Nrurophormu~nlu~~?. Vol. IX. pp. 865 to X69 Pergamon Press Ltd 1979. Punted m Great Britain
THE INFLUENCE OF ELECTROACUPUNCTURE ON NALOXONE-INDUCED MORPHINE WITHDRAWAL-III THE EFFECT OF CYCLIC-AMP W. K. K. Ho’, H. K. WONG’ and H. L. WENT ‘Department
of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong ‘The Neurosurgical Unit, Kwong Wah Hospital, Hong Kong (Accepred
2 May
Kong
and
1979)
Summary-The role of cyclic-AMP in the treatment of morphine addiction by electroacupuncture was studied in rats and mice. During addiction there was a significant drop of plasma cyclic-AMP level. On the other hand, when animals were going through withdrawal, a dramatic rise of plasma cyclic-AMP was observed. Electroacupuncture was effective in suppressing withdrawal behaviour but whether this was associated with a concomitant reduction of plasma cyclic-AMP level is uncertain, Pre-administration of a phosphodiesterase inhibitor subcutaneously or dibutyl-cyclic-AMP intracerebrally into addicted animals completely antagonized the effect of electroacupuncture. In view of the possibility that the effect of acupuncture may be mediated by the secretion of p-endorphin, these results suggest that cyclic-AMP may somehow be involved.
The role of cyclic-AMP in morphine addiction is still obscure. Nevertheless, several lines of evidence have provided support that this compound may play an important role in mediating tolerance and physical dependence (Collier, 1977; Simon and Hiller, 1978). For example, Collier and Francis (1975) have shown that the administration of cyclic-AMP or phosphodiesterase inhibitors to morphine-addicted mice can intensify naloxone precipitated withdrawal behaviours. In addition, Mehta and Johnson (1974) have demonstrated that brain cyclic-AMP level increases sharply during withdrawal and that the magnitude of the increase is proportional to the intensity of withdrawal. Under in vitro conditions, the presence of dibutyl-cyclic-AMP can prevent the development of tolerance in the guinea-pig ileum while cyclic-GMP tends to potentiate it (Collier, 1977). Electroacupuncture has been successfully used in the treatment of heroin addicts since 1972 (Wen and Cheung, 1973). Recently, this procedure has been applied to suppress withdrawal in experimentally addicted animals (Choy, Tso, Fung, Leung, Tsang, Lee, Tsang and Wen, 1978; Ho, Wen, Lam and Ma, 1978a). The results indicated that 30 min of treatment was effective in suppressing ‘naloxone-precipitated withdrawal behaviours. Accordiyg to a preliminary finding, the mechanism underlyng this method of treatment may be mediated by the secretion of /I-endorphin (Ho er al., 1978a). Since the endorphins are effective in inhibiting adenylate cyclase activity in both brain slices (Minneman and Iversen, 1976) cells (Wahlstrom, and neuroblastoma x glioma Brandt, Moroder, Wunsch, Linderberg, Ragnasson, Terenius and Hamprecth, 1977; Brandt, Buchen and Key words: acupuncture, drawal symptoms.
addiction,
cyclic-AMP,
with-
865
Hamprecth, 1977), it is possible that electroacupuncture may also exert an indirect action on the adenylate cyclase/cyclic-AMP system. The purpose of the present study was to ascertain whether there is an interrelationship between electroacupuncture, withdrawal symptoms and cyclic-AMP level.
METHODS Methods
of addiction
Female mice of WHT strain (British), weighing 2530g and female rats of Sprague-Dawley strain, weighing 20&25Og, were used throughout the study. Animals were kept in a temperature and humidity controlled room with a 12 hr light (6 a.m.6 p.m.) and I2 hr (6 p.m.-6 a.m.) dark cycle. Food and water were given ud libirum. Rats were addicted by intraperitoneal injection of morphine-HCI in saline according to the following schedules. Schedule A: rats were injected twice daily at 8 a.m. and 8 p.m. with an initial dose of 5 mg/kg per day. The dose was increased at 5 mg/kg increments per day until a final dose of 40mg/kg per day was reached. This dose was maintained for a total period of 8 weeks. Schedule B: rats were addicted for a period of 2f weeks. Two injections were given, one at 9 a.m. and the other at 5 p.m. The initial dose was 5 mg/kg per day and was doubled in consecutive days to a final dose of 40mg/kg per day. Thereafter, it was maintained at 4Omg/kg per day for 2 weeks. Mice were addicted for 2 days by morphine pellet implantation according to the procedure of Hui and Roberts (1975). The amount of morphine in a pellet was approx. 15 mg. Four weeks prior to the experiment, all mice were lighly addicted and scanned for their ability to jump when induced by naloxone.
