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Endogenous morphine and codeine
Brain Research 915 (2001) 155–160 www.elsevier.com / locate / bres
Research report
Endogenous morphine and codeine Possible role as endogenous anticonvulsants S. Spector*, I. Munjal, D.E. Schmidt Department of Psychiatry, Vanderbilt University Medical Center, Nashville, TN 37232, USA Accepted 12 July 2001
Abstract Exogenously administered morphine can have both convulsive or anticonvulsive effects, depending on the dose and species. The levels of the endogenous opiate alkaloids morphine and codeine were significantly elevated in specific rat brain regions by the convulsive drug, pentylenetetrazole, as well as by the anticonvulsant drugs, carbamazepine and phenytoin. Morphine and codeine levels in peripheral tissues (heart, lung, spleen and adrenal) were unaffected by these drugs. Maximal increases in morphine levels were seen in the hypothalamus and striatum (2–10-fold), while lesser increases occurred in the midbrain and brain stem (2–4-fold). Codeine levels were also markedly increased in hypothalamus (5–10 fold), In contrast to morphine, codeine levels were also increased in the hippocampus (2–10-fold), but were unchanged in the striatum. These studies suggest that the endogenous alkaloids morphine and codeine are involved in the modulation of convulsions and that morphine and / or codeine may act as an endogenous anticonvulsant. 2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Opioids: anatomy, physiology and behavior Keywords: Endogenous morphine and codeine; Pentylenetetrazole; Carbamazepine; Phenytoin; Convulsion; Anticonvulsant
1. Introduction The opiate alkaloids morphine and codeine have been identified in mammalian tissues [6,16,26] and mammalian tissues have the capacity to synthesize these alkaloids both in vivo [6,16] and in vitro [17]. It has also been reported that morphine is located in neurons [3,14] and can be released under conditions known to release other neurotransmitters [3]. The endogenous opiate alkaloids exhibit plasticity: their levels are altered by immuno-stimulants, and pain and fasting have been shown to modify the levels of morphine and codeine in brain [18,23,30]. Thus, it appears that a morphinergic neuronal pathway is present in mammalian brain. The effect of morphine administration is controversial. Exogenous morphine results in either a pro-convulsant or anticonvulsant effect in different animal models, depend*Corresponding author. Tel.: 11-615-343-7676; fax: 11-615-3438639. E-mail address: [email protected] (S. Spector).
ing on the dose. Low, sub-analgesic doses of morphine elevates the electroconvulsive threshold in rats and this anticonvulsive effect can be reversed by naloxone [1,4,12,13,29,31,34,35,37]. Ahmed and Pleuvry [2] reported that when seizures were induced with either bicuculline or kainic acid morphine exerted an anticonvulsant action. They also showed that morphine enhanced the anticonvulsant effect of phenytoin in the mouse electroshock model of seizures. Foote and Gale [10] also demonstrated that morphine exerted an anticonvulsant effect on electroshock seizures. Frey [11] reported that morphine suppressed blast-induced seizure. Using another animal model, the seizure-sensitive Mongolian gerbil, Lee and Bajorek [19] showed that morphine exerted an anticonvulsant effect. The brain levels of endogenous morphine or codeine are consistent with their possible role as an anticonvulsant [20]. Post and Weiss [27] have hypothesized the existence of compensatory and adaptive mechanisms that come into play to counterbalance pathological processes. They have reported that repeated amygdala-kindled seizures evoked
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the release of an endogenous substance that acts to enhance the efficacy of the anticonvulsant drug carbamazepine. Tortella and Long [36] have suggested that the endogenous anticonvulsant substance is an opioid peptide. However, since carbamazepine also exhibits antinocioceptive, anti-manic and antidepressant effects, the effects of carbamazepine and other anticonvulsant agents (phenytoin and phenobarbital) and the convulsant drug pentylenetetrazole on the endogenous levels of morphine and codeine were measured in brain and selected peripheral tissues.
(95.5%), 1-propanol (3%), pyridine (0.8%) and acetic acid (0.3%). The eluate was evaporated to dryness and the sample was resuspended in 0.250 ml of mobile phase (pH 5.2). The sample was then filtered and injected into a Lichrosorb RP-18 column at a flow-rate of 1.5 ml / min. The fractions corresponding to the elution times of morphine and codeine were collected and evaporated to dryness. The recovery, which was about 70–80%, was determined by parallel analysis of replicate samples spiked with authentic morphine and codeine and the values corrected accordingly.
