European Journal of Pharmacology, 55 (1979) 121--128 © Elsevier/North-Holland Biomedical Press
121
EFFECT OF MORPHINE ON THE ACCUMULATION OF DOPA AFTER DECARBOXYLASE INHIBITION IN THE RAT SVEN-AKE PERSSON
Department of Pharmacology, University of UmeJ, S-901 87 Ume~, Sweden Received 7 July 1978, revised MS received 18 October 1978, accepted 9 January 1979
S.-~. PERSSON, Effect of morphine on the accumulation of DOPA after decarboxylase inhibition in the rat, European J. Pharmacol. 55 (1979) 121--128. Acute systemic administration of morphine (10 mg/kg s.c.) to rats increased in vivo tyrosine hydroxylation in the striatum measured as the accumulation of DOPA after decarboxylase inhibition. DOPA accumulation reached a maximum 30--60 rain after morphine. The morphine antagonist naloxone (1, 10 or 100 mg/kg s.c.) did n o t significantly alter DOPA accumulation. However, naloxone completely antagonized the effect of morphine. The DA agonist apomorphine decreased and the DA antagonist haloperidol increased DOPA accumulation. The effect of apomorphine (0.05 mg/kg) was counteracted by morphine. Naloxone did not significantly change the accumulation of DOPA after apomorphine or after haloperidol. In rats treated with 7-butyrolactone (GBL) or with reserpine DOPA accumulation was not altered by treatment with morphine or naloxone. However, the inhibiting effect of apomorphine (0.5 mg/kg) on the accumulation of DOPA in rats treated with reserpine was weakly counteracted by morphine (10 mg/kg s.c.). Since the effects of morphine on the apomorphine-induced inhibition of DOPA accumulation were antagonized by naloxone, we suggest that the effects on striatal DOPA accumulation produced by morphine were mediated via opioid receptors and not directly via DA receptors. Dopamine receptors Opioid receptors
DOPA accumulation Haloperidol
1. Introduction The acute systemic administration of morphine increases the synthesis and turnover of dopamine (DA) in the striatum and produces muscular rigidity and catalepsy in rats (Clouet and Iwatsubo, 1975; Lal, 1975; Kuschinsky, 1976). Morphine has also been shown to block several effects produced by the DA agonist apomorphine (Lal et al., 1975). Systemic administration of morphine (Iwatsubo and Clouet, 1977; Nowycky et al., 1978) and local injection of morphine into the caudate nucleus increased the rate of spontaneous firing of DA neurons in the substantia nigra. These findings suggest that morphine, like haloperidol and other neuroleptics, blocks DA receptors. However, data have also been presented against DA receptor blockade after
Morphine
Naloxone
Striatum
morphine. Chlorpromazine but not morphine was found to inhibit the stereotyped behaviour induced by the DA agonist apomorphine (McKenzie and Sadof, 1974). Furthermore haloperidol but not morphine was found to inhibit the DA-induced stimulation of DA-sensitive adenylate cyclase (Iwatsubo and Clouet, 1975). In addition DA agonists were found to reverse the morphine-induced increase in the rate of spontaneous firing in DA neurons of the substantia nigra while the agonists were only partially successful in reversing the effect of haloperidol. The morphine antagonist naloxone blocked actions of morphine on firing rates, but not those of haloperidol (Iwatsubo and Clouet, 1977). Naloxone has also been shown to antagonize biochemical and behavioural effects of morphine (Lal, 1975; Kuschinsky, 1976). These findings suggest
122 that the effects of morphine on the nigroneostriatal pathway are mediated via receptors sensitive for morphine i.e. opioid receptors and not directly via DA receptors. However, the exact mechanism behind the effect of morphine on central dopaminergic pathways is still u n k n o w n . In the present study we report on the effects of a single dose of morphine on in vivo tyrosine h y d r o x y l a t i o n measured as the accumulation of DOPA after inhibition of neuronal decarboxylase. The effects of morphine and naloxone after DA receptor stimulation and DA receptor blockade were studied. Furthermore we have examined the effects of morphine and naloxone on DOPA accumulation after treatm e n t with ~/-butyrolactone (GBL) and after depletion of the granular DA stores by reserpine.
2. Materials and m e t h o d s
2.1. Animals Male Sprague-Dawley rats (Anticimex, Sollentuna, Sweden) weighing 200--299 g were used in all experiments. The rats were fed the Ewos-Anticimex commercial type pelleted diet, R3 and had access to water ad libitum. During the experiments t h e y were allowed free access only to water.
