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Additional complexities in the binding of muscarinic receptor antagonists
430 T I P S - N o v e m b e r 1985
13 Zeisberger, E., Merker, G., Bl/ihser, S. and Krannig, M. in Proc. 6th Int. Symp.
on the Pharmacology of Thermoregulation, Jasper, Karger, Basel, (in press) 14 Riedel, W. and Gray, D. (1984) Pfliig. Arch. Eur. J. Physiol. 402, (suppl.), R41 15 Epstein, Y., Horowitz, M., Bosin, E.,
m
I
J
Shapiro, Y., Glick, S.M. (1984) i n Thermal Physiology (Hales, J. R. S., ed.), pp. 137-140, Raven Press, New York 16 Szczepanska-Sadowska, E. (1974) Am. J. Physiol. 226, 155-161 17 Szczepanska-Sadowska, E., Gray, D. and Simon-Oppermann, Ch. (1983) Am.
r
~'
J. Physiol. 245, R549-555 18 Robinson, I. C. A. F. (1983) Prog. Brain. Res. 60, 129-145 19 Szelenyi, Z. (1983) J. Therm. Biol. 8, 185190 20 Clark, W. G. and Lipton, J.M. (1983) Pharmacol. Ther. 22, 249-297
and benzilates have been observed. For example, the dependence • "r+rLrpi,,+ + ,- of antagonist affinity on brain re.,1~_ I I L I ' k ~ . I t gion is more noticeable with tropates than with benzilates 2. It might be concluded that in most Additional c o m p l e x i t i e s in the binding of cases where binding of tertiary and quaternary amine muscarinic muscarinic r e c e p t o r antagonists receptor antagonists has been determined simultaneously, the Receptor binding studies have rements. For example, [3H]NMS is former compounds are capable of sulted in the accumulation of a capable of labeling only 65% of the labeling muscarinic receptor sites significant amount of valuable inreceptor population that is availthat are not easily accessible to the formation regarding the properties able to [3H]QNB, suggesting that latter ligands. These sites might be of muscarinic acetylcholine recep[3H]NMS might recognize only a embedded in the lipid milieu or tors in the CNS and PNS. It has subpopulation of [aH]QNB bindanother hydrophobic domain of generally been accepted that, ing sites4,5. This is reminiscent of the cell membrane. In addition, the while muscarinic agonists can disthe fact that the atypical muscardifference in binding capacity of criminate between multiple affininic receptor antagonist, pirenthese two groups of muscarinic ity states of the receptor, resulting zepine, also binds to a selective antagonists might be due to their in heterogeneity of agonist bindpopulation of [3H]QNB binding ability to recognize different coning sites 1, benzilate and tropate sites in the brain 6. Similar findings formations of the receptor. For muscarinic receptor antagonists that [3H]QNB labels double the example, it has been reported that bind to a homogeneous populanumber of receptors labeled by [3H]QNB is more efficient than tion of receptors according to the [aH]NMS in homogenates of [3H]NMS in converting the muslaw of mass action 2, with some 1321N1 astrocytoma cells have also carinic receptor into its high afregional variability in affinity3. been reported recently7. It should finity conformation s. Differentiabe noted however that contradiction between these hypotheses re0 OH tory reports have illustrated that quires further testing. Also, more [3H]QNB and [3H]NMS label the extensive experimentation with same receptor density in intact muscarinic receptor ligands bechick cardiac cells s. In addition, it longing to chemical classes other has been demonstrated that the than the benzilates and tropates is quaternary ammonium analog of required before further generaliQu/nuclidJnyl Benzilate [3H]QNB, [3H]QNB methiodide, zations are justified. binds only to half of the receptor We have also found that the population detected with [3H]QNB behavior of quaternary muscarinic in both dog ventricle homogenantagonists, when used to displace ates 9 and intact chick heart cells 10. [3H]QNB binding in rat brain o °-~1 = Much older reports suggesting a homogenates, is