2. Materials and methods
2.1. Radioimmunoassay for morphine and codeine
All research was conducted accordance with the NIH guide for the care and use of laboratory animals and was approved by the Vanderbilt Animal Care Committee. Male 225–250-g Sprague–Dawley rats were injected with either pentylenetetrazole (60 mg / kg, i.p.) or carbamazepine (25 mg / kg in 20% DMSO–saline, i.p.), phenytoin (25 mg / kg, i.p.) or phenobarbital (25 mg / kg, i.p.) dissolved in saline and the response and time course on the morphine and codeine content in specific brain regions and selected peripheral tissues was measured. Control rats received an equivalent volume of the appropriate vehicle and were sacrificed at the indicated times. At various times following drug administration, the rats were anesthetized by 50% carbon dioxide and decapitated. The brains were quickly removed, chilled in ice cold saline and dissected on ice. The peripheral tissues, lung, heart, spleen and adrenals were also removed and frozen at 2708C until assayed. Morphine and codeine were extracted from the tissues and assayed by RIA as described by Donnerer et al. [6]. The morphine antibody has been previously shown to be highly selective for morphine and does not cross react with opioid peptides, narcotic antagonists or the morphine precursors salutaridine, reticuline or thebaine [7,32]. Briefly, the tissues were washed free of blood or faeces, blotted and frozen immediately. At the time of assay, tissues were weighed and then homogenized in 10 mM HCl to yield 100 mg tissue / ml. The opiate conjugates were hydrolyzed by adding 6 ml of 12 N HCl to 10 ml of tissue homogenate and heating in a water bath (95–1008) for 30 min. The hydrolyzed samples were centrifuged 30 min at 10 0003g at 48C and filtered through glass wool. The pH was adjusted to 8.5–9.0. Five volumes of a 1:9 mixture of n-butanol in chloroform was added and the mixture was shaken for 5 min. The organic phase was collected and back-extracted into 10 mM HCl. The aqueous phase was evaporated to dryness in a Savant vacuum centrifuge. The dried samples were reconstituted in 5 ml of 10 mM HCl plus phosphate-buffered saline, pH 7.4, and the pH was adjusted to 8.5–9.0. The samples were passed through C 18 -Sep Pak cartridge which had been activated with methanol and then flushed with water. The samples were eluted with a mobile phase, containing water
The samples were resuspended in 200 ml of phosphate buffered saline (pH 7.4), 50 ml of the morphine antibody [33] and [ 125 I]iodo-morphine were added and the samples were incubated for 90 min at 48C. A standard curve of authentic morphine and codeine were similarly treated. Following equilibration, 250 ml of saturated ammonium sulfate was added and the samples are incubated for 45 min and filtered through GF / G glass fiber filter. The filters were counted in a gamma counter and concentrations of morphine and codeine established using Riacole Software. Significance was determined by one-way ANOVA followed by Bonferroni’s post-hoc testing of individual values.
3. Results The effect of pentyleneterazole (60 mg / kg, i.p.) on regional morphine and codeine levels in brain are presented in Fig. 1a and b. TonicBclonic convulsions occurred within 2–3 min of drug administration and the rats were sacrificed 15 and 30 min after the drug was given. There was about a three-fold increase in morphine content in the hypothalamus, striatum and midbrain within 15 min. The elevated levels persisted for more than 30 min in the hypothalamus, while morphine concentrations in the striatum and midbrain returned to control values by 30 min. A smaller, non-significant increase in morphine levels at 15 min was observed in the hippocampus.The morphine content in the brain stem, cortex and cerebellum was unaffected (Fig. 1a). Similar to morphine, codeine levels in the hypothalamus, striatum and midbrain were significantly elevated, while levels in the cortex and cerebellum were unchanged (Fig. 1b). In contrast to morphine, codeine concentrations were also increased in the hippocampus and brainstem. These regional differences and the increases observed persisted longer than seen with morphine. Peripheral tissues content of morphine and codeine in the heart, lung, spleen and adrenal levels were not changed (data not shown). Blocking the mu opiate receptor with naltrexone (5
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Fig. 1. (A) and (B) The effect of pentylenetetrazole on morphine and codeine levels in rat brain. Groups of rats (N56) were sacrificed 15 and 30 min following administration of pentylenetetrazole (60 mg / kg, i.p.) or vehicle only. In an additional group, naltrexone (5 mg / kg, i.p.) was administered 10 min prior to the pentylenetetrazole. Mean values are stated as pmol / g tissue6s.e.m. There was no differences between the 15- and 30-min vehicle groups and the values were combined. Significance (*P.0.05) was determined by one-way ANOVA followed by Bonferroni’s post-hoc testing of individual values.
