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D-2 dopamine receptor synthesis and turnover in rat striatum
European Journal of Pharmacology, 127 (1986) 291-294
291
Elsevier
Short communication
D-2 D O P A M I N E R E C E P T O R S Y N T H E S I S AND TURNOVER IN RAT STRIATUM ISAO FUKUCHI *, NORIHISA FUJITA, MASANOBU NAKAHIRO *, KIHACHI SAITO ** and HIROSHI YOSHIDA
Department of Pharmacology I, Osaka University School of Medicine, 4-3-57 Nakanoshima, Kita-ku, Osaka 530, and ** Department of Pharmacology, Osaka University School of Dentistry, 1-8 Yamadaoka, Suita 564, Japan Received 15 May 1986, accepted 27 May 1986
I. FUKUCHI, N. FUJITA, M. NAKAHIRO, K. SAITO and H. YOSHIDA, D-2 dopamine receptor synthesis and turnover in rat striaturrg European J. Pharmacol. 126 (1986) 291-294.
Direct injection of phenoxybenzamine into rat striatum inhibited apomorphine-induced stereotyped behavior. This inhibition corresponded well with the inhibition of D-2 dopamine receptor labelling with [3H]spiroperidol. Both the behavioral response and the receptor level were completely restored within 5 days after the injection. The recoveries of both were blocked by cycloheximide. The rate of synthesis and half-life of the D-2 receptor associated with the stereotyped behavior were calculated to be 6.9 fmol/mg protein per h and 28 h, respectively. D-2 dopamine receptor
Phenoxybenzamine
Striatum
1. Introduction
Long-term administration of neuroleptics or chemical denervation of the nigrostriatal dopamine pathway with 6-hydroxydopamine is known to result in the development of D-2 dopamine receptor supersensitivity, which is characterized by proliferation of 3H-neuroleptic specific binding sites in rat striatum and an enhanced stereotypy response to dopamine agonists (for review, see Seeman, 1980). Conversely, long-term interception of coupling between the D-2 dopamine receptor and the guanine .nucleotide binding protein reduces [3H]spiroperidol specific binding sites in rat striatum (Fujita et al.~ 1985). These results show that the concentration of D-2 dopamine receptors can be varied experimentally. However, the turnover rate of D-2 receptor is controversial. Hall et al. (1983) reported that the half-life of the D-2 receptor was about 8-9 h after blockade with the alkylating reagent phenoxybenzamine, which is an * Present address: Safety Research Laboratory, Tanabe Seiyaku Co. Ltd., 3-16-89 Kashima, Yodogawa-ku, Osaka 532, Japan. * To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Rat
irreversible blocker of the D-2 receptor as well as an al-adrenergic blocker (Marcharis and Bockaert, 1980; Hamblin and Creese, 1982). On the other hand, Left et al. (1984) reported that the half-life of the D-2 receptor was 45-119 h after irreversible blockade with the alkylating reagent N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ). In these studies, phenoxybenzamine and EEDQ were administered intraperitoneaUy (i.p.). We have been studying the dopamine receptor in the central nervous system by direct injection of drugs into the brain (Fujita et al., 1981; Fujita et al., 1985). This method is very useful for studying brain function pharmacologically and behaviorally without affecting the peripheral system. In the work described here, by direct injection of phenoxybenzamine into rat striatum through chronically implanted guide cannulas we obtained a value for the D-2 dopamine receptor turnover rate that corresponded well with the behavioral response to the dopamine agonist. 2. Materials and methods
Male Sprague-Dawley rats weighing 280-300 g were anesthetized with nembutal (35 m g / k g i.p.)