W. K. K. Ho.. H. K.
X66 Method
of electroacupuncture
und assessment
of with-
druwal behaviors
Electroacupuncture was performed on animals by inserting two 34G acupuncture needles into the ears at the region corresponding to the “lung point” of the human (Choy et a/., 1978; Ho et al., 1978a). The “lung point” of the human ear is a region, approx. 3 mm wide, circumscribing the centre of the deepest portion of the cavum conchae. The needles were anchored into place by rubber fasteners and after an hour of adaptation, the needles were connected to an electrical stimulator (Biopulse Ltd, Hong Kong). The frequency of the pulse used was 125 Hz and the voltage applied was from 0.5 to 4 V. Under such conditions, the animals were usually calm and not agitated. After 30min of stimulation, a single dose of naloxone (gift from Endo Laboratories, Inc., Garden City. New York, U.S.A.; 0.2mg/kg for rats and 10 mg/kg for ,mice) was injected. The withdrawal symptoms induced were scored for the next I5 min while electrical stimulation was maintained. Animals were killed by decapitation at the end of the observation period and trunk blood was collected in heparinized tubes. The number of occurrences of the following behaviors was used to calculate the final behavioural score: Rats-wet dog shake, abnormal posturing, teeth chattering, diarrhea and attempt to escape; Mice-jumping, body shake, diarrhea and abnormal posturing. In general, whenever a sign lasted for more than 15 set another score was given. Anulyticul
methods
Plasma cyclic-AMP level was determined by competitive protein binding assay using a commercially available kit (Catalog No., TRK432, Radiochemical Centre, Amersham, Bucks, U.K.). The procedures used for plasma extraction and assay were essentially as described by the manufacturer. The sensitivity of the assay ranged from 0.1 to 16 pmol.
RESULTS
The goal of the first experiment was to ascertain whether there is a change of plasma cyclic-AMP level during addiction and withdrawal. The findings are summarized in Table 1. Compared with normal rats, the plasma cyclic-AMP level of rats addicted for a period of 8 weeks was significantly lower. This result agrees with a previous observation in human addicts (Ho, Wen. Fung, Ng, Au and Ma, 1977). In contrast, when addicted rats were deprived of drug for a period of 14-16 hr, their plasma cyclic-AMP level went up from an average of 10 to 28 pmol/ml. This elevation of plasma cyclic-AMP level was observed to coincide with the onset of increasing signs of withdrawal. The above experiment clearly suggests that there may be a close relationship between plasma cyclicAMP level and morphine withdrawal. Since electroacupuncture is effective in suppressing withdrawal
WONG
and H. L.
WEN
symptoms, the next experiment was to ascertain whether this method can also suppress the elevation of plasma cyclic-AMP. The results of this cxperimcnt are presented in Table 2. Addicted rats treated with acupuncture for 30min prior to the injection of naloxone had a significant lower average score in withdrawal behaviours (8 vs 21). When this group’s plasma cyclic-AMP level was compared with that of the control. a 20% but insignificant decrease was observed (0.1 < P < 0.2). On the contrary. when normal rats were subjected to similar treatment, no apparent change of plasma cyclic-AMP could be detected. In view of the uncertainty of these results. whether there is an association between the effect of acupuncture in suppressing withdrawal symptoms and plasma cyclic-AMP level remains an open question. Since
an increase
of plasma
observed
during
withdrawal,
ascertain
if, by artificially
cyclic-AMP it
elevating
was
level
was
interesting
tissue
to
or plasma
level, the effectiveness of acupuncture in suppressing abstinence could be reduced. Figure I presents the results of such an experiment. Rats
cyclic-AMP
Table
1. Effect of morphine addiction and withdrawal plasma cyclic-AMP level*
Method
of treatment
n
Normal Addicted Natural withdrawalt
Cyclic-AMP
on
(pmoliml)
23 i 1 IO+- 1: 28 * 2,’
46 II 17
* Rats were addicted according to Schedule A. Results are presented as the mean plus or minus one standard error of the mean. t Rats in this group were killed 14-16 hr after the last injection of morphine. In orienting experiments, the highest number of “wet dog shake” was observed in this period. 1 Compared with the normal group. the difference is statistically significant (P < 0.05, t-test). $Compared with the addicted group. the difference is statistically significant (P < 0.001, t-test).
Table 2. Effect of electroacupuncture on withdrawal iors and plasma cyclic-AMP level*
Treatment Normal Control Acupunctured Addicted Control Acupunctured
n
Withdrawal
Cyclic-AMP (pmol/ml)
23 + I 27 + 2
46 27 22 26
Score
behav-
21 *3 8 + It
38 + 3 31 i3
*Rats were addicted according to Schedule B and treated as described in Methods. The normal rats were handled identically to the addicted ones, except that the injection of naloxone was omitted. In the addicted control group, acupuncture needles were tapped on the ears and no current was applied. Withdrawal was induced by intraperitoneal injection of naloxone (0.2 mg/kg). Results presented as mean +_ SEM. t Difference is statistically significant (0.001 < P < 0.01. t-test).
Effect of acupuncture on cyclic-AMP
n=5
.
