m.
-P
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Pctntcd InGreat
0278 - 684B/s2
199!& Vol. 16. pp. 783-789
SlS.00
Q I~R~n~~d
Brltaln. All II&W a-esawd
EFFECT OF SEDATXVEDRUGS UPON RECEPTOR BINDING INVMI OSAMU INOUE, KAORU KOBAYASHI, and TETSUYA SUHARA Division of Clinical Research, National Institute of Radiological Sciences, Anagawa, Chiba-shi, Japan (Final from, January 1992)
1.
2. 3. 4. 5.
Abstract lntr~uction Effect of Acute Treatment with Pentobarbitai (PB), Ethanol and 3en~~i~epines 3HSCH23390 Binding ln Vivo Differences in Receptor Binding between In Vitro and In Vivo Effect of PB and Ffunitrazepam on Other Receptor Binding In Vivo Conclusions References
on
783 784 784 785 785
787 788
Osamu Inoue, K.aoru Kobayashi and Tetsuya Suhara : Effect of Sedastive Drugs upon Receptor Binding In Vivo. Prog. Neuro-Psychopharmacoi. & Biol. Psychiat. 1992, 16(6): 783789 1. Acute treatment with pentobarbital (PB), ethanol and flunitrazepam significantly decreased 3H-SCH 23390 binding in mouse striatum in a dose dependent manner. In contrast, no significant alterations in 3H-SCH 23390 binding in the cerebral cortex have been observed in mice treated with these sedative and hypnotic drugs. 2. Flumazenil (Ro15-1788) reversed the effect of flunitrazepam suggesting the reduction in dopamine DI receptor binding in the striatum was mediated via GABA-Bz-Cl channel complex. 3. Using kinetic analysis, it was found that such changes in dopamine DI receptor binding in vivo were mainly due to changes in rates of ligand-receptor binding in vivo. 4. Other non-site-specific drugs such as propanol and buthanol also decreased 3H-SCH 23390 binding in vivo, depending on their lipophilicities. These results indicated that micro-environmental factors surrouwding receptors, including cellmembranes seem to have impo~ant roles in receptor binding in vivo. 5. Both PB and flunitrazepam decreased muscarinic acetylcholine receptor binding in mouse cortex, striatum, hippocampus and other regions. Together with the fact that PB also altered 3H-Ro15-1788 binding in vivo, this suggested global changes in microenvironmental factors may occur due to these sedative drugs. In vivo qauntitative analysis of neuroreceptors with positron emission tomography (PET) seems to have some potencies to reveal the neurochemical base of benzodiazepine dependence. Kevwor&:acetylcholine receptor, benzodiazepines, flunitrazepam, pentbarbital, sedative drugs.
ethanol,
flumatenil
(Ro15-1788),
Abbrevaitions: Y -Aminobutyric acid (GABA), Benzodi~epine (Bz), Chloride (Cl), N-methylpiperidil-Benzilate(NMPB), Positrone Emission Tomography (PET). 783
0. Inoue et at
784
1.
~ntrodu~ion
The neurochemicai mechanism for dependence on benrodiazepines is not well clarified (James et al, 1987). It has been well recognized that the withdrawal signs in experimental animals chronically treated with benzodiazepines are very similar to those of other classes of sedative drugs, such as ethanol and barbiturates, and crossdependence between these three types of sedative drugs has been reported (Wakasa et al,1985, Boisse et al.1981). These observations suggest a common neurochemical basis for dependence on sedative drugs. The pharmacological effects of benzodiazepines and barbiturates have been thought to be mediated via GABA-Bz receptor complex (Richard,1 981, Schwartz, 1988). Several lines of evidence have indicated that ethanol also may modify GABAnergic neurons (Allan and Harris, 1986, Ticku and Kulkarni, 1988). The reward pathway in the brain seems to be involved in d~gdependence probably through releasing dopamine (Mark et al, 1990.. Roy, 1987). Thus, it is valuable to study the interaction between GABAnergic and dopaminergic neurons in the brain for unde~tanding d~gdependence, and the intact brain seems to be an ideal system to study such neural interaction. In vivo quantitative analysis of receptor binding has been available both in the animai and human brain by using positron emission tomography (PET) (Suhara et al, 1991). In this paper, the authers describe the effect of sedative drugs upon 3HSCH 23390 binding in vivo, and discuss some possibilities for PET in drugdependence studies.