quite anomasimilar phenomenon can also be lous 4,s. Under these conditions, found in the literature. For unlabeled NMS and methylatroN-Methylscopolamine example, it has been shown that pine, but not their tertiary amine [3H]methylatropine binds with analogs, demonstrated very shalHowever, it has recently been prohigh affinity only to half of the low displacement curves 5. When posed that binding of antagonists sites labeled by [3H]atropine in these curves are resolved into to muscarinic receptors might not smooth muscle preparations11. high- and low-affinity componbe quite so simple. Collectively, these findings sugents, only the former correspond We have recently reported that gest that the differences in the closely to the sites labeled directly significant differences exist in the binding characteristics of [3H]by [3H]NMS (Ref. 5). Furthermore, nature of interaction of tertiary QNB and [3H]NMS might not be this anomalous receptor binding amine, e.g. [3H]quinuclidinyl due simply to the fact that they profile cannot be seen if [3H]NMS benzilate ([3H]QNB) and quaterbelong to different chemical is used as a ligand s. Similar findnary amine, e.g. [3H]N-methylgroups; these differences can also ings have been published recently scopolamine ([3H]NMS) ligands be seen when the binding of terby two other groups of investigawith the muscarinic receptors in tiary and quaternary analogs of a tors 1°,12. There are reasons to berat brain homogenates4,S. These particular ligand is examined. lieve that the high affinity muscardifferences can be revealed both in However, subtle differences in the inic receptors in rat brain labeled saturation and competition experibinding characteristics of tropates directly by [3H]NMS, or revealed
c'~'~/~/c"~L__~o clio.
1985,ElsevierSciencePublishersB.V.,Amsterdam 0165- 6147/85/$02.00
431
T I P S - N o v e m b e r 1985
in N M S / [ 3 H ] Q N B c o m p e t i t i o n exp e r i m e n t s , are not e q u i v a l e n t to the p u r p o r t e d M1 r e c e p t o r s sensit i v e to p i r e n z e p i n e . Firstly, p i r enzepine demonstrates receptor heterogeneity regardless of w h e t h e r [3H]QNB or [3H]NMS is u s e d for b i n d i n g e x p e r i m e n t s 5. Secondly, there is e v i d e n c e sugg e s t i n g that q u a t e r n a r y m u s c a r inic r e c e p t o r a n t a g o n i s t s do n o t possess the u n i q u e p h a r m a c o l o g i cal specificity of p i r e n z e p i n e (see Ref. 13). T h e i m p o r t a n c e of t h e r e c e n t f i n d i n g s of the u n e x p e c t e d h e t e r o g e n e i t y of the b i n d i n g sites for s o m e classical m u s c a r i n i c r e c e p t o r a n t a g o n i s t s is two-fold: (1) the p h y s i c o - c h e m i c a l p r o p e r t i e s of a tested a n t a g o n i s t s h o u l d b e t a k e n into c o n s i d e r a t i o n b e f o r e a t t e m p t ing to i n t e r p r e t c o m p l e x b i n d i n g data in t e r m s of m u l t i p l e m u s c a r inic r e c e p t o r (M1 a n d M2) s u b types; a n d (2) the a s s u m p t i o n that d i f f e r e n t m u s c a r i n i c r e c e p t o r lig-
a n d s label an e q u a l n u m b e r of receptors in a g i v e n tissue, w h i c h has b e e n s o m e t i m e s m a d e in calc u l a t i n g u n k n o w n specific activities of newly synthesized l i g a n d s 9,14, m i g h t b e a v e r y d a n g e r o u s practice. Acknowledgements
The a u t h o r w o u l d like to t h a n k Anita Saulsbury and Renee Powe for t y p i n g the m a n u s c r i p t . The w o r k f r o m the a u t h o r ' s l a b o r a t o r y cited h e r e w a s s u p p o r t e d b y a grant f r o m the N a t i o n a l Science F o u n d a t i o n (BNS-84-06357) a n d a contract f r o m the US A r m y Research Office (DAAG-29-85-K0123). ESAM E. EL-FAKAHANY Department of Pharmacology and Toxicology, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.
