845,198l. Horst, M.A., Sastry, B.V.R. and Landon, E.J., Arch. Int. Pharmacodyn. Therap. 288:87-99,1987. Stephan, CC. and Sastry, B.V.R., Pharmacologist 30(3):A183, 1988. Sastry, B.V.R. and Stephan, C.C., FASEBJ 2(5), A1401, 1988.
Abstracts
Vol. 60, Nos. 13/14, 1997
64 PRESYNAPTIC INHIBITORY MUSCARlNlC AUTORECEPTORS MODULATING 3H-ACETYLCHOLINE RELEASE IN HUMAN ISOLATED DETRUSOR MUSCLE GD’Agostino, ‘M.Tagliani, lS.M.Candura, E.Chiossa, *G.P.Franceschetti, C.Gregotti and # MTonini. Institute of Pharmacology, University of Pavia, l S.fvlaugeri Foundation IRCCS, Pavia, r)ivision of Urology, Hospital of Voghera (Pavia) and department of Internal Medicine and Therapeutics, Division of Pharmacology and Toxicology, University of Pavia, I -27100 Pavia, Italy The presence of presynaptic muscarinic mechanism(s) modulating the release of newly-synthesized tritiated acetylcholine @-I-ACh) from cholinergic nerves was investigated in human isolated detrusor muscle ships. Specimens from the anterior pad of the urinary bladder dome were obtained from patients undergoing total cystectomy due to bladder base malignancy. Muscular strips (20 mm long, 4 mm wide), prepared by removing serosal and mucosal layers and mounted isometrically in a modified Krebs solution (37 C), were preincubated with sH-choline and stimulated at 10 Hz to improve their neuronal sH-ACh content. Electrical field stimulation (EFS) induced contractile responses and tritium-outflow. These effects were prevented by TTX. Most of the evoked outflow was recognized as sH-ACh by HPLC analysis. When two EFSs (Sl and S2) were carried out at 5 Hz (000 pulses, 0.1 ms, 00 V) 30 min apart, an S2/St ratio of 0.84 was calculated. Muscarone (10-1000 nM), a potent muscarinic agonist, caused a concentration-dependent inhibition of the outflow of sH-ACh (pECy, 7.30) with maximal inhibition by about 50%. Physostigmine (100 nM) markedly reduced (by 50%) the evoked release whereas atropine (100 nM) increased it by 25%. Maximal (50%) facilitation of release was observed for atropine when six intermittent EFSs were applied at IO Hz (100 pulses, 1 ms, 12 V). Atropine and the subtype-selective antagonists CDAMP (M3), methoctramine (Md M4) and tripitramine (M2) increased the evoked release in a concentration-dependent fashion. The calculated pEC50 potencies were 9.07, 8.00, 7.97, and 8.73, respectively. These findings provide direct evidence that in the human urinary bladder acetylcholine release is under the local inhibitory control of muscarinic autoreceptors. The comparison between the observed rank order of pre- and postjunctional potency of muscarinic antagonists and their affinities at muscarinic receptor subtypes suggests that these autoreceptors may be regarded as Mz or a mixture of M2 and M4 receptors.
65 SPECIFIC AGONISTS FOR TWO SUBTYPES OF PRESYNAPTIC MUSCARINIC WHICH REGULATE ACETYLCHOLINE RELEASE IN GUINEA PIG A UERBACH B.V.R. Sastry and R.F. Ochillo; Vanderbilt
University
Medical Center, Nashville,
RECEPTORS PLEXUS
TN 37232-2125 U.S.A.
Several furan analogs of muscarine (M) were synthesized and evaluated pharmacologically to characterize the muscarinic receptors (MR) which regulate acetylcholine (ACh) release (1,2). Two furan analogs, 5methoxyfurmethide (5MOFT) and 5hydroxymethylfurmethide (5HMFT) indicated two subtypes of presynaptic MRs which regulate ACh release. 5-MOFT produced a contractile response of guinea pig longitudinal ileal muscle (EC,, 28mM), which disappeared upon depletion of tissue ACh with hemicholinium-3. 5-HMFT (4 x 1O‘aM) blocked nicotine-induced release of ACh and subsequent contraction of the muscle. The effects of both 5-MOFT and HMFT were blocked by atropine. These agonists did not exhibit significant effects on tissues containing M2 or M3 receptors. Both agonists were weak to induce phosphoinositol hydrolysis or attenuate CAMP accumulation in cultured human astrocytoma and neuroblastoma glioma cells, respectively (3,4). These results indicate that Auerbach plexus contains two novel muscarinic receptors (Mi, MS), one excitatory and the other inhibitory, which maintain homeostasis of ACh release. (Supported by The Smokeless Tobacco Research Council, Inc.; The Council for Tobacco Research, U.S.A., Inc.; and The Study Center for Anesthesia Toxicology of Vanderbilt University.) 1.) 2.) 3.) 4.)
Chaturvedi, A.K., Rowell, P.P. and Sastry, B.V.R., Pharmacol. Res. Comm. 13:829-845,198l. Horst, M.A., Sastry, B.V.R. and Landon, E.J., Arch. Int. Pharmacodyn. Therap. 288:87-99,1987. Stephan, CC. and Sastry, B.V.R., Pharmacologist 30(3):A183, 1988. Sastry, B.V.R. and Stephan, C.C., FASEBJ 2(5), A1401, 1988.