T earn
NON
ACU
SALINE
NON
ACU
6
IEIMX
Fig. 1. Effect of IBEX on the ability of ele~roa~pun~ture (ACU) to suppress withdrawal behaviours. Rats were addicted according to Schedule B. In the IBMX group, rats were first injected, subcutaneously, with 2.5 mg IBMX in saline and then randomly divided into two groups. One treated with electroacupuncture and the other not treated. After 45 min. a single dose of naloxone was injected intraperitoneally to induce withdrawal. Behaviours were scored as described in Methods. In the control group, rats were handled identically to the IBMX group except that saline was injected instead of IBMX.
addicted according to Schedule B were divided randomly into 4 groups and treated with or without 3-isobutyl-l-methyl xanthine (IBMX), a potent phosphodiesterase inhibitor. When IBMX was not preadministered, animals that were acupunctured had in general a lower withdrawal score (0.05 < P < 0.1). On the other hand, pre-treatment with IBMX completely abrogated the effect of acupuncture. Up to this point, attention was focused on plasma cyclic-AMP changes. In view of the fact that morphine acts preliminarily at the brain level, it was next necessary to ascertain if by injecting cyclic-AMP directly into the brain, the effect of acupuncture could be neutralized. For experimental convenience, mice were used in this study. The results obtained are summarized in Figure 2. In agreement with a previous observation (Choy et al., 1978; Ho et nl., 1978a), 30 min of acupuncture was effective in reducing withdrawal by as much as 75% (P = 0.05). On the contrary, when 10 pg of dibutyl-cyclic-AMP was administered intra~rebrally, no significant reduction of withdrawal behaviours could be demonstrated in the acupunctured group. DISCUSSION
Complementary to previous findings in human subjects (Ho et al., 1977), rats addicted to morphine
867
have in general lower plasma cyclic-AMP level than their normal counter-parts. The source or sources of plasma cyclic-AMP is still uncertain. It is well established that a number of hormonal and neural activities are mediated by the adenylate cyclase/cyclicAMP system. Since all extra-cellular cyclic-AMP are believed to be of tissue origin (Broadus, Hardman, Kamnsky, Ball, Sutherland and Liddle, 1971), variations of plasma cyclic-AMP level may then reflect changes of neural and/or endocrine activities. In this respect, the lower level of plasma cyclic-AMP found in addicted animals may be due to the effect of morphine on the nervous and/or the endocrine system. Nominal support for this contention comes from fact that morphine addiction has been shown to have suppressive effects on the pituitary-adrenal axis (Ho, Lam, Leung, Au, Wang, Tsang and Wen, 1978b; Eisenman, Fraser and Brooks, 1961). In vitro data obtained from neuroblastoma x glioma cells by Sharma, Klee and Nirenberg (1975) indicate that cellular cyclic-AMP level can be induced to rise when morphine-tolerant cells are deprived of the drug suddenly. The present observation that plasma cyclic-AMP was significantly elevated during withdrawal is in accord with these findings, What causes the sudden increase of cyclic-AMP during withdrawal is unclear. Since withdrawal may be considered as a state of stress, the increase of cyclicAMP levei may at least be partly amounted for by hyperneural activities such as an over release of neurotransmitters. Injection of phosphodiesterase inhibitors into naive rats has been shown to induce behavioural changes similar to those observed during morphine withdrawal (Collier, Francis, Henderson and Schneider, 1974). If these “quasi withdrawal symptoms” could be solely induced by cyclic-AMP alone, it is not unreasonable to speculate that cyclic-AMP acts at a point beyond the level of the opiate receptors. The observation that the administration of phosphodiesterase inhibitor and dibutyl-cyclic-AMP can reverse the effect of electroacupuncture provides partial sup port to this hypothesis. Thus, during acupunctire, P-endorphin secretion may be stimulated (Ho et al., 1978a) and as a result there is an increased amount of agonist available to bind to the opiate receptors. Since /?-endorphin has been shown to suppress adenylate cyclase activity (Minneman and Iversen, 1976; Wahlstrom et al., 1977; Brandt et al., 1977), it is probable that there will be a concomitant decrease of cyclic-AMP level in the opiate sensitive neurons. If an increase of cyclic-AMP level is required for the development of withdrawal, P-endorphin may then exert its primary effect at this level. When the inter- and intra-cellular level of cyclic-AMP is artificially elevated by treatment with phosphodiesterase inhibitor or dibutyl-cyclic-AMP, the ability of fi-endorphin to suppress cyclic-AMP is bypassed. Because of this, withdrawal symptoms may be induced to reappear. To confirm this hypothesis, the present authors are
W. K. K. Ho., H. K. WONU and H. L. WIN
NON
ACU
SALrNE
NON
ACU
CJE3cAMP
Fig. 2. The effect of dibutyl-cyclic-AMP (DBcAMP) on reversing acupuncture-suppressible withdrawal behaviours in mice. Mice were addicted by pellet implantatjon and were divided randomly into two groups: one treated with electroacupuncture for 60min and the other not treated. At the end of the 6Ornin. the a~upunctured and the Noel-a~upunctured groups were further subdivided into two groups. One injected intracerebrally with 2 pt saline, containing 10 pg dibutyl-cychc-AMP and the other a similar volume of plain saline. All mice were then observed for I.5min for possible signs of brain damage. Afterwards. I ml naloxone was administered, intraperitoneally, to induce withdrawal. Scores were counted as described in the text. In the acupunctured animals. electrical stimulation was maintained throughout the two observation periods.