References
1 Birdsall, N. J. M., Burgen, A. S. V. and Hulme, E. C. (1978) MoL PharmacoL 14,
723--736 2 Hulme, E. C., Birdsall, N. J. M., Burgen, A. S. V. and Mehta, P. (1978) Mol. PharmacoL 14, 737-750 3 Sokolovsky, M. (1984) Int. Rev. Neurobiol. 25, 139-183 4 Lee, J.-H. and EI-Fakahany, E. E. (1985) Eur. J. Pharmacol. 110, 263-266 5 Lee, J.-H. and El-Fakahany, E. E. (1985)J. Pharmacol. Exp. Ther. 233, 707-714 6 Luthin, G. R. and Wolfe, B. B. (1984) J. PharmacoL Exp. Ther. 228, 648-655 7 Evans, T., Smith, M.M., Tanner, L.I. and Harden, T. K. (1984) Mol. Pharmacol. 26, 395-404 8 Galper, J. B., Dziekan, L.C., O'Hara, D.S. and Smith, T.W. (1982) J. Biol. Chem. 257, 10344-10356 9 Gibson, R. E., Rzeszotarski, W. ]., Jagoda, E. M., Francis, B. E., Reba, R. C. and Eckelman, W. C. (1984) Life Sci. 34, 2287-2296 10 Goldstein, D. and Brown, J. H. (1985) Fed. Proc. 44, 1827 11 Paton, W. D. M. and Rang, H. P. (1965) Proc. Roy. Soc. Lond. B. 163, 1-44 12 Ellis, J. and Lenox, R. H. (1985) Biochem. Biophys. Res. Commun. 126, 1242-1250 13 Wess, J., Lambrecht, G., Moser, U. and Mutschler, E. (1984) Life Sci. 35, 553-560 14 Frey, K. A., Ehrenkaufer, R. L. E., Beaucage, S. and Agranoff, B.W. (1985) ]. NeuroscL 5, 421--428
13 Zeisberger, E., Merker, G., Bl/ihser, S. and Krannig, M. in Proc. 6th Int. Symp.
on the Pharmacology of Thermoregulation, Jasper, Karger, Basel, (in press) 14 Riedel, W. and Gray, D. (1984) Pfliig. Arch. Eur. J. Physiol. 402, (suppl.), R41 15 Epstein, Y., Horowitz, M., Bosin, E.,
m
I
J
Shapiro, Y., Glick, S.M. (1984) i n Thermal Physiology (Hales, J. R. S., ed.), pp. 137-140, Raven Press, New York 16 Szczepanska-Sadowska, E. (1974) Am. J. Physiol. 226, 155-161 17 Szczepanska-Sadowska, E., Gray, D. and Simon-Oppermann, Ch. (1983) Am.