2. Fffect of -Treatment SBindina
.
ital IPBI. Fthms
.
on 8J&
Mce were pretreated with varying doses of PB, ethanol and flunitrazepam 30 min prior to the administration of 3HSCH 23390. The ratio of radioactivity in the striatum or cerebral cortex to that in the cerebellum 15 min after the tracer injection is-shown in Fig 1,
s-ran 2
z 0
3
PB PwW
EWI WW
---
Cerebral
i Cerebellum
Strlatum
E
g
g
7
doa
u
FNZPOwW
cortex
*b-loo
.kNYIO
B
--0
PB bwW
g
.-
I
0
Cerebellum
n
L
0
EtQH(en(g)
c: ? c%,,
E
s
FNZP (mgncg)
Fig 1. Changes in 3H-SCH23390bindingin mousestriatumand cerebellum.
Micewere pretreated with varing dose of pentobarbital(PS),ethanoi (EtOH) or fulnilrazepam (FNZP)15 or 30 min prior to the tracer injection _ The ratiiS of m~~a~iv~ in the striatum or cerebral cortex to that in the cerebellum 15 min after injectionof sH-SCH 233% were determined. VakreS are means of three mice in each group.
Sedative drugs and receptor binding in ofw
785
Significant, doserelated decrease in 3H-SCH 23390 binding in the striatum was seen in mice treated with these drugs. In contrast, no changes in dopamine Di receptor binding in the cerebral cortex were observed. Flumazenil (Ro15-1788) reversed the effect of flunitrazepam on 3H-SCH 23390 binding in vivo, thus, changes in 3H-SCH 23390 binding by flunitrazepam seems to be mediated via GABA-Bz-Cl channel complex. Fig 2 shows the time course of radioactivity in the striatum, cerebral cortex and cerebellum following intravenous injection of 3H-SCH 23390 into untreated and PB-pretreated mice. As in vivo ligand-receptor binding does not reach equilibrium state, kinetic analysis is needed for quantification. The three compartment model has been used for the kinetic analysis, and the concept of the binding potential (BP=k3/k4) has been proposed as an index of receptor binding in vivo (Mintun et al, 1984). The results of the kinetic analysis, summarized in Table 1, indicate that changes in 3H-SCH 23390 binding in vivo caused by PB were mainly due to changes in binding rates (k3 and k4). Similar results were also obtained in mice treated with flunitrazepam (Inoue et al, 1991 b). In addition, the in vivo dissociation rate of 3H-SCH 23390 in the striatum was actually decreased by flunitrazepam (Inoue et al. 1991 b). One possible explanation is that micro-environmental factors might be altered by these sedative drugs. 3. Differences of Receptor Binding between In Vitro and In Vivo Several lines of evidence suggested significant discrepancies in receptor binding between in vitro and in vivo. For example, lnsel found region specific decrease in 3H-Ro15-1788 binding in socially-separated rat pup brain, whereas no significant changes in binding were observed when brain slices were used (Insel 1989). In vivo specific changes in 3H-Ro15 1788 binding were also reported in either single (Inoue et al, 1985) or repeated swimstressed mice (Weizman et al, 1989). These results indicate factors other than affinity (KD) and the number of binding sites available (Bmax) play important roles in in vivo receptor binding. These factors, which act as a rate-limiting step for receptor binding in vivo, may disappear in tissue preparations. The concept of the diffusion boundary or field effects as a mechanism for such discrepancies in receptor binding between in vitro and in vivo have been proposed (Perry et al, 1980). With regard to dopamine receptor binding, an increase in 3H-spiperone binding was reported with bupropion treatment and other dopamine reuptake inhibitors, despite competitive inhibition that may be expected (Bischoff et al, 1984). Chugani et al (1988) reported a decrease in 3H-spiperone binding in reserpine-treated rat striatum without any changes in. KD and Bmax. Our study also confirmed in vivo reserpine-induced changes in both dopamine DI and 02 receptor binding. Amphetamine, a dopamine releasing drug, increased 3H-spiperone binding and reversed the effect of reserpine in a dose-dependent manner (Inoue et al, 1991 a). These results indicated an important role for endogeneous dopamine on in vivo receptor binding. PB, flunitrazepam, and ethanol all drugs used here have been reported to release endogeneous dopamine (Gaetano and Assunta, 1986., Gaetano and Assunta, 1988., Mitchell and Martin, 1980). Although the relationship between releasing of dopamine and changes in 3H-SCH 23390 binding is unclea, it warrants future investigation. 4. Pffect of PR and Flu-
