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Affinity profile of S(-)-hyoscyamine for muscarinic receptor subtypes
568
Abstracts
Vol.
52,
No.s
5 & 6, 1993
36 CYCLIC AMP POTENTIATES MUSCARINIC RECEPTOR-STIMULATED PHOSPHOINOSITIDE HYDROLYSIS IN HUMAN NEUROEPITHELIOMA CELLS S.K. Fisher, E.L. McEwen, S.C. Lovell and R.E. Landon. University of Michigan, Ann Arbor, MI 48104, USA A stimulatory role for cAMP in the regulation of muscarinic receptor (mAChR)-stimulated phosphoinositide (PPI) hydrolysis has been examined in human neuroepithelioma cells. These cells elicit an increase in PPI hydrolysis in the presence of agonists specific for muscarinic, C~ladrenergic, endothelin and ATP receptors. All combinations of agonists resulted in a release of inositol phosphates that was at least additive. However, the addition of optimal concentrations of oxotremorine-M and norepinephrine to SK-N-MCIXC or SK-N-MCIIE cells resulted in a PP! hydrolysis that was 30% greater than additive. This potentiation of inositol lipid hydrolysis resulted from an increased activity of the mAChR after the addition of norepinephrine and persisted after C~l-adrenergic receptor blockade. The enhancement of mAChR-stimulated inositol phosphate release could be quantitatively mimicked by inclusion of the [3-adrenergic agonist, ~soproterenol, but not by C~l or c~2-adrenergic agonists. Potentiation of oxotremorine-M stimulated inositol lipid hydrolysis observed in the,, presence of either norepinephrine or isoproterenol was reduced in the absence of added Ca z~. Addition of either norepinephrine or isoproterenol to SK-N-MCIXC cells also resulted in a 16-fold increase in cAMP concentration. Inclusion of the cell permeant 8-chloro-4-phenyhhio-cAMP resulted in a 30% enhancement of mAChR-stimulated inositol phosphate release. We conclude that in SK-N-MCIXC cells, an increase in cAMP concentration potentiates mAChR-stimulated phosphoinositide hydrolysis. (Supported by NIMH Grant 46252).
37 AFFINITY PROFILE OF S(-)-HYOSCYAMINE FOR MUSCARINIC RECEPTOR SUBTYPES *S. Scapecchi, *C. Bellucci, C. Ghelardini, A. Gheri, A. Giotti, *F. Gualtieri & A. Bartolini. Departments of Pharmacology and *Pharmaceutical Sciences, Florence, Italy Ghelardini et al. (1992) reported that S-(-)-hyoscyamine, contrary to R-(+)-hyoscyamine, is not able either to induce analgesia in rodents or to potentiate electrically evoked guinea-pig ileum contractions. In order to clarify the reason for the htck of S-(-)-hyoscyamine in vivo and in vitro effects, we investigated its affinity profile for three muscarinic receptor subtypes. Antagonist potencies were determined by functional studies performed using field-stimulated rabbit vas deferens (M1), rat ileum (M3) and guinea pig uterus (M4). Concentration-response curves were constructed by the addition respectively of McN-A-343, ACh and carbachol in the absence or presence of antagonist, pA 2 values, obtained by using Arunlakshana-Schild plots are listed below. Preparation R a b b i t vas deferens Rat i l e u m G u i n e a - p i g uterus
Subtype
pA 2
(MI) (M3) (M 4)
9.28 + 0.21 9.01 _+ 0,01 8.71 + 0.02
Slope 1.21 + 0.52 0.95 + 0.01 0.91 + 0.02
S-(-)-hyoscyamine affinity for guinea-pig atrium (M2) has been already determined by Barlow et a1.(1986), pA 2 values revealed that S-(-)-hyoscyamine does not discriminate between M 1 and M 3 receptors and that it shows less affinity for the M 4 receptor subtype than R-(+)-hyoscyamine (Ghelardini et al. this meeting). The above reported results suggest that the lack of analgesic effect of S-(-)-hyoscyamine compared to its enantiomer R-(+)-hyoscyamine reflects their different affinities for both M 1 and M 4 receptors. This work was supported by grants from MURST and CNR.