currently testing the effect of phosphodiesterase inhibitors and dibutyl-c~c~~-AMP on ~-endorphin mediated physiological responses (e.g. suppression of withdrawal, catatonic posturing and analgesia). Acknow~~dge~~effts--We thank the Lee Foundation of Singapore and Hong Kong. Mr Lee Wing Tat, Mr Ko Fook Son and the Takshing Investment Co., Ltd. Hong Kong for financial support. We are also grateful to Professor Ma Lin for helpful discussions. The technical assistance of the staff of our Department was most invaluable.
REFERENCES Brandt, M., Buchen, C. and Hamprecht. B (1977). Endorphine exert opiate-like action on neuroblastoma x glioma hybrid cells. FEBS Lett. 80: 251-254. Broadus, A. E., Hardman, J. G., Kamnsky, N. I.. Ball. J. H.. Sutherland, E. W. and Liddle, G. W. (1971) Extracellular cyclic nucleotides. Ann. N.k: Acad. Sci. 185: 50-66. Choy, Y. M., Tso, W. W., Fung, K. P., Leung, K. C., Tsang, Y. F., Lee, C. Y., Tsang, D. and Wen, H. L. (1978). Suppression of narcotic withdrawaIs and plasma ACTH by auricular electroacupuncture. Biochrm. hiophys. Rrs. Comm. 82: 305-309.
Collier, H. 0. J. (1977). Consequences of interaction between opioid and receptor. Trans. Biochem. Sac. 5: 70-73.
Collier, H. 0. J. and Francis, D. L. (1975). Morphine abstinent: is associated with increased brain cAMP. Nature
255: 159-162.
Collier, H. 0. J., Francis, D. L., Henderson. G. and Schneider, S. (1974). Quasi morphine-abstinence syndrome. Nature 249: 471-473. Eisenman, A. J., Fraser, H. F. and Brooks, J. W. (1961). Urinary excretion and plasma levels of 17-hydroxycorticosteroids during a cycle of addiction to morphine. J. P~iurmuc. e.rp. Thrr. 132: 22423 1~ Ho, W. K. K., Wen, H. L., Lam, S. and Ma, L. (1978a). The intluence of electro-acupuncture on naloxoneinduced morphine withdrawal in mice: elevation of brain opiate-like activity. Eur. J. Pharmuc. 49: 197.-199. Ho, W. K. K.. Wen, H. L.. Fung, K. P., Ng, Y. H., Au. K. K. and Ma, L. (1977). Comparison of plasma hormonal levels hetween heroin-addicted and normat subjects. Clin. Chim. Acta 75: 415419. Ho, W. K. K., Lam, S., Leung, K. C.. Au, K. K.. Wong, H. K., Tsang. Y. F. and Wen. H. L. (1978b). Effect of naloxone on morphine induced changes in ACTH. corticosterone and cyclic nucleotides. ~ef~~~~~~ffrrnac~l~g~ 17: 397.400. Hui, K. S. and Roberts, M. B. (1975). An improved implantation pellet for rapid induciion of morphine dependence in mice. J. Phurm. Pharmuc. 27: 569-573. Mehta, C. S. and Johnson, W. (1974). Elevation of brain cyclic adenosine 3’,5’-monophosphate during naloxone precipitated withdrawal in morphine dependent rats. Fedn Proc. Fedn Am. Sots cup. Bioi. 33: 493.
Effect of acupuncture on cyclic-AMP Minneman, K. F. and Iversen, L. L. (1976). Enkephalin and opiate narcotics increase cyclic GMP accumulation in slices of rat neostriatum. Nature 262: 313-314. Sharma, S. K., Klee, W. A. and Nirenberg, M. (1975). Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proc. natn. Acad. Sci. U.S.A. 72: 3092-3096.
Simon, E. J. and Hiller, J. M. (1978). The opiate receptors. A. Rev. Pharmac. Tox. 18: 371-394.
869
Wahlstrom, A., Brandt, M., Moroder, W., Wunsch, E., Linderberg, G., Ragnasson, U., Terenius, L. and Hamprecth, B. (1977). Peptides related to fi-lipotropin with opioid activity. Effects on levels of CAMP in neuroblastoma x glioma hybrid cells. FEBS Lett. 77: 2832. Wen, H. L. and Cheung, S. Y. C. (1973). Treatment of drug addiction by acupuncture and electrical stimulation. Asian J. Mod. Med. 9: 138-140.