S.-A. PERSSON or used directly as the injectable form supplied by Endo laboratories. Reserpine was used as the injectable form supplied by CIBA. All other substances were dissolved in 0.9% NaC1. The drugs were administered s.c. or i.p. as described in the legends to the figures and tables.
2. 3. Experimental The experiments were performed between 9 am and 11 am. In some experiments drugs were injected at 6.30 am. The room temperature was 22--26°C. The b o d y temperature was checked by means of a telethermometer with the probe inserted 5 cm from the anal orifice. The lowest temperature observed was 36.0°C. In vivo tyrosine h y d r o x y l a t i o n was measured as the accumulation of DOPA after inhibition of the neuronal decarboxylase. The m e t h o d used was originally described by Carlsson et al. (1972). Dissection of the striata, isolation of DOPA from striatal tissue and the fluorimetric determination of DOPA are described in detail elsewhere (Persson, 1977). In the present study the recovery of 500 ng DOPA taken through the entire procedure was 77.4 + 0.9% (mean + S.E.M., n = 56). All values were corrected for recovery. The statistical significance of the results was assessed using the two-tailed Student's t-test. A P-value of less than 0.05 was considered significant.
2.2. Drugs The following drugs were used: apomorphine HC1 (Sandoz, Basle); gamma-butyrolactone GBL (Sigma, St. Louis, Mo.); haloperidol, Haldol® (Leo, Helsingborg); morphine HC1 (ACO, Solna); naloxone HC1, Nalone® (Endo, N.Y.); reserpine, Serpasil® (Ciba, Basle). 3-Hydroxybenzylhydrazine HC1, NSD 1015 was kindly synthesized by Dr. B. Magnusson, Department of Organic Chemistry, University of Ume~, Sweden. All doses refer to the salts. Haloperidol was dissolved in 5.5% glucose after acidification with 20 pl glacial acetic acid. Naloxone was either dissolved in saline
3. Results
3.1. Time- and dose-related changes in DOPA accumulation after a single dose of morphine The administration of morphine 10 mg/kg s.c. produced an initial decrease in the rate of DOPA accumulation in the striatum (fig. 1). However, 15 min after the administration of morphine the rate of DOPA accumulation was increased. DOPA accumulation reached a m a x i m u m 30 and 60 min after morphine, but
MORPHINE
AND
STRIATAL
200C 2 1000
ACCUMULATION
123
2000
121
EFFECT OF MORPHINE ON THE ACCUMULATION OF DOPA AFTER DECARBOXYLASE INHIBITION IN THE RAT SVEN-AKE PERSSON
Department of Pharmacology, University of UmeJ, S-901 87 Ume~, Sweden Received 7 July 1978, revised MS received 18 October 1978, accepted 9 January 1979
S.-~. PERSSON, Effect of morphine on the accumulation of DOPA after decarboxylase inhibition in the rat, European J. Pharmacol. 55 (1979) 121--128. Acute systemic administration of morphine (10 mg/kg s.c.) to rats increased in vivo tyrosine hydroxylation in the striatum measured as the accumulation of DOPA after decarboxylase inhibition. DOPA accumulation reached a maximum 30--60 rain after morphine. The morphine antagonist naloxone (1, 10 or 100 mg/kg s.c.) did n o t significantly alter DOPA accumulation. However, naloxone completely antagonized the effect of morphine. The DA agonist apomorphine decreased and the DA antagonist haloperidol increased DOPA accumulation. The effect of apomorphine (0.05 mg/kg) was counteracted by morphine. Naloxone did not significantly change the accumulation of DOPA after apomorphine or after haloperidol. In rats treated with 7-butyrolactone (GBL) or with reserpine DOPA accumulation was not altered by treatment with morphine or naloxone. However, the inhibiting effect of apomorphine (0.5 mg/kg) on the accumulation of DOPA in rats treated with reserpine was weakly counteracted by morphine (10 mg/kg s.c.). Since the effects of morphine on the apomorphine-induced inhibition of DOPA accumulation were antagonized by naloxone, we suggest that the effects on striatal DOPA accumulation produced by morphine were mediated via opioid receptors and not directly via DA receptors. Dopamine receptors Opioid receptors
DOPA accumulation Haloperidol
1. Introduction The acute systemic administration of morphine increases the synthesis and turnover of dopamine (DA) in the striatum and produces muscular rigidity and catalepsy in rats (Clouet and Iwatsubo, 1975; Lal, 1975; Kuschinsky, 1976). Morphine has also been shown to block several effects produced by the DA agonist apomorphine (Lal et al., 1975). Systemic administration of morphine (Iwatsubo and Clouet, 1977; Nowycky et al., 1978) and local injection of morphine into the caudate nucleus increased the rate of spontaneous firing of DA neurons in the substantia nigra. These findings suggest that morphine, like haloperidol and other neuroleptics, blocks DA receptors. However, data have also been presented against DA receptor blockade after
