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Changes in the D1 receptor-adenylate cyclase complex after priming
European Journal of Pharmacology, 180 (1990) 365-367
365
Elsevier EJP 20624 Short communication
Changes in the D~ receptor-adenylate cyclase complex after priming M i c a e l a Morelli, Graziella D e M o n t i s 1 a n d G a e t a n o Di C h i a r a Institute of Experimental Pharmacology and Toxicology, 1 Institute of Pharmacology and Biochemical Phatology, University of Cagliari, Cagliari, Italy
Received 13 March 1990, accepted 27 March 1990
The D 1 agonist, SKF 38393, failed to induce contralateral turning in drug-naive rats lesioned unilaterally with 6-hydroxydopamine from 17 days. Priming with a dopamine agonist, such as the D 2 agonist, LY 171555, three days before, made SKF 38393 fully effective in inducing contralateral turning. Analysis of D 1 receptor binding in striata of drug-naive and primed rats showed no change in the Bm,x and K d. In contrast, dopamine-stimulated adenylate cyclase showed a decrease in its K m for dopamine in the lesioned side of primed rats as compared with drug-naive rats. Thus, priming appears to elicit changes at the level of the transduction mechanism of D~ receptors rather than in the D 1 recognition site itself. Dopamine; Dopamine D 1 receptors; Adenylate-cyclase; 6-Hydroxydopamine (6-OHDA)
1. Introduction In the unilateral 6-hydroxydopamine (6-OHDA) model of denervation, administration of a dopamine (DA) agonist is known to elicit turning behavior contralateral to the lesioned side (Ungerstedt, 1971). We have shown recently that 17 days after 6 - O H D A lesion, the D 1 agonist, SKF 38393, is unable to induce contralateral turning in drug-naive rats (Morelli and Di Chiara, 1987; Morelli et al., 1989). However, administration of a D A agonist 3 days earlier makes the otherwise ineffective dose of SKF 38393 capable of inducing intense contralateral turning (priming), (Morelli and Di Chiara, 1987; Morelli et al., 1989). While these studies indicate that priming induces important adaptive changes, the biochemical mechanism underlying this phenomenon has not been
Correspondence to: M. Morelli, Institute of Experimental Pharmacology and Toxicology,Viale A. Diaz 182, 09100 Cagliari, Italy.
explored yet. The present study investigates the possibility that priming induces changes in DAstimulated adenylate cyclase and in D 1 receptor recognition sites.
2. Materials and methods Male Sprague-Dawley rats weighing 275-300 g were used in all the experiments. In order to lesion the DA nigro-striatal neurons the rats were injected unilaterally with 6 - O H D A (8/~g in 4 ml) in the left medial forebrain bundle (MFB) at coordinates A - 2 . 2 , L 1.5, V 7.9, according to the atlas Pellegrino et al. (1979). Spontaneous rotational behavior was tested for 5 days after the lesion; only rats showing tight ipsilateral posture and turning towards the side of the lesion were used for the experiments. Rats were divided in two groups 2 weeks after lesioning, and were placed in rotameters and injected with vehicle (controls) or with 0.2 m g / k g s.c. of the D E agonist, LY 171555 (primed). Three
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
366
days later, at a time when the behavioral response to the D 1 agonist is maximal (Morelli et al., 1989), the rats were killed and the striata of both sides were dissected out and used for the biochemical experiments. The binding of the D1 antagonist, [3H]SCH 23390, was performed in striatal homogenates according to the method of Billard et al. (1984). For autoradiography, coronal brain sections of 20 ,,tm were mounted onto glass slides, and the binding assay was performed according to the method of Dawson et al. (1985), using a concentration of 1 nM of [3H]SCH 23390 (Amersham). Optical densities of the autoradiograms were determined with an image analyzer system (ASBA Wild-Leitz). A concentration of 50 nM mianserine was added to the incubation buffer to prevent binding of SCH 23390 to 5-HT receptors (Billard et al., 1984). Non-specific binding was determined in the presence of 1/xM SCH 23390. DA-stimulated adenylate cyclase activity was assayed in membranes prepared from striata by a modification (Olianas et al., 1983) of the method of Solomon et al. (1974) in the presence of 1 ~M ( - ) - s u l p i r i d e to block D 2 receptors. Results are expressed as pmol of cAMP formed per mg of protein per min. The concentration of DA required to induce a half-maximal activation ( K m ) and the maximal increment obtained by stimulation (Vmax) were calculated by linear regression analysis of Eadie-Holfstee plots obtained from concentration-response curves.
Estimation of the DA content in the intact and lesioned striata was performed according to the method of Mefford (1981). Student's t-test was applied to evaluate the significance of the results obtained.