THE INFLUENCE OF ELECTROACUPUNCTURE ON NALOXONE-INDUCED MORPHINE WITHDRAWAL-III THE EFFECT OF CYCLIC-AMP W. K. K. Ho’, H. K. WONG’ and H. L. WENT ‘Department
of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong ‘The Neurosurgical Unit, Kwong Wah Hospital, Hong Kong (Accepred
2 May
Kong
and
1979)
Summary-The role of cyclic-AMP in the treatment of morphine addiction by electroacupuncture was studied in rats and mice. During addiction there was a significant drop of plasma cyclic-AMP level. On the other hand, when animals were going through withdrawal, a dramatic rise of plasma cyclic-AMP was observed. Electroacupuncture was effective in suppressing withdrawal behaviour but whether this was associated with a concomitant reduction of plasma cyclic-AMP level is uncertain, Pre-administration of a phosphodiesterase inhibitor subcutaneously or dibutyl-cyclic-AMP intracerebrally into addicted animals completely antagonized the effect of electroacupuncture. In view of the possibility that the effect of acupuncture may be mediated by the secretion of p-endorphin, these results suggest that cyclic-AMP may somehow be involved.
The role of cyclic-AMP in morphine addiction is still obscure. Nevertheless, several lines of evidence have provided support that this compound may play an important role in mediating tolerance and physical dependence (Collier, 1977; Simon and Hiller, 1978). For example, Collier and Francis (1975) have shown that the administration of cyclic-AMP or phosphodiesterase inhibitors to morphine-addicted mice can intensify naloxone precipitated withdrawal behaviours. In addition, Mehta and Johnson (1974) have demonstrated that brain cyclic-AMP level increases sharply during withdrawal and that the magnitude of the increase is proportional to the intensity of withdrawal. Under in vitro conditions, the presence of dibutyl-cyclic-AMP can prevent the development of tolerance in the guinea-pig ileum while cyclic-GMP tends to potentiate it (Collier, 1977). Electroacupuncture has been successfully used in the treatment of heroin addicts since 1972 (Wen and Cheung, 1973). Recently, this procedure has been applied to suppress withdrawal in experimentally addicted animals (Choy, Tso, Fung, Leung, Tsang, Lee, Tsang and Wen, 1978; Ho, Wen, Lam and Ma, 1978a). The results indicated that 30 min of treatment was effective in suppressing ‘naloxone-precipitated withdrawal behaviours. Accordiyg to a preliminary finding, the mechanism underlyng this method of treatment may be mediated by the secretion of /I-endorphin (Ho er al., 1978a). Since the endorphins are effective in inhibiting adenylate cyclase activity in both brain slices (Minneman and Iversen, 1976) cells (Wahlstrom, and neuroblastoma x glioma Brandt, Moroder, Wunsch, Linderberg, Ragnasson, Terenius and Hamprecth, 1977; Brandt, Buchen and Key words: acupuncture, drawal symptoms.
addiction,
cyclic-AMP,
with-
865
Hamprecth, 1977), it is possible that electroacupuncture may also exert an indirect action on the adenylate cyclase/cyclic-AMP system. The purpose of the present study was to ascertain whether there is an interrelationship between electroacupuncture, withdrawal symptoms and cyclic-AMP level.
METHODS Methods
of addiction
Female mice of WHT strain (British), weighing 2530g and female rats of Sprague-Dawley strain, weighing 20&25Og, were used throughout the study. Animals were kept in a temperature and humidity controlled room with a 12 hr light (6 a.m.6 p.m.) and I2 hr (6 p.m.-6 a.m.) dark cycle. Food and water were given ud libirum. Rats were addicted by intraperitoneal injection of morphine-HCI in saline according to the following schedules. Schedule A: rats were injected twice daily at 8 a.m. and 8 p.m. with an initial dose of 5 mg/kg per day. The dose was increased at 5 mg/kg increments per day until a final dose of 40mg/kg per day was reached. This dose was maintained for a total period of 8 weeks. Schedule B: rats were addicted for a period of 2f weeks. Two injections were given, one at 9 a.m. and the other at 5 p.m. The initial dose was 5 mg/kg per day and was doubled in consecutive days to a final dose of 40mg/kg per day. Thereafter, it was maintained at 4Omg/kg per day for 2 weeks. Mice were addicted for 2 days by morphine pellet implantation according to the procedure of Hui and Roberts (1975). The amount of morphine in a pellet was approx. 15 mg. Four weeks prior to the experiment, all mice were lighly addicted and scanned for their ability to jump when induced by naloxone.