mg / kg, i.p.) administered 10 min prior to the convulsant agent did not cause a feedback-induced increase in either morphine or codeine (Fig. 1a and b). Naltrexone alone did not alter the levels of morphine or codeine. Fig. 2a shows that the anticonvulsant agent carbamazepine markedly elevated the morphine content in the hypothalamus, striatum and midbrain within 15 min. There was also a significant increase in the cerebellum. Again, there was a different effect of carbamazepine on codeine content in the brain (Fig. 2b). The hypothalamus and hippocampus showed the greatest effect following carbamazepine administration, while the striatum, cerebellum and cortex were unchanged. The midbrain had an elevated codeine content at 30 min and the brainstem had a significant elevation at 15 min. The peripheral tissue content of morphine and codeine were unchanged (data not shown). Fig. 3 shows that phenytoin also increased the morphine levels in specific brain regions. The morphine levels in the hypothalamus was increased seven-fold within 15 min and returned to control levels by 30 min. The striatal levels of
morphine increased ten-fold within 15 min and the elevated levels persisted for the 30-min period. Midbrain, brainstem and cerebellum all showed significant increases in morphine content. The administration of vehicle alone did not change the morphine or codeine levels at any time point and the control values were combined from all time points. There also were no changes in morphine or codeine levels in the peripheral tissues (data not shown).
4. Discussion The data presented here demonstrates that the brain concentrations of both morphine and codeine are elevated following pentylenetetrazole-induced convulsions. The levels also increased following administration of the anticonvulsant drugs carbamazepine and phenytoin, drugs with different chemical structures. It has been previously reported that pain, stress, starvation and immunostimulants [18,23,30] also alter the levels of endogenous morphine and codeine in the brain. This plasticity in response to
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Fig. 2. (A) and (B) The effect of carbamazepine on morphine and codeine levels in rat brain. Groups of rats (N56) were sacrificed 15 and 30 min following administration of carbamazepine (25 mg / kg, i.p.) or vehicle only. Mean values are stated as pmol / g tissue6s.e.m. There was no differences between the 15- and 30-min vehicle groups and the values were combined. Significance (*P.0.05) was determined by one-way ANOVA followed by Bonferroni’s post-hoc testing of individual values.
various pharmacological and physiological stimuli strongly suggests that the endogenous opiate alkaloids are functionally significant within the brain. However, not all anticonvulsive agents are able to modify the CNS morphine content, since phenobarbital
had no effect (data not presented). Blocking the mu opiate receptor with naltrexone failed to produce further feedback increases in the opiate alkaloid content following convulsions. This could be interpreted that either the opiate levels achieved during convulsions were already maximal or that
Fig. 3. The effect of phenytoin on morphine levels in rat brain. Groups of rats (N56) were sacrificed 15 and 30 following phenytoin (25 mg / kg, i.p.) administration. Mean values are stated as pmol / g tissue6s.e.m. Significance (*P.0.05) was determined by one-way ANOVA followed by Bonferroni’s post-hoc testing of individual values.
S. Spector et al. / Brain Research 915 (2001) 155 – 160
15 min was too short a time for feedback-induced increases to occur. Since the evidence would indicate that the turnover of the opiate alkaloids is quite rapid, one would expect an effect by 15 min. Carbamazepine, although an effective anticonvulsant, also exerts antinocioceptive, antimanic and antidepressive actions [32]. Carbamazepine caused a robust elevation of morphine in the hypothalamus, striatum and midbrain within 15 min. Phenytoin, having a hydantoin structure in contrast to the iminostilbine structure of carbamazepine, showed an effect on CNS morphine levels that was similar to that seen with carbamazepine. Both phenytoin and carbamazepine affected the specific brain region without altering the levels of opiate alkaloids in peripheral tissues. Both anticonvulsant drugs elevated the morphine content in the hypothalamus. The hypothalamus is a multi-functional center for the control of vasomotor and endocrine activity and regulates endocrine function of the adrenohypophysis through a vascular portal system via the action of various releasing factors. Corticotropin releasing factor (CRF) has been reported to elicit, in a dose-related fashion, a spectrum of effects ranging from arousal to signs of convulsions [8,9]. Exogenously administered morphine inhibits the release of CRF [38,39]. We hypothesize that one of the mechanisms for the anticonvulsant actions of both carbamazepine and phenytoin is to produce an elevation of the endogenous morphine content in the hypothalamus, thereby inhibiting CRF secretion which, in turn, alters ACTH and corticosteroid secretion. Studies