Morphine
Naloxone
Striatum
morphine. Chlorpromazine but not morphine was found to inhibit the stereotyped behaviour induced by the DA agonist apomorphine (McKenzie and Sadof, 1974). Furthermore haloperidol but not morphine was found to inhibit the DA-induced stimulation of DA-sensitive adenylate cyclase (Iwatsubo and Clouet, 1975). In addition DA agonists were found to reverse the morphine-induced increase in the rate of spontaneous firing in DA neurons of the substantia nigra while the agonists were only partially successful in reversing the effect of haloperidol. The morphine antagonist naloxone blocked actions of morphine on firing rates, but not those of haloperidol (Iwatsubo and Clouet, 1977). Naloxone has also been shown to antagonize biochemical and behavioural effects of morphine (Lal, 1975; Kuschinsky, 1976). These findings suggest
122 that the effects of morphine on the nigroneostriatal pathway are mediated via receptors sensitive for morphine i.e. opioid receptors and not directly via DA receptors. However, the exact mechanism behind the effect of morphine on central dopaminergic pathways is still u n k n o w n . In the present study we report on the effects of a single dose of morphine on in vivo tyrosine h y d r o x y l a t i o n measured as the accumulation of DOPA after inhibition of neuronal decarboxylase. The effects of morphine and naloxone after DA receptor stimulation and DA receptor blockade were studied. Furthermore we have examined the effects of morphine and naloxone on DOPA accumulation after treatm e n t with ~/-butyrolactone (GBL) and after depletion of the granular DA stores by reserpine.
2. Materials and m e t h o d s
2.1. Animals Male Sprague-Dawley rats (Anticimex, Sollentuna, Sweden) weighing 200--299 g were used in all experiments. The rats were fed the Ewos-Anticimex commercial type pelleted diet, R3 and had access to water ad libitum. During the experiments t h e y were allowed free access only to water.
S.-A. PERSSON or used directly as the injectable form supplied by Endo laboratories. Reserpine was used as the injectable form supplied by CIBA. All other substances were dissolved in 0.9% NaC1. The drugs were administered s.c. or i.p. as described in the legends to the figures and tables.
2. 3. Experimental The experiments were performed between 9 am and 11 am. In some experiments drugs were injected at 6.30 am. The room temperature was 22--26°C. The b o d y temperature was checked by means of a telethermometer with the probe inserted 5 cm from the anal orifice. The lowest temperature observed was 36.0°C. In vivo tyrosine h y d r o x y l a t i o n was measured as the accumulation of DOPA after inhibition of the neuronal decarboxylase. The m e t h o d used was originally described by Carlsson et al. (1972). Dissection of the striata, isolation of DOPA from striatal tissue and the fluorimetric determination of DOPA are described in detail elsewhere (Persson, 1977). In the present study the recovery of 500 ng DOPA taken through the entire procedure was 77.4 + 0.9% (mean + S.E.M., n = 56). All values were corrected for recovery. The statistical significance of the results was assessed using the two-tailed Student's t-test. A P-value of less than 0.05 was considered significant.
2.2. Drugs The following drugs were used: apomorphine HC1 (Sandoz, Basle); gamma-butyrolactone GBL (Sigma, St. Louis, Mo.); haloperidol, Haldol® (Leo, Helsingborg); morphine HC1 (ACO, Solna); naloxone HC1, Nalone® (Endo, N.Y.); reserpine, Serpasil® (Ciba, Basle). 3-Hydroxybenzylhydrazine HC1, NSD 1015 was kindly synthesized by Dr. B. Magnusson, Department of Organic Chemistry, University of Ume~, Sweden. All doses refer to the salts. Haloperidol was dissolved in 5.5% glucose after acidification with 20 pl glacial acetic acid. Naloxone was either dissolved in saline
3. Results
3.1. Time- and dose-related changes in DOPA accumulation after a single dose of morphine The administration of morphine 10 mg/kg s.c. produced an initial decrease in the rate of DOPA accumulation in the striatum (fig. 1). However, 15 min after the administration of morphine the rate of DOPA accumulation was increased. DOPA accumulation reached a m a x i m u m 30 and 60 min after morphine, but
MORPHINE
AND
STRIATAL
200C 2 1000
ACCUMULATION
123
2000