. . on Other Receotor Rrndj_naIn Vivq
Our preliminary hypothesis is that dopamine alters the diffusion boundary resulting in rate changes of 3H-SCH 23390 binding in vivo. If our hypothesis is true, changes in binding rate of other receptors by these drugs could be expected. To clarify whether these sedative drugs also cause global changes in the brain, in vivo binding of muscarinic acetylcholine receptors were measured using 3H-N-methyl piperidil benzylate (3H-NMPB). As shown in Fig 3, both PB and flunitrazepam significantly decreased 3H-NMPB binding in various brain regions.
0. Inoue et QL
786
3
3 f
‘;
6
sol
$2 30 00 m3
4
o-
Specific
binding
In
the
strlatum)
G E
2
I
of 10
0
Time
40
30
20
(mln)
Fig 2. Time course of specific binding in the striatum and total radioactivity in the cerebellum following i.v. injection of 3HSCH23390 into normal and pentobarbital (50mg/kg, 30min Lp.) treated mice. The free ligand concentration in the brain was assumed to be parallel to radioactivity in the cerebellum. The kinetic analysis using using three compartment model was performed and results are summarized in table l.Values are means of three mice in each point.
Table
Kinetic Analysis of 3H SCH23390 in Mouse Striatum and Cerebral
1
Binding Cortex. l/min k3
Sttiatum
Cerebral
(Values
cortex
C C
k4
Control
0.262 + 0.016
0.0186 f 0.037
PB(50mg/kg)
0.107 rt 0.009
0.0120
Control
0.1144 + 0.0150
0.0937 f 0.0167
PB(50mg/kg)
0.0370 ? 0.0030
0.0255 f 0.0054
are mean It: S.D.
n=21)
* 0.0047
Sedative drugs and receptor binding in duo
787
Alteration in 3H-Ro15-1788 binding in vivo by PB was also reported (Miller et al. 1988). These results indicate global changes in the brain were induced by sedative drugs. Many factors such as blood flow, endogeneous ligand, internalization or externalization of receptors, and micro-environmental factors may effect receptor binding in vivo.
erebral
Cortex
n
10
2 2 =
a 5 $ Sg
Non-Specific Binding
8 6
oc 20 ‘L)
4
Cerebellum
: 2
Fig 3. Effect of various sedative drugs on rnuscarinic receptor binding in vivo. Mice were pretreated with pentbarbital (PB) , ethanol (EIOH) or flunitrazepam (FNZP) 15 or 30 min prior to the tracer injection and intravenously injected with 3H-N-methyl pipedilbenzilate. Radioactivfty, in the cerebellum and cerebral cortex at 30 min after injection of the tracer, was measured. The specific binding in each region was determined by the subtraction of radioactivity obtained in mice pretreated with 3mgkg of quinuclidinyl benzilate
In addition, micro-environmental factors including cellmembrane fluidity seem to have on important role on the diffusion boundary, since nonsite-specific drugs, such as isopropanol, and buthanol and ethanol also significantly effected 3H-SCH 23390 binding in vivo (unpublished data). The degree of changes in 3H-SCH 23390 binding was dependent on their lipophilicities. Changes in cellmembrane fluidity by ethanol, propanol and buthanol have been reported (Francoise et al, 1984). Further detailed studies are required to clarify the roles of membrane fluidity on receptor binding rates in vivo. 5.Conclusions In vivo quantitative analysis of various types of neuroreceptors in the human brain have been available by using PET. Since PET is based upon the in vivo tracer technique, only the binding process can be measured. Determination of the rates of receptor binding rather than static binding parameters such as KD and Bmax seems to be more important for the in vivo receptor studies. In vivo receptor binding is capable of being indirectly changed by various drugs. Our preliminary experiments concerning effects of sedative drugs on receptor binding in vivo suggested an important role for micro-environmental factors including membrane fluidity, on receptor binding in vivo. Clinical PET studies together with further animal experiments using such indirect effects of drugs are needed to reveal the neuro-chemical basis of benzodiazepine dependence.
