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Is nicotine sharing common molecular and anatomical substrates with cocaine?
ABSTRACTS
466
Structural
discrimination between the two agonist binding sites of the Torpedo nicotinic acetylcholine receptor using time resolved fluorescence K. Martinef’,
F?J. Corringerb, F. Merola”, J.-P. Changeux’
Muscle type nicotinic acetylcholine receptors (nAChR) are heteropentamers a2Py& They carry two binding sites for acetylcholine and competitive effecters at the al/y and a/6 subunit interfaces. To investigate for structural differences between the two binding sites. we used time resolved fluorescence (TRF). TRF consihts of exciting a fluorescent probe during an extremely short time, and measuring the time required by this molecule to reemit light (nanosecond time range). The re-emission speed depends on both the nature and chemical environment of the fluorescent probe. In the present work, we used the single photon counting methodle3, which establishes the re-emission probability of a photon versus time with very high accuracy. Mathematical analysis was performed using the maximum entropy method’.’ to give the fluorescence lifetime distribution, each lifetime putatively representing a particular state of the probe. We studied the interactions between the fluorescent probe dansyl-C6-choline6 (Dns-C6-Cho, excitation at 3.10 nm and emission at 520 nm) and the nAChR from Torpedo n~urn~~ruru, solubilized in CHAPS. The lifetime distribution of free Dns-C6-Cho in buffer containing CHAPS is quasi monoexponential with a lifetime of 3.7 ns. In contrast, the distribution of Dns-C6-Cho in the presence of the receptor is multi-exponential. with lifetimes of 0.25. 1.5, 5 and I8 ns (mean lifetime of 9 ns). Preincubation of the receptor with cc-bungarotoxin results in a distribution corresponding to free Dns-C6-Cho, showing that the signal is specific for the competitive agonist binding sites. To detect putative differences in binding aflinity and fluorescence fingerprint of the two binding sites, we measured the fluorescence decay at various occupation ratios of receptor by Dns-C6-Cho. No significant differences were found from 20 to 100% occupation of the binding sites, indicating that Dns-Cb-Cho
anatomical
displays similar binding affinities for the two sites or that the microenvironment of Dna-Cb-Cho in both sites is similar. In order to discrimate between these two hypotheses, we used the u-conotoxin MI 7.8, which has a 2%fold higher affinity for the al/u agonist binding site as compared to the c(/6 binding site on our receptor preparation. We studied the fluorescence decay of bound Dns-C6-Cho at various occupation ratios of the a-conotoxin Ml on the receptor. Preliminary experiments show different distributions at low and high occupation ratios, indicating that Dns-C6-Cho displays different fluorescent distributions depending on the binding sites. These data suggest that Dns-Cb-Cho remains in different microenvironments when bound to the two sites. possibly due to the contributions of the y and F subunits.
References (I)
Phillips, Analytical
(2)
Wahl. P In New techniques Pun and B. Smith, Ed.: 233-285.
(3) (41
Yguerabide,
J. Methods
Livesey, A. 198.5, AJI.
K.; Skilhng. 113-122.
(5)
Livesey. 693-706.
K.;
(6)
Waksman, tnacology
(7)
Hum, R. M.; Pag.m, 33, 14058-14063
(8)
Groebe, Molecular
D.Drake. R. C.: Instrumentation
A.
O‘Connor, D. V.; Christensen, 1985, 14, 267-292. in biophysics Wiley: London, in
Enzymology
J. Acta
Brochon,
J. C.
G.; Changeux. J. P; 1980. IX. 20-27.
D. R.; Dumm, Pharmacology
0.
R.;
and
cell 1975;
1972.
26,
Crystallographica. Bmphysical Roquei,
Eterovic,
R. L.
bicblogy; H. Vol. 2; pp 49X--578. Section
B.
1987,
52,
Journal B.
P. Molecular
phar-
V. A.
Bmchemistry
1994,
J. M.: Levitan, E. S.: Abramson. 1995. 48. 105-l I I.
S. N.
substrates with cocaine?
