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GTP-γ-S modulation of agonist affinity at m1 receptors expressed in A9 L cells
Vol.
52, No.s
5 & 6, 1993
Abstracts
575
5O DIFFERENCE IN THE AFFINITY OF MeN-A-343 FOR M U S C A R I N E RECEPTORS IN SMOOTH A N D CARDIAC MUSCLE. A. Elnatan and F. Mitchelson, School o f Pharmacology, Victorian College of Pharmacy (Monash University), Parkville, Australia. Studies with antagonists and receptor antibodies have suggested that the majority of the muscarine receptors in intestinal smooth muscle are of the M 2 subtype similar to those in cardiac muscle. Investigations were undertaken with a partial agonist at muscarine receptors, MeN-A-343, to compare ils affinity for muscarine receptors in the smooth muscle of the guinea pig taenia caecum with those in the atrium. In the taenia, suspended in McEwen's solution, McN-A-343 produced only 72.3% of tile maximal response Io carbacbol with an ECs0 of 1.2 (aM. The K A (dissociation constant) for McN-A-343 delermined by compari,g eqniactive concentrations of the two agonists was 4.6 (aM. Displacement binding sludies with MeN-A-343 versus 13HIQNB in McEwen's solution containing Gpp(NH)p (50 [aM) gave a K I value of 5.6 [aM for binding to a single site and this value was not altered significantly (P > 0.05) when binding was performed i , 5(1 mM phosphate buffer. M e N - A - 3 4 3 in concentratiotls up to 0.3 mM failed to affect oxotremorine-iuduced inhibition of the increase in c A M P produced by isoprenaline in the taenia. In guinea pig isolated left atrium set up in McEwen's solution MeN-A-343 (50 to 3(10 (aM) was used as an antagonist o f the negative inotropic response to carbachol. The K B for McN-A-343 estimated by the method of Kaumarm and Blinks (1) was 13.1 (aM. Binding studies with [3HIQNB and MeN-A-343 ill McEweu's solution containing Gpp(NH)p (50 [aM) gave a K l value of 14.8 [AM. In phosphate buffer this value was 7.0 (aM. Both the atrial K B value and K 1 estimate in McEwen's solution were significantly different (P < (I.05) from the K A and K 1 values obtained in the taenia. It is concluded that M e N - A - 3 4 3 exhibits a different afl-mity for muscarine receptors in the two tissues and thai it is unable to detect a binding site in the taenia similar to that observed in cardiac muscle. 1. A.J. K A U M A N N and J.R. BLINKS. Naunyn-Schmiedeberg's Arch. Pharmacol. 311 241-248 (1980).
51 GTP-y-S M O D U L A T I O N
OF AGONIST
AFFINITY
AT ml R E C E P T O R S
EXPRESSED
IN A9 L CELLS
W.S. Messer, Jr., Y. Liu, Y.F. A b u h and P.G. D u n b a r The U n i v e r s i t y of Toledo, Toledo, OH, U.S.A. W a s h i n g t o n State University, Spokane, WA, U.S.A. The r e c e p t o r b i n d i n g properties of m u s c a r i n i c a g o n i s t s were e x a m i n e d in A9 L cells e x p r e s s i n g ml r e c e p t o r s .
with
varying
efficacy
A g o n i s t a f f i n i t y was d e t e r m i n e d by the inhibition of [3H]-(R)-QNB binding. Activity was a s s e s s e d by e x a m i n i n g the e f f e c t s of GTP-y-S on a g o n i s t affinity (Potter & Ferrendelli. J. Pharmacol. Exp. Therap. 248: 974-978, 1989). IC50 v a l u e s a n d Hill s l o p e s were d e t e r m i n e d from Hill plots of the inhibition data. GTP-y-S l o w e r e d the affinity of carbachol by four fold from 17 ± 9.8 ~M to 51 ± 15 ~M. GTP-y--S was less e f f e c t i v e in l o w e r i n g the a f f i n i t y of a r e c o l i n e from 5.6 ± 1.2 ~M to 8.3 ± 1.0 ~M. The 3 - m e t h y l - l , 2 , 4 - o x a d i a z o l e derivative of a r e c o l i n e was s y n t h e s i z e d a n d s h o w e d h i g h e r a f f i n i t y t h a n a r e c o l i n e , yet the m a g n i t u d e of t h e GTP-y-S effect was c o m p a r a b l e (IC50 shift from 0.71 ± 0.14 ~M to 1.6 ± 0.67 ~M). The d a t a i n d i c a t e that GTP-T-S d e c r e a s e s a g o n i s t a f f i n i t y for ml receptors, a n d t h a t the m a g n i t u d e of the shift m a y be r e l a t e d to efficacy. The partial a g o n i s t c h a r a c t e r of a r e c o l i n e is c o n s i s t e n t with PI m e t a b o l i s m d a t a o b t a i n e d in the CNS a n d in m e a s u r e s of c A M P f o r m a t i o n in A9 L c e l l s e x p r e s s i n g ml receptors (Novotny & Brann, TIPS supplement, 116, 1989). Furthermore, the d a t a i n d i c a t e that s u b s t i t u t i n g the e s t e r with a 1 , 2 , 4 - o x a d i a z o l e moiety i n c r e a s e s l i g a n d affinity but not efficacy at ml receptors. In summary, GTPy-S m o d u l a t i o n of a g o n i s t a f f i n i t y p r o v i d e s a useful measure of a g o n i s t activity in c e l l s e x p r e s s i n g s i n g l e subtypes of muscarinic receptors.
Work was s u p p o r t e d by Lilly Ohio Department of Aging.