788
0.
Inoue et al.
References ALLAN, A. M. and HARRIS, Ft. A. (1986) Gamma-aminobutyric Acid and Alcohol Actions: Neurochemical Studies of Long Sleep and Short Sleep Mice. Life Sci. 3: 20052015. BISCHOFF, S., BITTIGER, H., KARAUSS, J., VASSOUT, A. and WALDMEIER, P. (1984) Affinity Changes of Rat Striatal Dopamine Receptors In Vivo. Eur. J. Pharmacol. 1p4: 173176. BOISSE, N. R., RYAN, G. P., GUARINO, J. J. and GAY, M. H. (1981) Comparison of Benzodiazepine and Barbituate Tolerance and Physical Dependence in the Rat. Pharmacologist B: 192. CHUGANI, D. C., ACKERMANN, R. F. and PHELPS, M. E. (1988) Evidence for Accumulation In Corpus Striatum by Agonist Mediated Receptor Internalization. J.Cereb. Blood Flow Metab. 8: 291-303. FRANCOISE, B., CATHERINE, F., FRANCOISE, B. and ROGER, N. (1984) Brain Membrane Disordering After Acute In Vivo Administration of Ethanol, lsopropanol or t-Butanol in Rats. Biochem. Pharmacol. a: 3591-3595. GAETANO, D. C. and ASSUNTA, I. (1986) Preferential Stimulation of Dopamine Release in the Nucleus Accumbens by Opiates,Alcohol,and Barbiturates: Studies with Transcerebral Dialysis in Freely Moving Rats. Ann. N. Y. Acad. Sci. m: 367-381. GAETANO, D. C. and ASSUNTA, I. (1988) Drugs Abused by Humans Preferentially Increase Synaptic Dopamine Concentration in the Mesolimbic System of Freely Moving Rats. Proc. Natl. Acad. Sci. USA. 85: 5274-5278. INOUE, O., AKIMOTO, Y., HASHIMOTO, K. and YAMASAKI, T. (1985) Alteration in Biodistribution of 3HRol51788 in Mice by Acute Stress: Possible Changes in In Vivo Binding Availability of Brain Benzodiazepine Receptor. Int. J. Nucl. Med. Biol. 12: 369-374. INOUE, O., TSUKADA, H., YONEZAWA, H., SUHARA, T. and LANGSTROM, B (1991 a) Reserpine Induced Reduction of In Vivo Binding of SCH 23390 and N-methylspiperone and its Reversal by d-amphetamine. Eur. J. Pharmacol. w: 143-l 49. INOUE, 0.. KOBAYASHI, K., YOJIRO, S., and SUZUKI, T. (1991 b) The Effect of Benzodiazepine on 3H-SCH 23390 Binding In Vivo . Neuropharmacol. in press. INSEL, T. R. (1989) Decreased In Vivo Binding to Brain Benzodiazepine Social Isolation. Psychopharmacology 9z: 142-l 44.