A.M. Mathieu-Kia,
M. Rogard, M.J. Besson :, UMR
Nicotine and cocaine are both psychostimulant drugs which activate dopamine (DA) neurons in the ventral mesencephalon. These neurons project massively to the striatal complex, the dorsal part (or atriatum) which is involved in movement co-ordination and the ventral part (or nucleus accumbenh, NAc) which is part of the limbic system. To investigate possible common molecular targets between these two drugs we have examined the effects produced by repeated injection5 (3 times a day during I5 days) of either nicotine or cocaine on peptide expression in the atriatal complex and by a single or repeated injections on immediate early gene expression in various cerebral regions.
7624.
9, qua;
Suurt-Bernard,
75005
Pa+
France
In the dorsal striatum, cocaine increased mRNAs encoding for substance P (PPT-A) and dynorphin (PPDYN) whereas nicotine did not change the levels of these mRNAs. The mRNA encoding for preproenkephalin (PPE), remained unchanged with both treatments. Similarly, in the ventral striatum (or NAc), nicotine did not affect the peptide mRNA expression except in the shell where it produced a decrease of PPDYN mRNA. In contrast, cocaine treatment induced increases of PPT-A mRNA in the core and PPDYN and PPE mRN.4s in the rostra] pole of the NAc Thus in the striatal complex, peptides which are expressed by the great majority of neurons did appear to be common substrates for these
Xth International Symposium on Cholinegic
Mechanisms
467
tivity was also examined. Forty minutes after an acute injection of nicotine (0.4 mg/kg, s.c.) c-fos and zif 268 mRNA were increased in visual, visuo-motor and retino-limbic structures as well as in amygdala and septum but not in the dorsal striatum. In contrast, c-fos and c.if 268 mRNAs were markedly increased but onI) in the striatal complex followin g an acute cocaine injection (I? mglkg, i.p.) (see figure I). The c-Fos protein analysed 90 min after an acute (0.4 mg/kg) and the last injection of a chronic nicotine (0.4 mg/kg. 3 time< a day for 15 days, s.c.) administration showed a pattern of induction similar to that found with c-fos mRNA analysis and in addition pointed to the involvement of various cortical areas. The acute versus chronic treatment comparison indicated a maintrnancc of c-Fos inducibility and in some structures a larger rccruitment of c-Fos positive neurons after chronic nicotine injections (Math&-Kia et al., 199X). The persistence of a c-F+ induction was generally not observed after a chronic cocaine treatment which instead generates chronic fos related antigens (Chen et al., 1995; Nye et al., 1997). While the striatal complex appears a\ a preferential target for cocaine. visual. retino-limbic and limhic areas are preferential targets for nicotine indicating that nicotine and cocaine share few common anatomical substrates
References
wwnr
m
Figure 1. Rats were treated with nicotine (0.4 m&g, LC.) or COCB~X (I? m&g. Lp.) and killed 40 min later. Brains were cut sagitally and sections wrre processed for zif 268 and c-fos mRNA using 3% riboprobes. It can be noted that nicotine mainly induced lif 268 and cmfos mainly in the superior collicuius and associated structnres as well as some thalamic nucleus (in particular the antero-rentral nucleus and the septum. In contrast, cocaine produced an Induction of zif 268 and c-F07 mRNA mamly in the strlatal complex.