292
and placed in a David Kopf stereotaxic holder. Guide cannulas constructed from 23 gauge stainless-steel tubing were implanted into the striatum according to the brain atlas of Pellegrino and Cushman (2.2 mm anterior to the bregma, 2.8 mm bilateral to the sagittal suture, 5.2 mm ventral to the skull). The cannulas were fastened to the skull with dental cement and acrylic resin. Five days later, drugs were injected bilaterally into the striaturn in a volume of 1-3 #1 and at a rate of 1 /~l/min through the injection cannulas. Phenoxybenzamine and cycloheximide (Nakarai, Japan) were dissolved in saline at final concentrations of 25-75 #g//~l and 10 # g / # l , respectively. Apomorphine HC1 (Sigma) was dissolved in saline and was injected subcutaneously (s.c.) at a dose of 280 #g/kg. Stereotyped behaviours were rated as described previously (Fujita et al., 1985). Striata were homogenized in 25 volumes of 25 mM Tris-HCl buffer (pH 7.3) containing 5 mM EDTA Na 2. The homogenate was centrifuged at 100 000 x g for 10 min, and the pellet was washed twice with the same buffer. In binding assays, membrane suspensions were incubated with [3H] spiroperidol (31 Ci/mmol, NEN) at 37°C for 20 min or with [3H]prazosin (27 Ci/mmol, NEN) at 25°C for 60 min. The reactions were terminated by filtering the incubation medium through Whatman glass filters ( G F / F ) . The filters were washed four times with 2 ml of homogenization medium. The difference between the amounts of [3H]spiroperidol bound to striatal membranes in the presence of ( - ) - and (+)-butaclamol (10 -7 M) was designated as specific binding of [3H]spiroperidol to D-2 dopamine receptors. The difference in the amounts of [3H]prazosin bound to striatal membranes in the presence and absence of phentolamine (10-5 M) was designated as specific binding of [3H]prazosin to al-adrenoceptors.
3. Results
Direct injection of phenoxybenzamine (100-300 vg) into the rat striatum resulted in significant inhibition of apomorphine-induced stereotyped behavior and [3H]spiroperidol specific binding to striatal membrane preparations. These inhibitions
showed similar dose-dependence (fig. 1). Injection of 50 #g of phenoxybenzamine completely blocked [3H]prazosin specific binding but had no effect on either apomorphine-induced stereotyped behavior or [3H]spiroperidol specific binding (fig. 1). The inhibition of [3H]spiroperidol binding by phenoxybenzamine treatment in vivo was not accompanied by any significant change in the K d value of the residual binding; the dissociation constants of control and 200 /~g phenoxybenzamine-treated membrane preparations were 0.10 + 0.02 (n = 6) and 0.11 + 0.03 nM (n = 3), respectively. Furthermore, the inhibition was not affected by extensive washing of the membrane preparation treated with phenoxybenzamine in vivo. As we have reported previously, the effect of injecting haloperidol into the rat striatum, i.e. inhibition of the behavioral response, lasted for several hours and disappeared within 24 h (Fujita et al., 1985). The reductions of both apomorphineinduced behavior and [3H]spiroperidol binding
g
'oj
POB 501Jg
ali 0
IO0.uo
200JJg
30090
Fig. 1. Dose-dependence of inhibition by phenoxybenzamine of apomorphine-induced stereotyped behavior and [3H]spiroperidol binding to rat striatal membrane preparations. Stereotyped behavior was measured every 5 n'fin for 60 min after apomorphine injection (280 v g / k g s.c.). Apom0rphine was injected 24 h after phenoxybenzamine treatment. Binding assays were performed 4 h after apomorphine injection using 0.6 nM [3 H]prazosin as described in Materials and methods. With this concentration of 3H-ligands, approximately 85% of the total binding was specific binding. Columns and bars show means+S.D, in 3-5 experiments. The total score for stereotyped behavior was 50+6 in control rats. The values for binding of 0.6 nM [3H]spiroperidol and 0.8 nM [3H]prazosin were 276+31 and 48+10 f m o l / m g protein, respectively, in control rat striatal membranes.