Receptors During
JAMES, H. W., JONATHAN, L. K. and GAIL, W. (1987) Abuse Liability of Benzodiazepines. Pharmacol. Rev. 2: 251-390. MARK, S. B., SARAH, A. S. and THOMAS, V. D. (1990) Ethanol Increases the Firing Rate of Dopamine Neurons of the Rat Ventral Tegmental Area In Vitro. Brain Res. a: 65-69. MILLER, L. G., DEUTSH, S. I., GREENBLATT, D. J., PAUL, S. M. and SHADER, R.I. (1988) Acute Barbituate Administration Increases Benzodiazepine Receptor Binding In Vivo. Psychopharmacology s: 385-390. MINTUN, M. A., RAICHLE, M. E., KILBOURN, M. R., WOOTEN, G. F. and WELCH, M. (1984) A Quantitative Model for the In Vivo Assessment of Drug Binding Sites with Positron Emission Tomography. Ann. Neurol. j!j: 217-227.
Sedattvc drugs
and receptor binding in u&o
789
MITCHELL, P. Ft. and MARTIN, I. L. (1980) Facilitation of Striatal Potassium-Induced Dopamine Release-Novel Structural Requirements for a Presynaptic Action of Benzodiazepines. Neuropharmacol. 19: 147-l 50. PERRY, D. C., MULLIS, K. B., OIE, S. and SADEE, W. (1980) Opiate Antagonist Receptor Binding In Vivo: Evidence for a new Receptor Binding Model. Brain Res. m: 49-61. SCHWARTZ, R. D. (1988) The GABA A Receptor-Gated Ion Channel: Biochemical and Pharmacological Studies of Structure and Function. Biochem. Pharmacol. a: 3369-3375. RICHARD, W. 0. (1981) GABA-Benzodiazepine-Barbiturate Neurochem. z: l-l 3.
Receptor
Interactions.
J.
ROY, A. W. (1987) The Role of Reward Pathways in the Development of Drug Dependence. Pharmacol. Ther. 3: 227-263. SUHARA, T., FUKUDA, H., INOUE, O., ITOH, T., SUZUKI, K., YAMASAKI, T. and TATENO, Y. (1991) Age-Related Changes in Human DI Dopamine Receptors Measured by Positron Emission Tomography. Psychopharmacology m: 41-45. TICKU, M. K. and KULKARNI, S. K. (1988) Molecular Interaction of Ethanol with GABAergic System and Potential of Rol5-4513 as an Ethanol Antagonist. Pharmacl. Biochem. Behav. ;Lp: 501-510. WAKASA, Y., KATO, S. and YANAGITA, T. (1985) Comparison of Physical DependenceProducing Mechanism Between Barbiturates and Bentodiazepines. Natl. Inst. Drug Abuse Res. Monogr. Ser. %: 276-282. WEIZMAN, R., WEIZMAN, A., KOOK, K. A., VOCCI, F., DENTSH, S. I. and PAUL, S. M. (1989) Repeated Swim Stress Alters Brain Benzodiazepine Receptors Measured In Vivo. J. Pharmacol. Exp. Ther. a: 701-707. Inquiries and reprint requests should be addressed to: Osamu lnoue Ph.D. Division of Clinical Research, National lnsutitute of Radiological 9-1, Anagawa 4-chome, Chiba-shi Japan
Sciences, 260
-P
8 E&IL Pq&iat
Pctntcd InGreat
0278 - 684B/s2
199!& Vol. 16. pp. 783-789
SlS.00
Q I~R~n~~d
Brltaln. All II&W a-esawd
EFFECT OF SEDATXVEDRUGS UPON RECEPTOR BINDING INVMI OSAMU INOUE, KAORU KOBAYASHI, and TETSUYA SUHARA Division of Clinical Research, National Institute of Radiological Sciences, Anagawa, Chiba-shi, Japan (Final from, January 1992)
1.
2. 3. 4. 5.