W. K. K. Ho.. H. K.
X66 Method
of electroacupuncture
und assessment
of with-
druwal behaviors
Electroacupuncture was performed on animals by inserting two 34G acupuncture needles into the ears at the region corresponding to the “lung point” of the human (Choy et a/., 1978; Ho et al., 1978a). The “lung point” of the human ear is a region, approx. 3 mm wide, circumscribing the centre of the deepest portion of the cavum conchae. The needles were anchored into place by rubber fasteners and after an hour of adaptation, the needles were connected to an electrical stimulator (Biopulse Ltd, Hong Kong). The frequency of the pulse used was 125 Hz and the voltage applied was from 0.5 to 4 V. Under such conditions, the animals were usually calm and not agitated. After 30min of stimulation, a single dose of naloxone (gift from Endo Laboratories, Inc., Garden City. New York, U.S.A.; 0.2mg/kg for rats and 10 mg/kg for ,mice) was injected. The withdrawal symptoms induced were scored for the next I5 min while electrical stimulation was maintained. Animals were killed by decapitation at the end of the observation period and trunk blood was collected in heparinized tubes. The number of occurrences of the following behaviors was used to calculate the final behavioural score: Rats-wet dog shake, abnormal posturing, teeth chattering, diarrhea and attempt to escape; Mice-jumping, body shake, diarrhea and abnormal posturing. In general, whenever a sign lasted for more than 15 set another score was given. Anulyticul
methods
Plasma cyclic-AMP level was determined by competitive protein binding assay using a commercially available kit (Catalog No., TRK432, Radiochemical Centre, Amersham, Bucks, U.K.). The procedures used for plasma extraction and assay were essentially as described by the manufacturer. The sensitivity of the assay ranged from 0.1 to 16 pmol.
RESULTS
The goal of the first experiment was to ascertain whether there is a change of plasma cyclic-AMP level during addiction and withdrawal. The findings are summarized in Table 1. Compared with normal rats, the plasma cyclic-AMP level of rats addicted for a period of 8 weeks was significantly lower. This result agrees with a previous observation in human addicts (Ho, Wen. Fung, Ng, Au and Ma, 1977). In contrast, when addicted rats were deprived of drug for a period of 14-16 hr, their plasma cyclic-AMP level went up from an average of 10 to 28 pmol/ml. This elevation of plasma cyclic-AMP level was observed to coincide with the onset of increasing signs of withdrawal. The above experiment clearly suggests that there may be a close relationship between plasma cyclicAMP level and morphine withdrawal. Since electroacupuncture is effective in suppressing withdrawal
WONG
and H. L.
WEN
symptoms, the next experiment was to ascertain whether this method can also suppress the elevation of plasma cyclic-AMP. The results of this cxperimcnt are presented in Table 2. Addicted rats treated with acupuncture for 30min prior to the injection of naloxone had a significant lower average score in withdrawal behaviours (8 vs 21). When this group’s plasma cyclic-AMP level was compared with that of the control. a 20% but insignificant decrease was observed (0.1 < P < 0.2). On the contrary. when normal rats were subjected to similar treatment, no apparent change of plasma cyclic-AMP could be detected. In view of the uncertainty of these results. whether there is an association between the effect of acupuncture in suppressing withdrawal symptoms and plasma cyclic-AMP level remains an open question. Since
an increase
of plasma
observed
during
withdrawal,
ascertain
if, by artificially
cyclic-AMP it
elevating
was
level
was
interesting
tissue
to
or plasma
level, the effectiveness of acupuncture in suppressing abstinence could be reduced. Figure I presents the results of such an experiment. Rats
cyclic-AMP
Table
1. Effect of morphine addiction and withdrawal plasma cyclic-AMP level*
Method
of treatment
n
Normal Addicted Natural withdrawalt
Cyclic-AMP
on
(pmoliml)
23 i 1 IO+- 1: 28 * 2,’
46 II 17
* Rats were addicted according to Schedule A. Results are presented as the mean plus or minus one standard error of the mean. t Rats in this group were killed 14-16 hr after the last injection of morphine. In orienting experiments, the highest number of “wet dog shake” was observed in this period. 1 Compared with the normal group. the difference is statistically significant (P < 0.05, t-test). $Compared with the addicted group. the difference is statistically significant (P < 0.001, t-test).
Table 2. Effect of electroacupuncture on withdrawal iors and plasma cyclic-AMP level*
Treatment Normal Control Acupunctured Addicted Control Acupunctured
n
Withdrawal
Cyclic-AMP (pmol/ml)
23 + I 27 + 2
46 27 22 26
Score
behav-
21 *3 8 + It
38 + 3 31 i3
*Rats were addicted according to Schedule B and treated as described in Methods. The normal rats were handled identically to the addicted ones, except that the injection of naloxone was omitted. In the addicted control group, acupuncture needles were tapped on the ears and no current was applied. Withdrawal was induced by intraperitoneal injection of naloxone (0.2 mg/kg). Results presented as mean +_ SEM. t Difference is statistically significant (0.001 < P < 0.01. t-test).
Effect of acupuncture on cyclic-AMP
n=5
.