293
however were nearly maximal 24 h after phenoxybenzamine injection and lasted for several days (fig. 2a). These observations w.ere consistent with those of others (Hamblin and Creese, 1983). [3H]Spiroperidol binding and apomorphine-induced behavior were restored within 5 days after phenoxybenzamine injection and showed similar time courses of recovery (fig. 2a). The recovery of binding sites was accompanied by an increase in the Bmax from 108 + 18 fmol/mg protein at 24 h to 325 + 26 fmol/mg protein at 120 h with no significant alteration in the K d value (0.11 + 0.03, n = 3, and 0.12 + 0.03 nM, n = 3, at 24 h and 120 h after phenoxybenzamine treatment, respectively). To demonstrate that protein synthesis was necessary for the recovery of binding and behavior, we examined the effect of the inhibitor of protein synthesis cycloheximide after phenoxybenzamine injection. The broken line in fig. 2a shows that repeated injections of cycloheximide (30 #g, at 24, 48 and 72 h) inhibited the recovery of [3H]spiroperidol binding. Moreover rats treated with cycloheximide after phenoxybenzamine injection
i
(a)
0
],-!. "0
days after administration
- -
(b)
0
2~l
72 120 time(hr)
Fig. 2. (a) Time courses of the recovery of apomorphine-induced stereotyped behavior and [3 H]spiroperidol specific binding. Apomorphine-induced stereotyped behavior and [3H]spiroperidol specific binding (0.6 nM) were measured at the times indicated after injection of 200 #g of phenoxybenzamine into the striatum. The broken line ( O ) showed the result obtained on injection of 30 #g of cycloheximide at 24, 48 and 72 h after phenoxybenzamine administration. Results are means + S.D. in 3-5 experiments. * Significantly different from value obtained in rats not treated with cycloheximide (P < 0.01). (b) Semilogarithmic plot of D-2 dopamine receptor recovery. (Rss) = steady state receptor concentration, ( R t ) = receptor concentration at time t. The slope of this line is equal to the rate constant for degradation of the D-2 receptor (k).
showed no recovery of behavior (data not shown). Sladeczek and Bockaert (1983) reported the following equations: (Rt) = r / k ( 1 - e -kt)
(1)
In
(2)
(Rss) = kt (Rss) - (Rt)
where (Rt)= receptor concentration at time t, (Rss) = receptor concentration at steady state, k = rate constant for receptor degradation, r = receptor production rate. The degradation rate constant k was calculated from the recovery data (fig. 2b) by eq. (2) to be 0.025/h. Using this value for k in eq (1) the receptor production rate (r; when t is infinite time) was calculated to be 6.9 fmol/mg protein per h. Furthermore, based on the k value, the half-life of the D-2 dopamine receptor was calculated to be 28 h.
4. D i s c u s s i o n
Non-specific effects of high doses of phenoxybenzamine on receptor recovery have been reported (Sladeczek and Bockaert, 1983). But the time course of recovery of the D-2 dopamine receptor after injection of 300 /xg of phenoxybenzamine was essentially the same as that after injection of 200/~g of the drug (data not shown). Thus, the doses of the drug used in this study were not high enough to affect receptor recovery. We previously reported that the D-2 dopamine receptor labelled with [3H]spiroperidol in rat striatum is coupled with the Ni regulatory protein and that the dissociation of the receptor from the Ni regulatory protein resulted in disappearance of the behavioral response of rats to dopamine agonists. In the present study, we have shown that the inhibition by phenoxybenzamine of apomorphine-induced stereotyped behavior and of [3H]spiroperidol specific binding showed similar dose-dependence and time courses. These results indicate that the change in the number of [3H]spiroperidol binding sites corresponds well with the change in the behavioral response caused by the activation of the D-2 dopamine receptor. Furthermore, experiments with cycloheximide
294
showed that de novo synthesis of receptor was involved in the recovery process after phenoxybenzamine treatment. The half-life of D-2 dopamine receptor in the striatum calculatea from our results was 28 h. This value is intermediate between those of 8-9 and 45-119 h reported by Hall et al. (1983) and Left et al. (1984), respectively. The reason for this discrepancy is unknown, but differences in experimental conditions such as the site of injection, amount of drug administered for inactivation of the receptor and age of the animals, may be important factors. Under our conditions at least the D-2 dopamine receptor was synthesized at a rate of 6.9 f m o l / m g protein per h, and had a degradation rate constant of 0.025/h and half-life of 28 h in rat striatum.