Abstract lntr~uction Effect of Acute Treatment with Pentobarbitai (PB), Ethanol and 3en~~i~epines 3HSCH23390 Binding ln Vivo Differences in Receptor Binding between In Vitro and In Vivo Effect of PB and Ffunitrazepam on Other Receptor Binding In Vivo Conclusions References
on
783 784 784 785 785
787 788
Osamu Inoue, K.aoru Kobayashi and Tetsuya Suhara : Effect of Sedastive Drugs upon Receptor Binding In Vivo. Prog. Neuro-Psychopharmacoi. & Biol. Psychiat. 1992, 16(6): 783789 1. Acute treatment with pentobarbital (PB), ethanol and flunitrazepam significantly decreased 3H-SCH 23390 binding in mouse striatum in a dose dependent manner. In contrast, no significant alterations in 3H-SCH 23390 binding in the cerebral cortex have been observed in mice treated with these sedative and hypnotic drugs. 2. Flumazenil (Ro15-1788) reversed the effect of flunitrazepam suggesting the reduction in dopamine DI receptor binding in the striatum was mediated via GABA-Bz-Cl channel complex. 3. Using kinetic analysis, it was found that such changes in dopamine DI receptor binding in vivo were mainly due to changes in rates of ligand-receptor binding in vivo. 4. Other non-site-specific drugs such as propanol and buthanol also decreased 3H-SCH 23390 binding in vivo, depending on their lipophilicities. These results indicated that micro-environmental factors surrouwding receptors, including cellmembranes seem to have impo~ant roles in receptor binding in vivo. 5. Both PB and flunitrazepam decreased muscarinic acetylcholine receptor binding in mouse cortex, striatum, hippocampus and other regions. Together with the fact that PB also altered 3H-Ro15-1788 binding in vivo, this suggested global changes in microenvironmental factors may occur due to these sedative drugs. In vivo qauntitative analysis of neuroreceptors with positron emission tomography (PET) seems to have some potencies to reveal the neurochemical base of benzodiazepine dependence. Kevwor&:acetylcholine receptor, benzodiazepines, flunitrazepam, pentbarbital, sedative drugs.
ethanol,
flumatenil
(Ro15-1788),
Abbrevaitions: Y -Aminobutyric acid (GABA), Benzodi~epine (Bz), Chloride (Cl), N-methylpiperidil-Benzilate(NMPB), Positrone Emission Tomography (PET). 783
0. Inoue et at
784
1.
~ntrodu~ion
The neurochemicai mechanism for dependence on benrodiazepines is not well clarified (James et al, 1987). It has been well recognized that the withdrawal signs in experimental animals chronically treated with benzodiazepines are very similar to those of other classes of sedative drugs, such as ethanol and barbiturates, and crossdependence between these three types of sedative drugs has been reported (Wakasa et al,1985, Boisse et al.1981). These observations suggest a common neurochemical basis for dependence on sedative drugs. The pharmacological effects of benzodiazepines and barbiturates have been thought to be mediated via GABA-Bz receptor complex (Richard,1 981, Schwartz, 1988). Several lines of evidence have indicated that ethanol also may modify GABAnergic neurons (Allan and Harris, 1986, Ticku and Kulkarni, 1988). The reward pathway in the brain seems to be involved in d~gdependence probably through releasing dopamine (Mark et al, 1990.. Roy, 1987). Thus, it is valuable to study the interaction between GABAnergic and dopaminergic neurons in the brain for unde~tanding d~gdependence, and the intact brain seems to be an ideal system to study such neural interaction. In vivo quantitative analysis of receptor binding has been available both in the animai and human brain by using positron emission tomography (PET) (Suhara et al, 1991). In this paper, the authers describe the effect of sedative drugs upon 3HSCH 23390 binding in vivo, and discuss some possibilities for PET in drugdependence studies.
2. Fffect of -Treatment SBindina
.
ital IPBI. Fthms
.
on 8J&
Mce were pretreated with varying doses of PB, ethanol and flunitrazepam 30 min prior to the administration of 3HSCH 23390. The ratio of radioactivity in the striatum or cerebral cortex to that in the cerebellum 15 min after the tracer injection is-shown in Fig 1,
s-ran 2
z 0
3
PB PwW
EWI WW
---
Cerebral
i Cerebellum
Strlatum
E
g
g
7
doa
u
FNZPOwW
cortex
*b-loo
.kNYIO
B
--0
PB bwW
g
.-
I
0
Cerebellum
n
L
0
EtQH(en(g)
c: ? c%,,
E
s
FNZP (mgncg)
Fig 1. Changes in 3H-SCH23390bindingin mousestriatumand cerebellum.