two drugs; their expression was preferentially changed by cocaine (Mathieu-Kia et al., 1998). The induction of immediate early genes (c-fos and zif 2681 which can be considered as markers of changes in neuronal ac-
Chen
J, Krlr MB, Hope BT, Nakabeppu Y, Nestler EJ (1997) Chronic Fowelatcd antlgena: stable variants of deltaFoaB induced in hrnln by chronic treatments. J Neurosc~ 17-4933-4941 Mathieu-Kia AM, Beason MJ (1998) Repeated admmistration of cocaine. nicotine and ethanol: effects on preprodynorphin, preprot;,. chykinin A and preproenkephalin mRN.4 expression in the dorsal and the ventral \trwtum of the rat. Bran Res Mel Brain Kes 54.141-151 Mathicu-Kia AM, Pages. C.. Besson MJ 11998) Induclhrlity of c-l+ protein m viwn-motor system and llmbac structures after acute and repeated administration of n~cnt~nr in the rat. Synspre (1” press,. Nye HE, Hope BT, Kelz MB, Iadarnla M, Nestler EJ (19951 Pharmacological studies of the regulahon of chronic FOS-related antigen mduction by cocame in the aiatum and nuclew accumbens. I Pharmucol Exp Ther. 275:1671-1680
466
Structural
discrimination between the two agonist binding sites of the Torpedo nicotinic acetylcholine receptor using time resolved fluorescence K. Martinef’,
F?J. Corringerb, F. Merola”, J.-P. Changeux’
Muscle type nicotinic acetylcholine receptors (nAChR) are heteropentamers a2Py& They carry two binding sites for acetylcholine and competitive effecters at the al/y and a/6 subunit interfaces. To investigate for structural differences between the two binding sites. we used time resolved fluorescence (TRF). TRF consihts of exciting a fluorescent probe during an extremely short time, and measuring the time required by this molecule to reemit light (nanosecond time range). The re-emission speed depends on both the nature and chemical environment of the fluorescent probe. In the present work, we used the single photon counting methodle3, which establishes the re-emission probability of a photon versus time with very high accuracy. Mathematical analysis was performed using the maximum entropy method’.’ to give the fluorescence lifetime distribution, each lifetime putatively representing a particular state of the probe. We studied the interactions between the fluorescent probe dansyl-C6-choline6 (Dns-C6-Cho, excitation at 3.10 nm and emission at 520 nm) and the nAChR from Torpedo n~urn~~ruru, solubilized in CHAPS. The lifetime distribution of free Dns-C6-Cho in buffer containing CHAPS is quasi monoexponential with a lifetime of 3.7 ns. In contrast, the distribution of Dns-C6-Cho in the presence of the receptor is multi-exponential. with lifetimes of 0.25. 1.5, 5 and I8 ns (mean lifetime of 9 ns). Preincubation of the receptor with cc-bungarotoxin results in a distribution corresponding to free Dns-C6-Cho, showing that the signal is specific for the competitive agonist binding sites. To detect putative differences in binding aflinity and fluorescence fingerprint of the two binding sites, we measured the fluorescence decay at various occupation ratios of receptor by Dns-C6-Cho. No significant differences were found from 20 to 100% occupation of the binding sites, indicating that Dns-Cb-Cho
anatomical
displays similar binding affinities for the two sites or that the microenvironment of Dna-Cb-Cho in both sites is similar. In order to discrimate between these two hypotheses, we used the u-conotoxin MI 7.8, which has a 2%fold higher affinity for the al/u agonist binding site as compared to the c(/6 binding site on our receptor preparation. We studied the fluorescence decay of bound Dns-C6-Cho at various occupation ratios of the a-conotoxin Ml on the receptor. Preliminary experiments show different distributions at low and high occupation ratios, indicating that Dns-C6-Cho displays different fluorescent distributions depending on the binding sites. These data suggest that Dns-Cb-Cho remains in different microenvironments when bound to the two sites. possibly due to the contributions of the y and F subunits.
References (I)
Phillips, Analytical
(2)
Wahl. P In New techniques Pun and B. Smith, Ed.: 233-285.
(3) (41
Yguerabide,
J. Methods
Livesey, A. 198.5, AJI.
K.; Skilhng. 113-122.
(5)
Livesey. 693-706.
K.;
(6)
Waksman, tnacology
(7)
Hum, R. M.; Pag.m, 33, 14058-14063
(8)
Groebe, Molecular
D.Drake. R. C.: Instrumentation
A.
O‘Connor, D. V.; Christensen, 1985, 14, 267-292. in biophysics Wiley: London, in
Enzymology
J. Acta
Brochon,
J. C.
G.; Changeux. J. P; 1980. IX. 20-27.
D. R.; Dumm, Pharmacology
0.
R.;
and
cell 1975;
1972.
26,
Crystallographica. Bmphysical Roquei,
Eterovic,
R. L.
bicblogy; H. Vol. 2; pp 49X--578. Section
B.
1987,
52,
Journal B.
P. Molecular
phar-
V. A.
Bmchemistry
1994,
J. M.: Levitan, E. S.: Abramson. 1995. 48. 105-l I I.
S. N.
substrates with cocaine?