3. Results
Table 1 shows the Brnax and the K d of the specific [3H]SCH 23390 binding in homogenates of striata from the intact or 6-OHDA-injected side. No significant differences were found between the striata of the two sides and between the drug-naive and primed group of rats. Similarly, the Wmax of DA-stimulated adenylate cyclase did not show any difference between the intact and lesioned side and between drug-naive and primed rats, whereas the K m value was lower ( - 4 1 % ) in the lesioned striatum of primed rats than in drugnaive rats (table 1). The basal activity of the enzyme was not modified in the two experimental groups. Figure 1 shows a coronal section of [3H]SCH 23390-labelled striata from drug-naive (upper panel) and primed rats (lower panel). The amount of label bound, in the f m o l / m g of tissue equivalent, was 38.9 + 1.5 (intact), 39.6 + 1.2 (lesioned) and 4 1 . 6 _ 1.8 (intact), 40.1 + 1.9 (lesioned) for the two groups, respectively, thus confirming that the binding of label to D 1 receptors was not modified in the two experimental groups. DA
TABLE 1 [3H]SCH 23390 binding and adenylate cyclase activity in striata of drug-naive and primed rats. Rats were lesioned with 6-OHDA in the left MFB and injected on day 14 with vehicle s.c. (drug-naive), or with 0.2 m g / k g s.c. of LY 171555 (primed) and then killed 3 days later, cAMP formation is expressed as p m o l / m g protein per rain. The values are in units of ~M. Results are the m e a n s _ S.E.M. of at least five experiments in which there were two rats per group. L = left, R = right [3H]SCH 23390 binding
cAMP
Vmax
Km
0.79+0.07 0.8 +0.07
112+12 102+10
4.51+0.41 4.97+0.5
0.82+0.09 0.8 +0.08
111±11 110+10
2.66+0.3 a 4.85+0.42
Bmax
Kd
( f m o l / m g protein)
(nM)
Drug-naive
L R
1198+89 1110+90
Primed
L R
1150+99 1080+79
P < 0.01.
367
late cyclase for DA in the lesioned striatum, suggesting that the behavioral expression of D 1 receptor supersensitivity is associated with an alteration in the D 1 receptor transduction mechanism, rather than with a modification of the recognition site. These data can explain why priming increases the potency of SKF 38393 in eliciting contralateral turning in 6-OHDA-lesioned rats. In conclusion, stimulation of D 2 receptors elicits an adaptive change in the D1 receptor transduction mechanism. Since this modification corresponds to a potentiation of the behavioral expression of D~ receptor stimulation, it could explain the changes in sensitivity to L - D O P A that occur in Parkinsonian patients during treatment.
References
Fig. 1. Bright-field photomicrograph at striatal level of [3H]SCH 23390 binding after 6-OHDA-induced degeneration of MFB (right side). Sections were incubated with 1 nM [3H]SCH 23390. Upper panel, drug-naive rat; lower panel, primed rat.
levels in the striata ranged from 6850 to 7310 (intact) and from 305 to 348 (lesioned) n g / g wet weight.
4. Discussion
The results of this study show that the affinity (Bmax) of D~ receptor sites did not differ between drug-naive and primed rats. In contrast, priming increased the affinity of adeny( K d ) and number
Billard, W., V. Ruperto, G. Crosby, L.C. Iorio and A. Barnett, 1984, Characterization of the binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum, Life Sci. 35, 1885. Dawson, I.M., D.R. Gehlert, H.I. Yamamura, A. Barnett and J.K. Wamsley, 1985, D-1 dopamine receptors in the rat brain: autoradiographic localization using [3H]SCH 23390, European J. Pharmacol. 108, 323. Mefford, I.N., 1981, Application of high performance liquid chromatography with electrochemical detection to neurochemical analysis: measurement of catecholamine, serotonin and metabolites in rat brain, J. Neurosci. Meth. 3, 207. Morelli, M. and G. Di Chiara, 1987, Agonist-induced homologous and heterologous sensitization to D-1 and D-2 dependent contraversive turning, European J. Pharmacol. 141, 101. Morelli, M., S. Fenu, L. Garau and G. Di Chiara, 1989, Time and dose dependence of the 'priming' of the expression of dopamine receptor supersensitivity, European J. Pharmacol. 162, 329. Olianas, M.C., P. Onali, N.H. Neff and E. Costa, 1983, Adenylate cyclase activity of synaptic membranes from rat striatum, Mol. Pharmacol. 23, 393. Pellegrino, L.J., A.S. Pellegrino and A.J. Cushman, 1979, Stereotaxic Atlas of the Rat Brain (Plenum Press, New York). Solomon, Y., C. Londos and M. Rodbell, 1974, A highly sensitive adenylate cyclase assay, Anal. Biochem. 58, 541. Ungerstedt, U., 1971, Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system, Acta Physiol. Scand. (suppl.) 367, 69.