Abstracts
Vol.
52,
No.s
5 & 6, 1993
36 CYCLIC AMP POTENTIATES MUSCARINIC RECEPTOR-STIMULATED PHOSPHOINOSITIDE HYDROLYSIS IN HUMAN NEUROEPITHELIOMA CELLS S.K. Fisher, E.L. McEwen, S.C. Lovell and R.E. Landon. University of Michigan, Ann Arbor, MI 48104, USA A stimulatory role for cAMP in the regulation of muscarinic receptor (mAChR)-stimulated phosphoinositide (PPI) hydrolysis has been examined in human neuroepithelioma cells. These cells elicit an increase in PPI hydrolysis in the presence of agonists specific for muscarinic, C~ladrenergic, endothelin and ATP receptors. All combinations of agonists resulted in a release of inositol phosphates that was at least additive. However, the addition of optimal concentrations of oxotremorine-M and norepinephrine to SK-N-MCIXC or SK-N-MCIIE cells resulted in a PP! hydrolysis that was 30% greater than additive. This potentiation of inositol lipid hydrolysis resulted from an increased activity of the mAChR after the addition of norepinephrine and persisted after C~l-adrenergic receptor blockade. The enhancement of mAChR-stimulated inositol phosphate release could be quantitatively mimicked by inclusion of the [3-adrenergic agonist, ~soproterenol, but not by C~l or c~2-adrenergic agonists. Potentiation of oxotremorine-M stimulated inositol lipid hydrolysis observed in the,, presence of either norepinephrine or isoproterenol was reduced in the absence of added Ca z~. Addition of either norepinephrine or isoproterenol to SK-N-MCIXC cells also resulted in a 16-fold increase in cAMP concentration. Inclusion of the cell permeant 8-chloro-4-phenyhhio-cAMP resulted in a 30% enhancement of mAChR-stimulated inositol phosphate release. We conclude that in SK-N-MCIXC cells, an increase in cAMP concentration potentiates mAChR-stimulated phosphoinositide hydrolysis. (Supported by NIMH Grant 46252).
37 AFFINITY PROFILE OF S(-)-HYOSCYAMINE FOR MUSCARINIC RECEPTOR SUBTYPES *S. Scapecchi, *C. Bellucci, C. Ghelardini, A. Gheri, A. Giotti, *F. Gualtieri & A. Bartolini. Departments of Pharmacology and *Pharmaceutical Sciences, Florence, Italy Ghelardini et al. (1992) reported that S-(-)-hyoscyamine, contrary to R-(+)-hyoscyamine, is not able either to induce analgesia in rodents or to potentiate electrically evoked guinea-pig ileum contractions. In order to clarify the reason for the htck of S-(-)-hyoscyamine in vivo and in vitro effects, we investigated its affinity profile for three muscarinic receptor subtypes. Antagonist potencies were determined by functional studies performed using field-stimulated rabbit vas deferens (M1), rat ileum (M3) and guinea pig uterus (M4). Concentration-response curves were constructed by the addition respectively of McN-A-343, ACh and carbachol in the absence or presence of antagonist, pA 2 values, obtained by using Arunlakshana-Schild plots are listed below. Preparation R a b b i t vas deferens Rat i l e u m G u i n e a - p i g uterus
Subtype
pA 2
(MI) (M3) (M 4)
9.28 + 0.21 9.01 _+ 0,01 8.71 + 0.02
Slope 1.21 + 0.52 0.95 + 0.01 0.91 + 0.02
S-(-)-hyoscyamine affinity for guinea-pig atrium (M2) has been already determined by Barlow et a1.(1986), pA 2 values revealed that S-(-)-hyoscyamine does not discriminate between M 1 and M 3 receptors and that it shows less affinity for the M 4 receptor subtype than R-(+)-hyoscyamine (Ghelardini et al. this meeting). The above reported results suggest that the lack of analgesic effect of S-(-)-hyoscyamine compared to its enantiomer R-(+)-hyoscyamine reflects their different affinities for both M 1 and M 4 receptors. This work was supported by grants from MURST and CNR.