r
~'
J. Physiol. 245, R549-555 18 Robinson, I. C. A. F. (1983) Prog. Brain. Res. 60, 129-145 19 Szelenyi, Z. (1983) J. Therm. Biol. 8, 185190 20 Clark, W. G. and Lipton, J.M. (1983) Pharmacol. Ther. 22, 249-297
and benzilates have been observed. For example, the dependence • "r+rLrpi,,+ + ,- of antagonist affinity on brain re.,1~_ I I L I ' k ~ . I t gion is more noticeable with tropates than with benzilates 2. It might be concluded that in most Additional c o m p l e x i t i e s in the binding of cases where binding of tertiary and quaternary amine muscarinic muscarinic r e c e p t o r antagonists receptor antagonists has been determined simultaneously, the Receptor binding studies have rements. For example, [3H]NMS is former compounds are capable of sulted in the accumulation of a capable of labeling only 65% of the labeling muscarinic receptor sites significant amount of valuable inreceptor population that is availthat are not easily accessible to the formation regarding the properties able to [3H]QNB, suggesting that latter ligands. These sites might be of muscarinic acetylcholine recep[3H]NMS might recognize only a embedded in the lipid milieu or tors in the CNS and PNS. It has subpopulation of [aH]QNB bindanother hydrophobic domain of generally been accepted that, ing sites4,5. This is reminiscent of the cell membrane. In addition, the while muscarinic agonists can disthe fact that the atypical muscardifference in binding capacity of criminate between multiple affininic receptor antagonist, pirenthese two groups of muscarinic ity states of the receptor, resulting zepine, also binds to a selective antagonists might be due to their in heterogeneity of agonist bindpopulation of [3H]QNB binding ability to recognize different coning sites 1, benzilate and tropate sites in the brain 6. Similar findings formations of the receptor. For muscarinic receptor antagonists that [3H]QNB labels double the example, it has been reported that bind to a homogeneous populanumber of receptors labeled by [3H]QNB is more efficient than tion of receptors according to the [aH]NMS in homogenates of [3H]NMS in converting the muslaw of mass action 2, with some 1321N1 astrocytoma cells have also carinic receptor into its high afregional variability in affinity3. been reported recently7. It should finity conformation s. Differentiabe noted however that contradiction between these hypotheses re0 OH tory reports have illustrated that quires further testing. Also, more [3H]QNB and [3H]NMS label the extensive experimentation with same receptor density in intact muscarinic receptor ligands bechick cardiac cells s. In addition, it longing to chemical classes other has been demonstrated that the than the benzilates and tropates is quaternary ammonium analog of required before further generaliQu/nuclidJnyl Benzilate [3H]QNB, [3H]QNB methiodide, zations are justified. binds only to half of the receptor We have also found that the population detected with [3H]QNB behavior of quaternary muscarinic in both dog ventricle homogenantagonists, when used to displace ates 9 and intact chick heart cells 10. [3H]QNB binding in rat brain o °-~1 = Much older reports suggesting a homogenates, is quite anomasimilar phenomenon can also be lous 4,s. Under these conditions, found in the literature. For unlabeled NMS and methylatroN-Methylscopolamine example, it has been shown that pine, but not their tertiary amine [3H]methylatropine binds with analogs, demonstrated very shalHowever, it has recently been prohigh affinity only to half of the low displacement curves 5. When posed that binding of antagonists sites labeled by [3H]atropine in these curves are resolved into to muscarinic receptors might not smooth muscle preparations11. high- and low-affinity componbe quite so simple. Collectively, these findings sugents, only the former correspond We have recently reported that gest that the differences in the closely to the sites labeled directly significant differences exist in the binding characteristics of [3H]by [3H]NMS (Ref. 5). Furthermore, nature of interaction of tertiary QNB and [3H]NMS might not be this anomalous receptor binding amine, e.g. [3H]quinuclidinyl due simply to the fact that they profile cannot be seen if [3H]NMS benzilate ([3H]QNB) and quaterbelong to different chemical is used as a ligand s. Similar findnary amine, e.g. [3H]N-methylgroups; these differences can also ings have been published recently scopolamine ([3H]NMS) ligands be seen when the binding of terby two other groups of investigawith the muscarinic receptors in tiary and quaternary analogs of a tors 1°,12. There are reasons to berat brain homogenates4,S. These particular ligand is examined. lieve that the high affinity muscardifferences can be revealed both in However, subtle differences in the inic receptors in rat brain labeled saturation and competition experibinding characteristics of tropates directly by [3H]NMS, or revealed
c'~'~/~/c"~L__~o clio.