Micewere pretreated with varing dose of pentobarbital(PS),ethanoi (EtOH) or fulnilrazepam (FNZP)15 or 30 min prior to the tracer injection _ The ratiiS of m~~a~iv~ in the striatum or cerebral cortex to that in the cerebellum 15 min after injectionof sH-SCH 233% were determined. VakreS are means of three mice in each group.
Sedative drugs and receptor binding in ofw
785
Significant, doserelated decrease in 3H-SCH 23390 binding in the striatum was seen in mice treated with these drugs. In contrast, no changes in dopamine Di receptor binding in the cerebral cortex were observed. Flumazenil (Ro15-1788) reversed the effect of flunitrazepam on 3H-SCH 23390 binding in vivo, thus, changes in 3H-SCH 23390 binding by flunitrazepam seems to be mediated via GABA-Bz-Cl channel complex. Fig 2 shows the time course of radioactivity in the striatum, cerebral cortex and cerebellum following intravenous injection of 3H-SCH 23390 into untreated and PB-pretreated mice. As in vivo ligand-receptor binding does not reach equilibrium state, kinetic analysis is needed for quantification. The three compartment model has been used for the kinetic analysis, and the concept of the binding potential (BP=k3/k4) has been proposed as an index of receptor binding in vivo (Mintun et al, 1984). The results of the kinetic analysis, summarized in Table 1, indicate that changes in 3H-SCH 23390 binding in vivo caused by PB were mainly due to changes in binding rates (k3 and k4). Similar results were also obtained in mice treated with flunitrazepam (Inoue et al, 1991 b). In addition, the in vivo dissociation rate of 3H-SCH 23390 in the striatum was actually decreased by flunitrazepam (Inoue et al. 1991 b). One possible explanation is that micro-environmental factors might be altered by these sedative drugs. 3. Differences of Receptor Binding between In Vitro and In Vivo Several lines of evidence suggested significant discrepancies in receptor binding between in vitro and in vivo. For example, lnsel found region specific decrease in 3H-Ro15-1788 binding in socially-separated rat pup brain, whereas no significant changes in binding were observed when brain slices were used (Insel 1989). In vivo specific changes in 3H-Ro15 1788 binding were also reported in either single (Inoue et al, 1985) or repeated swimstressed mice (Weizman et al, 1989). These results indicate factors other than affinity (KD) and the number of binding sites available (Bmax) play important roles in in vivo receptor binding. These factors, which act as a rate-limiting step for receptor binding in vivo, may disappear in tissue preparations. The concept of the diffusion boundary or field effects as a mechanism for such discrepancies in receptor binding between in vitro and in vivo have been proposed (Perry et al, 1980). With regard to dopamine receptor binding, an increase in 3H-spiperone binding was reported with bupropion treatment and other dopamine reuptake inhibitors, despite competitive inhibition that may be expected (Bischoff et al, 1984). Chugani et al (1988) reported a decrease in 3H-spiperone binding in reserpine-treated rat striatum without any changes in. KD and Bmax. Our study also confirmed in vivo reserpine-induced changes in both dopamine DI and 02 receptor binding. Amphetamine, a dopamine releasing drug, increased 3H-spiperone binding and reversed the effect of reserpine in a dose-dependent manner (Inoue et al, 1991 a). These results indicated an important role for endogeneous dopamine on in vivo receptor binding. PB, flunitrazepam, and ethanol all drugs used here have been reported to release endogeneous dopamine (Gaetano and Assunta, 1986., Gaetano and Assunta, 1988., Mitchell and Martin, 1980). Although the relationship between releasing of dopamine and changes in 3H-SCH 23390 binding is unclea, it warrants future investigation. 4. Pffect of PR and Flu-
. . on Other Receotor Rrndj_naIn Vivq
Our preliminary hypothesis is that dopamine alters the diffusion boundary resulting in rate changes of 3H-SCH 23390 binding in vivo. If our hypothesis is true, changes in binding rate of other receptors by these drugs could be expected. To clarify whether these sedative drugs also cause global changes in the brain, in vivo binding of muscarinic acetylcholine receptors were measured using 3H-N-methyl piperidil benzylate (3H-NMPB). As shown in Fig 3, both PB and flunitrazepam significantly decreased 3H-NMPB binding in various brain regions.