A.M. Mathieu-Kia,
M. Rogard, M.J. Besson :, UMR
Nicotine and cocaine are both psychostimulant drugs which activate dopamine (DA) neurons in the ventral mesencephalon. These neurons project massively to the striatal complex, the dorsal part (or atriatum) which is involved in movement co-ordination and the ventral part (or nucleus accumbenh, NAc) which is part of the limbic system. To investigate possible common molecular targets between these two drugs we have examined the effects produced by repeated injection5 (3 times a day during I5 days) of either nicotine or cocaine on peptide expression in the atriatal complex and by a single or repeated injections on immediate early gene expression in various cerebral regions.
7624.
9, qua;
Suurt-Bernard,
75005
Pa+
France
In the dorsal striatum, cocaine increased mRNAs encoding for substance P (PPT-A) and dynorphin (PPDYN) whereas nicotine did not change the levels of these mRNAs. The mRNA encoding for preproenkephalin (PPE), remained unchanged with both treatments. Similarly, in the ventral striatum (or NAc), nicotine did not affect the peptide mRNA expression except in the shell where it produced a decrease of PPDYN mRNA. In contrast, cocaine treatment induced increases of PPT-A mRNA in the core and PPDYN and PPE mRN.4s in the rostra] pole of the NAc Thus in the striatal complex, peptides which are expressed by the great majority of neurons did appear to be common substrates for these
Xth International Symposium on Cholinegic
Mechanisms
467
tivity was also examined. Forty minutes after an acute injection of nicotine (0.4 mg/kg, s.c.) c-fos and zif 268 mRNA were increased in visual, visuo-motor and retino-limbic structures as well as in amygdala and septum but not in the dorsal striatum. In contrast, c-fos and c.if 268 mRNAs were markedly increased but onI) in the striatal complex followin g an acute cocaine injection (I? mglkg, i.p.) (see figure I). The c-Fos protein analysed 90 min after an acute (0.4 mg/kg) and the last injection of a chronic nicotine (0.4 mg/kg. 3 time< a day for 15 days, s.c.) administration showed a pattern of induction similar to that found with c-fos mRNA analysis and in addition pointed to the involvement of various cortical areas. The acute versus chronic treatment comparison indicated a maintrnancc of c-Fos inducibility and in some structures a larger rccruitment of c-Fos positive neurons after chronic nicotine injections (Math&-Kia et al., 199X). The persistence of a c-F+ induction was generally not observed after a chronic cocaine treatment which instead generates chronic fos related antigens (Chen et al., 1995; Nye et al., 1997). While the striatal complex appears a\ a preferential target for cocaine. visual. retino-limbic and limhic areas are preferential targets for nicotine indicating that nicotine and cocaine share few common anatomical substrates
References
wwnr
m
Figure 1. Rats were treated with nicotine (0.4 m&g, LC.) or COCB~X (I? m&g. Lp.) and killed 40 min later. Brains were cut sagitally and sections wrre processed for zif 268 and c-fos mRNA using 3% riboprobes. It can be noted that nicotine mainly induced lif 268 and cmfos mainly in the superior collicuius and associated structnres as well as some thalamic nucleus (in particular the antero-rentral nucleus and the septum. In contrast, cocaine produced an Induction of zif 268 and c-F07 mRNA mamly in the strlatal complex.
two drugs; their expression was preferentially changed by cocaine (Mathieu-Kia et al., 1998). The induction of immediate early genes (c-fos and zif 2681 which can be considered as markers of changes in neuronal ac-
Chen
J, Krlr MB, Hope BT, Nakabeppu Y, Nestler EJ (1997) Chronic Fowelatcd antlgena: stable variants of deltaFoaB induced in hrnln by chronic treatments. J Neurosc~ 17-4933-4941 Mathieu-Kia AM, Beason MJ (1998) Repeated admmistration of cocaine. nicotine and ethanol: effects on preprodynorphin, preprot;,. chykinin A and preproenkephalin mRN.4 expression in the dorsal and the ventral \trwtum of the rat. Bran Res Mel Brain Kes 54.141-151 Mathicu-Kia AM, Pages. C.. Besson MJ 11998) Induclhrlity of c-l+ protein m viwn-motor system and llmbac structures after acute and repeated administration of n~cnt~nr in the rat. Synspre (1” press,. Nye HE, Hope BT, Kelz MB, Iadarnla M, Nestler EJ (19951 Pharmacological studies of the regulahon of chronic FOS-related antigen mduction by cocame in the aiatum and nuclew accumbens. I Pharmucol Exp Ther. 275:1671-1680