365
Elsevier EJP 20624 Short communication
Changes in the D~ receptor-adenylate cyclase complex after priming M i c a e l a Morelli, Graziella D e M o n t i s 1 a n d G a e t a n o Di C h i a r a Institute of Experimental Pharmacology and Toxicology, 1 Institute of Pharmacology and Biochemical Phatology, University of Cagliari, Cagliari, Italy
Received 13 March 1990, accepted 27 March 1990
The D 1 agonist, SKF 38393, failed to induce contralateral turning in drug-naive rats lesioned unilaterally with 6-hydroxydopamine from 17 days. Priming with a dopamine agonist, such as the D 2 agonist, LY 171555, three days before, made SKF 38393 fully effective in inducing contralateral turning. Analysis of D 1 receptor binding in striata of drug-naive and primed rats showed no change in the Bm,x and K d. In contrast, dopamine-stimulated adenylate cyclase showed a decrease in its K m for dopamine in the lesioned side of primed rats as compared with drug-naive rats. Thus, priming appears to elicit changes at the level of the transduction mechanism of D~ receptors rather than in the D 1 recognition site itself. Dopamine; Dopamine D 1 receptors; Adenylate-cyclase; 6-Hydroxydopamine (6-OHDA)
1. Introduction In the unilateral 6-hydroxydopamine (6-OHDA) model of denervation, administration of a dopamine (DA) agonist is known to elicit turning behavior contralateral to the lesioned side (Ungerstedt, 1971). We have shown recently that 17 days after 6 - O H D A lesion, the D 1 agonist, SKF 38393, is unable to induce contralateral turning in drug-naive rats (Morelli and Di Chiara, 1987; Morelli et al., 1989). However, administration of a D A agonist 3 days earlier makes the otherwise ineffective dose of SKF 38393 capable of inducing intense contralateral turning (priming), (Morelli and Di Chiara, 1987; Morelli et al., 1989). While these studies indicate that priming induces important adaptive changes, the biochemical mechanism underlying this phenomenon has not been
Correspondence to: M. Morelli, Institute of Experimental Pharmacology and Toxicology,Viale A. Diaz 182, 09100 Cagliari, Italy.
explored yet. The present study investigates the possibility that priming induces changes in DAstimulated adenylate cyclase and in D 1 receptor recognition sites.
2. Materials and methods Male Sprague-Dawley rats weighing 275-300 g were used in all the experiments. In order to lesion the DA nigro-striatal neurons the rats were injected unilaterally with 6 - O H D A (8/~g in 4 ml) in the left medial forebrain bundle (MFB) at coordinates A - 2 . 2 , L 1.5, V 7.9, according to the atlas Pellegrino et al. (1979). Spontaneous rotational behavior was tested for 5 days after the lesion; only rats showing tight ipsilateral posture and turning towards the side of the lesion were used for the experiments. Rats were divided in two groups 2 weeks after lesioning, and were placed in rotameters and injected with vehicle (controls) or with 0.2 m g / k g s.c. of the D E agonist, LY 171555 (primed). Three
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
366
days later, at a time when the behavioral response to the D 1 agonist is maximal (Morelli et al., 1989), the rats were killed and the striata of both sides were dissected out and used for the biochemical experiments. The binding of the D1 antagonist, [3H]SCH 23390, was performed in striatal homogenates according to the method of Billard et al. (1984). For autoradiography, coronal brain sections of 20 ,,tm were mounted onto glass slides, and the binding assay was performed according to the method of Dawson et al. (1985), using a concentration of 1 nM of [3H]SCH 23390 (Amersham). Optical densities of the autoradiograms were determined with an image analyzer system (ASBA Wild-Leitz). A concentration of 50 nM mianserine was added to the incubation buffer to prevent binding of SCH 23390 to 5-HT receptors (Billard et al., 1984). Non-specific binding was determined in the presence of 1/xM SCH 23390. DA-stimulated adenylate cyclase activity was assayed in membranes prepared from striata by a modification (Olianas et al., 1983) of the method of Solomon et al. (1974) in the presence of 1 ~M ( - ) - s u l p i r i d e to block D 2 receptors. Results are expressed as pmol of cAMP formed per mg of protein per min. The concentration of DA required to induce a half-maximal activation ( K m ) and the maximal increment obtained by stimulation (Vmax) were calculated by linear regression analysis of Eadie-Holfstee plots obtained from concentration-response curves.
Estimation of the DA content in the intact and lesioned striata was performed according to the method of Mefford (1981). Student's t-test was applied to evaluate the significance of the results obtained.