Acknowledgements The authors wish to acknowledge the secretarial assistance of Mieko Nakamura. This work was supported by a Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References Fujita, N., M. Nakahiro, I. Fukuchi, K. Saito and H. Yoshida, 1985, Effects of pertussis toxin on D-2 dopamine receptor in rat striatum: Evidence for coupling of Ni regulatory protein with D-2 receptor, Brain Res. 333, 231. Fujita, N., K. Saito, A. Hirata, K. lwatsubo, Y. Nogucbi and H. Yoshida, 1981, Effects of dopamine agonists and antagonists on [3H]apomorphine binding to striatal membranes, Brain Res. 199, 335. Hall, D.M., P. Jenner and D. Marsden, 1983, Turn over rate of specific [3H]spiroperidol and .3H-N,n-propylnorapomorphine binding sites in rat striatum following phenoxybenzamine administration, Biochem. Pharmacol. 32, 2973. Hamblin, M.W. and I. Creese, 1982, Phenoxybenzamine treatment differentiates dopaminergic 3H-ligand-binding sites in bovine caudate membranes, Mol. Pharmacol. 21, 44. Hamblin, M.W. and 1. Creese, 1983, Behavioral and radioligand binding evidence for irreversible dopamine receptor blocking by N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline, Life SCi. 32, 2247. Left, E.S., P. Gariano and 1. Creese, 1984, Dopamine receptor turn over rates in rat striatum are age dependent, Proc. Natl. Acad. Sci. 81, 3910. Marcharis, D. and J. Bockaert, 1980, Is there a connection between high affinity [3H]spiperone binding sites and DAsensitive adenylate cyclase in corpus striatum?, Biochem. Pharmacol. 29, 1331. Seeman, P., 1980, Brain dopamine receptors, Pharmacol. Rev. 32, 229. Sladeczek, F. and J. Bockaert, 1983, Turnover in vivo of a~-adrenergic receptors in rat submaximally glands, Mol. Pharmacol. 23, 282.
291
Elsevier
Short communication
D-2 D O P A M I N E R E C E P T O R S Y N T H E S I S AND TURNOVER IN RAT STRIATUM ISAO FUKUCHI *, NORIHISA FUJITA, MASANOBU NAKAHIRO *, KIHACHI SAITO ** and HIROSHI YOSHIDA
Department of Pharmacology I, Osaka University School of Medicine, 4-3-57 Nakanoshima, Kita-ku, Osaka 530, and ** Department of Pharmacology, Osaka University School of Dentistry, 1-8 Yamadaoka, Suita 564, Japan Received 15 May 1986, accepted 27 May 1986
I. FUKUCHI, N. FUJITA, M. NAKAHIRO, K. SAITO and H. YOSHIDA, D-2 dopamine receptor synthesis and turnover in rat striaturrg European J. Pharmacol. 126 (1986) 291-294.