T earn
NON
ACU
SALINE
NON
ACU
6
IEIMX
Fig. 1. Effect of IBEX on the ability of ele~roa~pun~ture (ACU) to suppress withdrawal behaviours. Rats were addicted according to Schedule B. In the IBMX group, rats were first injected, subcutaneously, with 2.5 mg IBMX in saline and then randomly divided into two groups. One treated with electroacupuncture and the other not treated. After 45 min. a single dose of naloxone was injected intraperitoneally to induce withdrawal. Behaviours were scored as described in Methods. In the control group, rats were handled identically to the IBMX group except that saline was injected instead of IBMX.
addicted according to Schedule B were divided randomly into 4 groups and treated with or without 3-isobutyl-l-methyl xanthine (IBMX), a potent phosphodiesterase inhibitor. When IBMX was not preadministered, animals that were acupunctured had in general a lower withdrawal score (0.05 < P < 0.1). On the other hand, pre-treatment with IBMX completely abrogated the effect of acupuncture. Up to this point, attention was focused on plasma cyclic-AMP changes. In view of the fact that morphine acts preliminarily at the brain level, it was next necessary to ascertain if by injecting cyclic-AMP directly into the brain, the effect of acupuncture could be neutralized. For experimental convenience, mice were used in this study. The results obtained are summarized in Figure 2. In agreement with a previous observation (Choy et al., 1978; Ho et nl., 1978a), 30 min of acupuncture was effective in reducing withdrawal by as much as 75% (P = 0.05). On the contrary, when 10 pg of dibutyl-cyclic-AMP was administered intra~rebrally, no significant reduction of withdrawal behaviours could be demonstrated in the acupunctured group. DISCUSSION
Complementary to previous findings in human subjects (Ho et al., 1977), rats addicted to morphine
867
have in general lower plasma cyclic-AMP level than their normal counter-parts. The source or sources of plasma cyclic-AMP is still uncertain. It is well established that a number of hormonal and neural activities are mediated by the adenylate cyclase/cyclicAMP system. Since all extra-cellular cyclic-AMP are believed to be of tissue origin (Broadus, Hardman, Kamnsky, Ball, Sutherland and Liddle, 1971), variations of plasma cyclic-AMP level may then reflect changes of neural and/or endocrine activities. In this respect, the lower level of plasma cyclic-AMP found in addicted animals may be due to the effect of morphine on the nervous and/or the endocrine system. Nominal support for this contention comes from fact that morphine addiction has been shown to have suppressive effects on the pituitary-adrenal axis (Ho, Lam, Leung, Au, Wang, Tsang and Wen, 1978b; Eisenman, Fraser and Brooks, 1961). In vitro data obtained from neuroblastoma x glioma cells by Sharma, Klee and Nirenberg (1975) indicate that cellular cyclic-AMP level can be induced to rise when morphine-tolerant cells are deprived of the drug suddenly. The present observation that plasma cyclic-AMP was significantly elevated during withdrawal is in accord with these findings, What causes the sudden increase of cyclic-AMP during withdrawal is unclear. Since withdrawal may be considered as a state of stress, the increase of cyclicAMP levei may at least be partly amounted for by hyperneural activities such as an over release of neurotransmitters. Injection of phosphodiesterase inhibitors into naive rats has been shown to induce behavioural changes similar to those observed during morphine withdrawal (Collier, Francis, Henderson and Schneider, 1974). If these “quasi withdrawal symptoms” could be solely induced by cyclic-AMP alone, it is not unreasonable to speculate that cyclic-AMP acts at a point beyond the level of the opiate receptors. The observation that the administration of phosphodiesterase inhibitor and dibutyl-cyclic-AMP can reverse the effect of electroacupuncture provides partial sup port to this hypothesis. Thus, during acupunctire, P-endorphin secretion may be stimulated (Ho et al., 1978a) and as a result there is an increased amount of agonist available to bind to the opiate receptors. Since /?-endorphin has been shown to suppress adenylate cyclase activity (Minneman and Iversen, 1976; Wahlstrom et al., 1977; Brandt et al., 1977), it is probable that there will be a concomitant decrease of cyclic-AMP level in the opiate sensitive neurons. If an increase of cyclic-AMP level is required for the development of withdrawal, P-endorphin may then exert its primary effect at this level. When the inter- and intra-cellular level of cyclic-AMP is artificially elevated by treatment with phosphodiesterase inhibitor or dibutyl-cyclic-AMP, the ability of fi-endorphin to suppress cyclic-AMP is bypassed. Because of this, withdrawal symptoms may be induced to reappear. To confirm this hypothesis, the present authors are
W. K. K. Ho., H. K. WONU and H. L. WIN
NON
ACU
SALrNE
NON
ACU
CJE3cAMP
Fig. 2. The effect of dibutyl-cyclic-AMP (DBcAMP) on reversing acupuncture-suppressible withdrawal behaviours in mice. Mice were addicted by pellet implantatjon and were divided randomly into two groups: one treated with electroacupuncture for 60min and the other not treated. At the end of the 6Ornin. the a~upunctured and the Noel-a~upunctured groups were further subdivided into two groups. One injected intracerebrally with 2 pt saline, containing 10 pg dibutyl-cychc-AMP and the other a similar volume of plain saline. All mice were then observed for I.5min for possible signs of brain damage. Afterwards. I ml naloxone was administered, intraperitoneally, to induce withdrawal. Scores were counted as described in the text. In the acupunctured animals. electrical stimulation was maintained throughout the two observation periods.