1985,ElsevierSciencePublishersB.V.,Amsterdam 0165- 6147/85/$02.00
431
T I P S - N o v e m b e r 1985
in N M S / [ 3 H ] Q N B c o m p e t i t i o n exp e r i m e n t s , are not e q u i v a l e n t to the p u r p o r t e d M1 r e c e p t o r s sensit i v e to p i r e n z e p i n e . Firstly, p i r enzepine demonstrates receptor heterogeneity regardless of w h e t h e r [3H]QNB or [3H]NMS is u s e d for b i n d i n g e x p e r i m e n t s 5. Secondly, there is e v i d e n c e sugg e s t i n g that q u a t e r n a r y m u s c a r inic r e c e p t o r a n t a g o n i s t s do n o t possess the u n i q u e p h a r m a c o l o g i cal specificity of p i r e n z e p i n e (see Ref. 13). T h e i m p o r t a n c e of t h e r e c e n t f i n d i n g s of the u n e x p e c t e d h e t e r o g e n e i t y of the b i n d i n g sites for s o m e classical m u s c a r i n i c r e c e p t o r a n t a g o n i s t s is two-fold: (1) the p h y s i c o - c h e m i c a l p r o p e r t i e s of a tested a n t a g o n i s t s h o u l d b e t a k e n into c o n s i d e r a t i o n b e f o r e a t t e m p t ing to i n t e r p r e t c o m p l e x b i n d i n g data in t e r m s of m u l t i p l e m u s c a r inic r e c e p t o r (M1 a n d M2) s u b types; a n d (2) the a s s u m p t i o n that d i f f e r e n t m u s c a r i n i c r e c e p t o r lig-
a n d s label an e q u a l n u m b e r of receptors in a g i v e n tissue, w h i c h has b e e n s o m e t i m e s m a d e in calc u l a t i n g u n k n o w n specific activities of newly synthesized l i g a n d s 9,14, m i g h t b e a v e r y d a n g e r o u s practice. Acknowledgements
The a u t h o r w o u l d like to t h a n k Anita Saulsbury and Renee Powe for t y p i n g the m a n u s c r i p t . The w o r k f r o m the a u t h o r ' s l a b o r a t o r y cited h e r e w a s s u p p o r t e d b y a grant f r o m the N a t i o n a l Science F o u n d a t i o n (BNS-84-06357) a n d a contract f r o m the US A r m y Research Office (DAAG-29-85-K0123). ESAM E. EL-FAKAHANY Department of Pharmacology and Toxicology, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.
References
1 Birdsall, N. J. M., Burgen, A. S. V. and Hulme, E. C. (1978) MoL PharmacoL 14,
723--736 2 Hulme, E. C., Birdsall, N. J. M., Burgen, A. S. V. and Mehta, P. (1978) Mol. PharmacoL 14, 737-750 3 Sokolovsky, M. (1984) Int. Rev. Neurobiol. 25, 139-183 4 Lee, J.-H. and EI-Fakahany, E. E. (1985) Eur. J. Pharmacol. 110, 263-266 5 Lee, J.-H. and El-Fakahany, E. E. (1985)J. Pharmacol. Exp. Ther. 233, 707-714 6 Luthin, G. R. and Wolfe, B. B. (1984) J. PharmacoL Exp. Ther. 228, 648-655 7 Evans, T., Smith, M.M., Tanner, L.I. and Harden, T. K. (1984) Mol. Pharmacol. 26, 395-404 8 Galper, J. B., Dziekan, L.C., O'Hara, D.S. and Smith, T.W. (1982) J. Biol. Chem. 257, 10344-10356 9 Gibson, R. E., Rzeszotarski, W. ]., Jagoda, E. M., Francis, B. E., Reba, R. C. and Eckelman, W. C. (1984) Life Sci. 34, 2287-2296 10 Goldstein, D. and Brown, J. H. (1985) Fed. Proc. 44, 1827 11 Paton, W. D. M. and Rang, H. P. (1965) Proc. Roy. Soc. Lond. B. 163, 1-44 12 Ellis, J. and Lenox, R. H. (1985) Biochem. Biophys. Res. Commun. 126, 1242-1250 13 Wess, J., Lambrecht, G., Moser, U. and Mutschler, E. (1984) Life Sci. 35, 553-560 14 Frey, K. A., Ehrenkaufer, R. L. E., Beaucage, S. and Agranoff, B.W. (1985) ]. NeuroscL 5, 421--428