3. Results
Table 1 shows the Brnax and the K d of the specific [3H]SCH 23390 binding in homogenates of striata from the intact or 6-OHDA-injected side. No significant differences were found between the striata of the two sides and between the drug-naive and primed group of rats. Similarly, the Wmax of DA-stimulated adenylate cyclase did not show any difference between the intact and lesioned side and between drug-naive and primed rats, whereas the K m value was lower ( - 4 1 % ) in the lesioned striatum of primed rats than in drugnaive rats (table 1). The basal activity of the enzyme was not modified in the two experimental groups. Figure 1 shows a coronal section of [3H]SCH 23390-labelled striata from drug-naive (upper panel) and primed rats (lower panel). The amount of label bound, in the f m o l / m g of tissue equivalent, was 38.9 + 1.5 (intact), 39.6 + 1.2 (lesioned) and 4 1 . 6 _ 1.8 (intact), 40.1 + 1.9 (lesioned) for the two groups, respectively, thus confirming that the binding of label to D 1 receptors was not modified in the two experimental groups. DA
TABLE 1 [3H]SCH 23390 binding and adenylate cyclase activity in striata of drug-naive and primed rats. Rats were lesioned with 6-OHDA in the left MFB and injected on day 14 with vehicle s.c. (drug-naive), or with 0.2 m g / k g s.c. of LY 171555 (primed) and then killed 3 days later, cAMP formation is expressed as p m o l / m g protein per rain. The values are in units of ~M. Results are the m e a n s _ S.E.M. of at least five experiments in which there were two rats per group. L = left, R = right [3H]SCH 23390 binding
cAMP
Vmax
Km
0.79+0.07 0.8 +0.07
112+12 102+10
4.51+0.41 4.97+0.5
0.82+0.09 0.8 +0.08
111±11 110+10
2.66+0.3 a 4.85+0.42
Bmax
Kd
( f m o l / m g protein)
(nM)
Drug-naive
L R
1198+89 1110+90
Primed
L R
1150+99 1080+79
P < 0.01.
367
late cyclase for DA in the lesioned striatum, suggesting that the behavioral expression of D 1 receptor supersensitivity is associated with an alteration in the D 1 receptor transduction mechanism, rather than with a modification of the recognition site. These data can explain why priming increases the potency of SKF 38393 in eliciting contralateral turning in 6-OHDA-lesioned rats. In conclusion, stimulation of D 2 receptors elicits an adaptive change in the D1 receptor transduction mechanism. Since this modification corresponds to a potentiation of the behavioral expression of D~ receptor stimulation, it could explain the changes in sensitivity to L - D O P A that occur in Parkinsonian patients during treatment.
References
Fig. 1. Bright-field photomicrograph at striatal level of [3H]SCH 23390 binding after 6-OHDA-induced degeneration of MFB (right side). Sections were incubated with 1 nM [3H]SCH 23390. Upper panel, drug-naive rat; lower panel, primed rat.
levels in the striata ranged from 6850 to 7310 (intact) and from 305 to 348 (lesioned) n g / g wet weight.
4. Discussion
The results of this study show that the affinity (Bmax) of D~ receptor sites did not differ between drug-naive and primed rats. In contrast, priming increased the affinity of adeny( K d ) and number
Billard, W., V. Ruperto, G. Crosby, L.C. Iorio and A. Barnett, 1984, Characterization of the binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum, Life Sci. 35, 1885. Dawson, I.M., D.R. Gehlert, H.I. Yamamura, A. Barnett and J.K. Wamsley, 1985, D-1 dopamine receptors in the rat brain: autoradiographic localization using [3H]SCH 23390, European J. Pharmacol. 108, 323. Mefford, I.N., 1981, Application of high performance liquid chromatography with electrochemical detection to neurochemical analysis: measurement of catecholamine, serotonin and metabolites in rat brain, J. Neurosci. Meth. 3, 207. Morelli, M. and G. Di Chiara, 1987, Agonist-induced homologous and heterologous sensitization to D-1 and D-2 dependent contraversive turning, European J. Pharmacol. 141, 101. Morelli, M., S. Fenu, L. Garau and G. Di Chiara, 1989, Time and dose dependence of the 'priming' of the expression of dopamine receptor supersensitivity, European J. Pharmacol. 162, 329. Olianas, M.C., P. Onali, N.H. Neff and E. Costa, 1983, Adenylate cyclase activity of synaptic membranes from rat striatum, Mol. Pharmacol. 23, 393. Pellegrino, L.J., A.S. Pellegrino and A.J. Cushman, 1979, Stereotaxic Atlas of the Rat Brain (Plenum Press, New York). Solomon, Y., C. Londos and M. Rodbell, 1974, A highly sensitive adenylate cyclase assay, Anal. Biochem. 58, 541. Ungerstedt, U., 1971, Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system, Acta Physiol. Scand. (suppl.) 367, 69.