0. Inoue et QL
786
3
3 f
‘;
6
sol
$2 30 00 m3
4
o-
Specific
binding
In
the
strlatum)
G E
2
I
of 10
0
Time
40
30
20
(mln)
Fig 2. Time course of specific binding in the striatum and total radioactivity in the cerebellum following i.v. injection of 3HSCH23390 into normal and pentobarbital (50mg/kg, 30min Lp.) treated mice. The free ligand concentration in the brain was assumed to be parallel to radioactivity in the cerebellum. The kinetic analysis using using three compartment model was performed and results are summarized in table l.Values are means of three mice in each point.
Table
Kinetic Analysis of 3H SCH23390 in Mouse Striatum and Cerebral
1
Binding Cortex. l/min k3
Sttiatum
Cerebral
(Values
cortex
C C
k4
Control
0.262 + 0.016
0.0186 f 0.037
PB(50mg/kg)
0.107 rt 0.009
0.0120
Control
0.1144 + 0.0150
0.0937 f 0.0167
PB(50mg/kg)
0.0370 ? 0.0030
0.0255 f 0.0054
are mean It: S.D.
n=21)
* 0.0047
Sedative drugs and receptor binding in duo
787
Alteration in 3H-Ro15-1788 binding in vivo by PB was also reported (Miller et al. 1988). These results indicate global changes in the brain were induced by sedative drugs. Many factors such as blood flow, endogeneous ligand, internalization or externalization of receptors, and micro-environmental factors may effect receptor binding in vivo.
erebral
Cortex
n
10
2 2 =
a 5 $ Sg
Non-Specific Binding
8 6
oc 20 ‘L)
4
Cerebellum
: 2
Fig 3. Effect of various sedative drugs on rnuscarinic receptor binding in vivo. Mice were pretreated with pentbarbital (PB) , ethanol (EIOH) or flunitrazepam (FNZP) 15 or 30 min prior to the tracer injection and intravenously injected with 3H-N-methyl pipedilbenzilate. Radioactivfty, in the cerebellum and cerebral cortex at 30 min after injection of the tracer, was measured. The specific binding in each region was determined by the subtraction of radioactivity obtained in mice pretreated with 3mgkg of quinuclidinyl benzilate
In addition, micro-environmental factors including cellmembrane fluidity seem to have on important role on the diffusion boundary, since nonsite-specific drugs, such as isopropanol, and buthanol and ethanol also significantly effected 3H-SCH 23390 binding in vivo (unpublished data). The degree of changes in 3H-SCH 23390 binding was dependent on their lipophilicities. Changes in cellmembrane fluidity by ethanol, propanol and buthanol have been reported (Francoise et al, 1984). Further detailed studies are required to clarify the roles of membrane fluidity on receptor binding rates in vivo. 5.Conclusions In vivo quantitative analysis of various types of neuroreceptors in the human brain have been available by using PET. Since PET is based upon the in vivo tracer technique, only the binding process can be measured. Determination of the rates of receptor binding rather than static binding parameters such as KD and Bmax seems to be more important for the in vivo receptor studies. In vivo receptor binding is capable of being indirectly changed by various drugs. Our preliminary experiments concerning effects of sedative drugs on receptor binding in vivo suggested an important role for micro-environmental factors including membrane fluidity, on receptor binding in vivo. Clinical PET studies together with further animal experiments using such indirect effects of drugs are needed to reveal the neuro-chemical basis of benzodiazepine dependence.
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