Research
Laboratories,
HEW
grant
NS
01493
& the
52, No.s
5 & 6, 1993
Abstracts
575
5O DIFFERENCE IN THE AFFINITY OF MeN-A-343 FOR M U S C A R I N E RECEPTORS IN SMOOTH A N D CARDIAC MUSCLE. A. Elnatan and F. Mitchelson, School o f Pharmacology, Victorian College of Pharmacy (Monash University), Parkville, Australia. Studies with antagonists and receptor antibodies have suggested that the majority of the muscarine receptors in intestinal smooth muscle are of the M 2 subtype similar to those in cardiac muscle. Investigations were undertaken with a partial agonist at muscarine receptors, MeN-A-343, to compare ils affinity for muscarine receptors in the smooth muscle of the guinea pig taenia caecum with those in the atrium. In the taenia, suspended in McEwen's solution, McN-A-343 produced only 72.3% of tile maximal response Io carbacbol with an ECs0 of 1.2 (aM. The K A (dissociation constant) for McN-A-343 delermined by compari,g eqniactive concentrations of the two agonists was 4.6 (aM. Displacement binding sludies with MeN-A-343 versus 13HIQNB in McEwen's solution containing Gpp(NH)p (50 [aM) gave a K I value of 5.6 [aM for binding to a single site and this value was not altered significantly (P > 0.05) when binding was performed i , 5(1 mM phosphate buffer. M e N - A - 3 4 3 in concentratiotls up to 0.3 mM failed to affect oxotremorine-iuduced inhibition of the increase in c A M P produced by isoprenaline in the taenia. In guinea pig isolated left atrium set up in McEwen's solution MeN-A-343 (50 to 3(10 (aM) was used as an antagonist o f the negative inotropic response to carbachol. The K B for McN-A-343 estimated by the method of Kaumarm and Blinks (1) was 13.1 (aM. Binding studies with [3HIQNB and MeN-A-343 ill McEweu's solution containing Gpp(NH)p (50 [aM) gave a K l value of 14.8 [AM. In phosphate buffer this value was 7.0 (aM. Both the atrial K B value and K 1 estimate in McEwen's solution were significantly different (P < (I.05) from the K A and K 1 values obtained in the taenia. It is concluded that M e N - A - 3 4 3 exhibits a different afl-mity for muscarine receptors in the two tissues and thai it is unable to detect a binding site in the taenia similar to that observed in cardiac muscle. 1. A.J. K A U M A N N and J.R. BLINKS. Naunyn-Schmiedeberg's Arch. Pharmacol. 311 241-248 (1980).
51 GTP-y-S M O D U L A T I O N
OF AGONIST
AFFINITY
AT ml R E C E P T O R S
EXPRESSED
IN A9 L CELLS
W.S. Messer, Jr., Y. Liu, Y.F. A b u h and P.G. D u n b a r The U n i v e r s i t y of Toledo, Toledo, OH, U.S.A. W a s h i n g t o n State University, Spokane, WA, U.S.A. The r e c e p t o r b i n d i n g properties of m u s c a r i n i c a g o n i s t s were e x a m i n e d in A9 L cells e x p r e s s i n g ml r e c e p t o r s .
with
varying
efficacy
A g o n i s t a f f i n i t y was d e t e r m i n e d by the inhibition of [3H]-(R)-QNB binding. Activity was a s s e s s e d by e x a m i n i n g the e f f e c t s of GTP-y-S on a g o n i s t affinity (Potter & Ferrendelli. J. Pharmacol. Exp. Therap. 248: 974-978, 1989). IC50 v a l u e s a n d Hill s l o p e s were d e t e r m i n e d from Hill plots of the inhibition data. GTP-y-S l o w e r e d the affinity of carbachol by four fold from 17 ± 9.8 ~M to 51 ± 15 ~M. GTP-y--S was less e f f e c t i v e in l o w e r i n g the a f f i n i t y of a r e c o l i n e from 5.6 ± 1.2 ~M to 8.3 ± 1.0 ~M. The 3 - m e t h y l - l , 2 , 4 - o x a d i a z o l e derivative of a r e c o l i n e was s y n t h e s i z e d a n d s h o w e d h i g h e r a f f i n i t y t h a n a r e c o l i n e , yet the m a g n i t u d e of t h e GTP-y-S effect was c o m p a r a b l e (IC50 shift from 0.71 ± 0.14 ~M to 1.6 ± 0.67 ~M). The d a t a i n d i c a t e that GTP-T-S d e c r e a s e s a g o n i s t a f f i n i t y for ml receptors, a n d t h a t the m a g n i t u d e of the shift m a y be r e l a t e d to efficacy. The partial a g o n i s t c h a r a c t e r of a r e c o l i n e is c o n s i s t e n t with PI m e t a b o l i s m d a t a o b t a i n e d in the CNS a n d in m e a s u r e s of c A M P f o r m a t i o n in A9 L c e l l s e x p r e s s i n g ml receptors (Novotny & Brann, TIPS supplement, 116, 1989). Furthermore, the d a t a i n d i c a t e that s u b s t i t u t i n g the e s t e r with a 1 , 2 , 4 - o x a d i a z o l e moiety i n c r e a s e s l i g a n d affinity but not efficacy at ml receptors. In summary, GTPy-S m o d u l a t i o n of a g o n i s t a f f i n i t y p r o v i d e s a useful measure of a g o n i s t activity in c e l l s e x p r e s s i n g s i n g l e subtypes of muscarinic receptors.
Work was s u p p o r t e d by Lilly Ohio Department of Aging.
Research
Laboratories,
HEW
grant
NS
01493
& the