currently testing the effect of phosphodiesterase inhibitors and dibutyl-c~c~~-AMP on ~-endorphin mediated physiological responses (e.g. suppression of withdrawal, catatonic posturing and analgesia). Acknow~~dge~~effts--We thank the Lee Foundation of Singapore and Hong Kong. Mr Lee Wing Tat, Mr Ko Fook Son and the Takshing Investment Co., Ltd. Hong Kong for financial support. We are also grateful to Professor Ma Lin for helpful discussions. The technical assistance of the staff of our Department was most invaluable.
REFERENCES Brandt, M., Buchen, C. and Hamprecht. B (1977). Endorphine exert opiate-like action on neuroblastoma x glioma hybrid cells. FEBS Lett. 80: 251-254. Broadus, A. E., Hardman, J. G., Kamnsky, N. I.. Ball. J. H.. Sutherland, E. W. and Liddle, G. W. (1971) Extracellular cyclic nucleotides. Ann. N.k: Acad. Sci. 185: 50-66. Choy, Y. M., Tso, W. W., Fung, K. P., Leung, K. C., Tsang, Y. F., Lee, C. Y., Tsang, D. and Wen, H. L. (1978). Suppression of narcotic withdrawaIs and plasma ACTH by auricular electroacupuncture. Biochrm. hiophys. Rrs. Comm. 82: 305-309.
Collier, H. 0. J. (1977). Consequences of interaction between opioid and receptor. Trans. Biochem. Sac. 5: 70-73.
Collier, H. 0. J. and Francis, D. L. (1975). Morphine abstinent: is associated with increased brain cAMP. Nature
255: 159-162.
Collier, H. 0. J., Francis, D. L., Henderson. G. and Schneider, S. (1974). Quasi morphine-abstinence syndrome. Nature 249: 471-473. Eisenman, A. J., Fraser, H. F. and Brooks, J. W. (1961). Urinary excretion and plasma levels of 17-hydroxycorticosteroids during a cycle of addiction to morphine. J. P~iurmuc. e.rp. Thrr. 132: 22423 1~ Ho, W. K. K., Wen, H. L., Lam, S. and Ma, L. (1978a). The intluence of electro-acupuncture on naloxoneinduced morphine withdrawal in mice: elevation of brain opiate-like activity. Eur. J. Pharmuc. 49: 197.-199. Ho, W. K. K.. Wen, H. L.. Fung, K. P., Ng, Y. H., Au. K. K. and Ma, L. (1977). Comparison of plasma hormonal levels hetween heroin-addicted and normat subjects. Clin. Chim. Acta 75: 415419. Ho, W. K. K., Lam, S., Leung, K. C.. Au, K. K.. Wong, H. K., Tsang. Y. F. and Wen. H. L. (1978b). Effect of naloxone on morphine induced changes in ACTH. corticosterone and cyclic nucleotides. ~ef~~~~~~ffrrnac~l~g~ 17: 397.400. Hui, K. S. and Roberts, M. B. (1975). An improved implantation pellet for rapid induciion of morphine dependence in mice. J. Phurm. Pharmuc. 27: 569-573. Mehta, C. S. and Johnson, W. (1974). Elevation of brain cyclic adenosine 3’,5’-monophosphate during naloxone precipitated withdrawal in morphine dependent rats. Fedn Proc. Fedn Am. Sots cup. Bioi. 33: 493.
Effect of acupuncture on cyclic-AMP Minneman, K. F. and Iversen, L. L. (1976). Enkephalin and opiate narcotics increase cyclic GMP accumulation in slices of rat neostriatum. Nature 262: 313-314. Sharma, S. K., Klee, W. A. and Nirenberg, M. (1975). Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proc. natn. Acad. Sci. U.S.A. 72: 3092-3096.
Simon, E. J. and Hiller, J. M. (1978). The opiate receptors. A. Rev. Pharmac. Tox. 18: 371-394.
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Wahlstrom, A., Brandt, M., Moroder, W., Wunsch, E., Linderberg, G., Ragnasson, U., Terenius, L. and Hamprecth, B. (1977). Peptides related to fi-lipotropin with opioid activity. Effects on levels of CAMP in neuroblastoma x glioma hybrid cells. FEBS Lett. 77: 2832. Wen, H. L. and Cheung, S. Y. C. (1973). Treatment of drug addiction by acupuncture and electrical stimulation. Asian J. Mod. Med. 9: 138-140.