Direct injection of phenoxybenzamine into rat striatum inhibited apomorphine-induced stereotyped behavior. This inhibition corresponded well with the inhibition of D-2 dopamine receptor labelling with [3H]spiroperidol. Both the behavioral response and the receptor level were completely restored within 5 days after the injection. The recoveries of both were blocked by cycloheximide. The rate of synthesis and half-life of the D-2 receptor associated with the stereotyped behavior were calculated to be 6.9 fmol/mg protein per h and 28 h, respectively. D-2 dopamine receptor
Phenoxybenzamine
Striatum
1. Introduction
Long-term administration of neuroleptics or chemical denervation of the nigrostriatal dopamine pathway with 6-hydroxydopamine is known to result in the development of D-2 dopamine receptor supersensitivity, which is characterized by proliferation of 3H-neuroleptic specific binding sites in rat striatum and an enhanced stereotypy response to dopamine agonists (for review, see Seeman, 1980). Conversely, long-term interception of coupling between the D-2 dopamine receptor and the guanine .nucleotide binding protein reduces [3H]spiroperidol specific binding sites in rat striatum (Fujita et al.~ 1985). These results show that the concentration of D-2 dopamine receptors can be varied experimentally. However, the turnover rate of D-2 receptor is controversial. Hall et al. (1983) reported that the half-life of the D-2 receptor was about 8-9 h after blockade with the alkylating reagent phenoxybenzamine, which is an * Present address: Safety Research Laboratory, Tanabe Seiyaku Co. Ltd., 3-16-89 Kashima, Yodogawa-ku, Osaka 532, Japan. * To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Rat
irreversible blocker of the D-2 receptor as well as an al-adrenergic blocker (Marcharis and Bockaert, 1980; Hamblin and Creese, 1982). On the other hand, Left et al. (1984) reported that the half-life of the D-2 receptor was 45-119 h after irreversible blockade with the alkylating reagent N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (EEDQ). In these studies, phenoxybenzamine and EEDQ were administered intraperitoneaUy (i.p.). We have been studying the dopamine receptor in the central nervous system by direct injection of drugs into the brain (Fujita et al., 1981; Fujita et al., 1985). This method is very useful for studying brain function pharmacologically and behaviorally without affecting the peripheral system. In the work described here, by direct injection of phenoxybenzamine into rat striatum through chronically implanted guide cannulas we obtained a value for the D-2 dopamine receptor turnover rate that corresponded well with the behavioral response to the dopamine agonist. 2. Materials and methods
Male Sprague-Dawley rats weighing 280-300 g were anesthetized with nembutal (35 m g / k g i.p.)
292
and placed in a David Kopf stereotaxic holder. Guide cannulas constructed from 23 gauge stainless-steel tubing were implanted into the striatum according to the brain atlas of Pellegrino and Cushman (2.2 mm anterior to the bregma, 2.8 mm bilateral to the sagittal suture, 5.2 mm ventral to the skull). The cannulas were fastened to the skull with dental cement and acrylic resin. Five days later, drugs were injected bilaterally into the striaturn in a volume of 1-3 #1 and at a rate of 1 /~l/min through the injection cannulas. Phenoxybenzamine and cycloheximide (Nakarai, Japan) were dissolved in saline at final concentrations of 25-75 #g//~l and 10 # g / # l , respectively. Apomorphine HC1 (Sigma) was dissolved in saline and was injected subcutaneously (s.c.) at a dose of 280 #g/kg. Stereotyped behaviours were rated as described previously (Fujita et al., 1985). Striata were homogenized in 25 volumes of 25 mM Tris-HCl buffer (pH 7.3) containing 5 mM EDTA Na 2. The homogenate was centrifuged at 100 000 x g for 10 min, and the pellet was washed twice with the same buffer. In binding assays, membrane suspensions were incubated with [3H] spiroperidol (31 Ci/mmol, NEN) at 37°C for 20 min or with [3H]prazosin (27 Ci/mmol, NEN) at 25°C for 60 min. The reactions were terminated by filtering the incubation medium through Whatman glass filters ( G F / F ) . The filters were washed four times with 2 ml of homogenization medium. The difference between the amounts of [3H]spiroperidol bound to striatal membranes in the presence of ( - ) - and (+)-butaclamol (10 -7 M) was designated as specific binding of [3H]spiroperidol to D-2 dopamine receptors. The difference in the amounts of [3H]prazosin bound to striatal membranes in the presence and absence of phentolamine (10-5 M) was designated as specific binding of [3H]prazosin to al-adrenoceptors.
3. Results
Direct injection of phenoxybenzamine (100-300 vg) into the rat striatum resulted in significant inhibition of apomorphine-induced stereotyped behavior and [3H]spiroperidol specific binding to striatal membrane preparations. These inhibitions
showed similar dose-dependence (fig. 1). Injection of 50 #g of phenoxybenzamine completely blocked [3H]prazosin specific binding but had no effect on either apomorphine-induced stereotyped behavior or [3H]spiroperidol specific binding (fig. 1). The inhibition of [3H]spiroperidol binding by phenoxybenzamine treatment in vivo was not accompanied by any significant change in the K d value of the residual binding; the dissociation constants of control and 200 /~g phenoxybenzamine-treated membrane preparations were 0.10 + 0.02 (n = 6) and 0.11 + 0.03 nM (n = 3), respectively. Furthermore, the inhibition was not affected by extensive washing of the membrane preparation treated with phenoxybenzamine in vivo. As we have reported previously, the effect of injecting haloperidol into the rat striatum, i.e. inhibition of the behavioral response, lasted for several hours and disappeared within 24 h (Fujita et al., 1985). The reductions of both apomorphineinduced behavior and [3H]spiroperidol binding
g
'oj
POB 501Jg
ali 0
IO0.uo
200JJg
30090
Fig. 1. Dose-dependence of inhibition by phenoxybenzamine of apomorphine-induced stereotyped behavior and [3H]spiroperidol binding to rat striatal membrane preparations. Stereotyped behavior was measured every 5 n'fin for 60 min after apomorphine injection (280 v g / k g s.c.). Apom0rphine was injected 24 h after phenoxybenzamine treatment. Binding assays were performed 4 h after apomorphine injection using 0.6 nM [3 H]prazosin as described in Materials and methods. With this concentration of 3H-ligands, approximately 85% of the total binding was specific binding. Columns and bars show means+S.D, in 3-5 experiments. The total score for stereotyped behavior was 50+6 in control rats. The values for binding of 0.6 nM [3H]spiroperidol and 0.8 nM [3H]prazosin were 276+31 and 48+10 f m o l / m g protein, respectively, in control rat striatal membranes.
293
however were nearly maximal 24 h after phenoxybenzamine injection and lasted for several days (fig. 2a). These observations w.ere consistent with those of others (Hamblin and Creese, 1983). [3H]Spiroperidol binding and apomorphine-induced behavior were restored within 5 days after phenoxybenzamine injection and showed similar time courses of recovery (fig. 2a). The recovery of binding sites was accompanied by an increase in the Bmax from 108 + 18 fmol/mg protein at 24 h to 325 + 26 fmol/mg protein at 120 h with no significant alteration in the K d value (0.11 + 0.03, n = 3, and 0.12 + 0.03 nM, n = 3, at 24 h and 120 h after phenoxybenzamine treatment, respectively). To demonstrate that protein synthesis was necessary for the recovery of binding and behavior, we examined the effect of the inhibitor of protein synthesis cycloheximide after phenoxybenzamine injection. The broken line in fig. 2a shows that repeated injections of cycloheximide (30 #g, at 24, 48 and 72 h) inhibited the recovery of [3H]spiroperidol binding. Moreover rats treated with cycloheximide after phenoxybenzamine injection
i
(a)
0
],-!. "0
days after administration
- -
(b)
0
2~l
72 120 time(hr)
Fig. 2. (a) Time courses of the recovery of apomorphine-induced stereotyped behavior and [3 H]spiroperidol specific binding. Apomorphine-induced stereotyped behavior and [3H]spiroperidol specific binding (0.6 nM) were measured at the times indicated after injection of 200 #g of phenoxybenzamine into the striatum. The broken line ( O ) showed the result obtained on injection of 30 #g of cycloheximide at 24, 48 and 72 h after phenoxybenzamine administration. Results are means + S.D. in 3-5 experiments. * Significantly different from value obtained in rats not treated with cycloheximide (P < 0.01). (b) Semilogarithmic plot of D-2 dopamine receptor recovery. (Rss) = steady state receptor concentration, ( R t ) = receptor concentration at time t. The slope of this line is equal to the rate constant for degradation of the D-2 receptor (k).
showed no recovery of behavior (data not shown). Sladeczek and Bockaert (1983) reported the following equations: (Rt) = r / k ( 1 - e -kt)
(1)
In
(2)
(Rss) = kt (Rss) - (Rt)
where (Rt)= receptor concentration at time t, (Rss) = receptor concentration at steady state, k = rate constant for receptor degradation, r = receptor production rate. The degradation rate constant k was calculated from the recovery data (fig. 2b) by eq. (2) to be 0.025/h. Using this value for k in eq (1) the receptor production rate (r; when t is infinite time) was calculated to be 6.9 fmol/mg protein per h. Furthermore, based on the k value, the half-life of the D-2 dopamine receptor was calculated to be 28 h.
4. D i s c u s s i o n
Non-specific effects of high doses of phenoxybenzamine on receptor recovery have been reported (Sladeczek and Bockaert, 1983). But the time course of recovery of the D-2 dopamine receptor after injection of 300 /xg of phenoxybenzamine was essentially the same as that after injection of 200/~g of the drug (data not shown). Thus, the doses of the drug used in this study were not high enough to affect receptor recovery. We previously reported that the D-2 dopamine receptor labelled with [3H]spiroperidol in rat striatum is coupled with the Ni regulatory protein and that the dissociation of the receptor from the Ni regulatory protein resulted in disappearance of the behavioral response of rats to dopamine agonists. In the present study, we have shown that the inhibition by phenoxybenzamine of apomorphine-induced stereotyped behavior and of [3H]spiroperidol specific binding showed similar dose-dependence and time courses. These results indicate that the change in the number of [3H]spiroperidol binding sites corresponds well with the change in the behavioral response caused by the activation of the D-2 dopamine receptor. Furthermore, experiments with cycloheximide
294
showed that de novo synthesis of receptor was involved in the recovery process after phenoxybenzamine treatment. The half-life of D-2 dopamine receptor in the striatum calculatea from our results was 28 h. This value is intermediate between those of 8-9 and 45-119 h reported by Hall et al. (1983) and Left et al. (1984), respectively. The reason for this discrepancy is unknown, but differences in experimental conditions such as the site of injection, amount of drug administered for inactivation of the receptor and age of the animals, may be important factors. Under our conditions at least the D-2 dopamine receptor was synthesized at a rate of 6.9 f m o l / m g protein per h, and had a degradation rate constant of 0.025/h and half-life of 28 h in rat striatum.
Acknowledgements The authors wish to acknowledge the secretarial assistance of Mieko Nakamura. This work was supported by a Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References Fujita, N., M. Nakahiro, I. Fukuchi, K. Saito and H. Yoshida, 1985, Effects of pertussis toxin on D-2 dopamine receptor in rat striatum: Evidence for coupling of Ni regulatory protein with D-2 receptor, Brain Res. 333, 231. Fujita, N., K. Saito, A. Hirata, K. lwatsubo, Y. Nogucbi and H. Yoshida, 1981, Effects of dopamine agonists and antagonists on [3H]apomorphine binding to striatal membranes, Brain Res. 199, 335. Hall, D.M., P. Jenner and D. Marsden, 1983, Turn over rate of specific [3H]spiroperidol and .3H-N,n-propylnorapomorphine binding sites in rat striatum following phenoxybenzamine administration, Biochem. Pharmacol. 32, 2973. Hamblin, M.W. and I. Creese, 1982, Phenoxybenzamine treatment differentiates dopaminergic 3H-ligand-binding sites in bovine caudate membranes, Mol. Pharmacol. 21, 44. Hamblin, M.W. and 1. Creese, 1983, Behavioral and radioligand binding evidence for irreversible dopamine receptor blocking by N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline, Life SCi. 32, 2247. Left, E.S., P. Gariano and 1. Creese, 1984, Dopamine receptor turn over rates in rat striatum are age dependent, Proc. Natl. Acad. Sci. 81, 3910. Marcharis, D. and J. Bockaert, 1980, Is there a connection between high affinity [3H]spiperone binding sites and DAsensitive adenylate cyclase in corpus striatum?, Biochem. Pharmacol. 29, 1331. Seeman, P., 1980, Brain dopamine receptors, Pharmacol. Rev. 32, 229. Sladeczek, F. and J. Bockaert, 1983, Turnover in vivo of a~-adrenergic receptors in rat submaximally glands, Mol. Pharmacol. 23, 282.