also have shown that ACTH and corticosteroids can increase susceptibility to seizures [5,9,15,28,35,39]. We have demonstrated that both pentylenetetrazole convulsions and the anticonvulsant drugs causes an increase in striatal morphine levels. Exogenous morphine has been shown to decrease GABAergic turnover in the caudate [22], while increasing activity in the GAGAergic pathway from the caudate to the globus pallidus [21]. GABA turnover in the substantia nigra is also increased [22]. The efferent neurons from the substantia nigra inhibit thalamic neurons that project to the cortex and thereby inhibit excitatory fibers to the cortex. The effects of carbamazepine on codeine levels are very interesting. Codeine content in the hypothalamus and hippocampus was elevated 7–10-fold, while levels in the striatum remained unchanged. The midbrain showed smaller, but statistically significant increases. Codeine is a precursor for morphine synthesis and normally a precursor would not be expected both to accumulate and also to show a different brain distribution than its product. Based on these findings, we suggest that codeine has an independent role in the CNS, as well as acting as a precursor to morphine. We have seen a similar relationship between dopamine and norepinephrine. The other interesting finding is that both the convulsant and the anticonvulsant drugs altered hippocampal codeine levels without altering striatal levels. This is markedly different than the effect of these
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drugs on morphine levels. The hippocampal formation is integral to many functions. One such function is the regulation of corticosteroid release from the adrenal gland that accompany changes in mood and behavioral states. Under conditions of stress the paraventricular neurosecretory cells are driven by signals from the hippocampus. To date, there is no evidence that codeine has a role as a neurotransmitter. The results of this study raises that possibility. Elevated levels of brain morphine, and possibly codeine, following both convulsions and the administration of anticonvulsant drugs suggest a new role for the endogenous opiate alkaloids, i.e., they play a modulatory role in promoting seizure arrest and refractoriness. We suggest that during a convulsion morphine levels are elevated as part of the homeostaic mechanisms present in the brain to counteract and / or terminate the convulsion. We also propose that the endogenous anticonvulsant morphine is involved in the mechanism of action of some anticonvulsant drugs and, thus, the brain levels of morphine increase in response to their administration. North and Williams [25] have proposed that potassium conductance is increased by opiates and some anticonvulsant drugs, which results in a reduction of the action potential duration. Nakamura et al. [24] reported that morphine reduced excitability due to membrane hyperpolarization and / or conductance increases. We recognize that carbamazepine and phenytoin exert an action on sodium channels and consequently we are not proposing that the two anticonvulsant drugs studied act only through the opiate alkaloid system, particularly since both have long half lives and exert their anticonvulsant action for many hours. However, the ability of both convulsant and anticonvulsant drugs to increase the levels of the endogenous opiate alkaloids only in specific brain regions suggests a new approach to the pathophysiology of seizures by exerting an effect of the propagation of action potentials in nerve trunks. We propose that the opiate alkaloids be considered as neurotransmitters that play a role in modulation of convulsions and introduce the possibility that drugs that raise brain opiate alkaloid concentrations offer a new therapeutic approach to certain forms of epilepsy.
References [1] M.W. Adler, C.H. Lin, S.H. Keimath, S. Braverman, E.B. Geller, Anticonvulsant action of acute morphine administration in rats, J. Pharmacol. Exp. Ther. 195 (1976) 655–660. [2] I. Ahmad, B.J. Pleuvry, Interactions between opioid drugs and propofol in laboratory models of seizures, Brit. J. Aneth. 74 (1995) 311–314. [3] E. Bianchi, M. Guana, A. Tagliamont, Immunocytochemical localization of endogenous codeine and morphine, Adv. Neuroimmunol. 4 (1999) 83–92. [4] J.E. Chapman, E.J. Walesek, Antagonism of some toxic effects of dextro-propoxyphene by morphine, Toxicol. Appl. Pharmacol. 4 (1962) 752–758.
160
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[5] N. Conforti, S. Feldman, Effect of cortisol on the excitability of limbic structure of the brain in freely moving rats, J. Neurol. Sci. 26 (1975) 29–38. [6] J. Donnerer, G. Cardinale, J. Coffey, C.A. Lisek, I. Tardine, S. Spector, Chemical characterization and regulation of endogenous morphine and codeine in rat, J. Pharmacol. Exp.Ther. 212 (1989) 583–587. [7] J. Donnerer, K. Oka, A. Brossi, K.C. Rice, S. Spector, Presence and formation of codeine and morphine in the rat, Proc. Natl. Acad. Sci. USA 83 (1986) 4566–4567. [8] C.L. Ehlers, S.J. Henriksen, M. Wang, J. Riviera, W. Vale, F.E. Bloom, Corticotropin releasing factor increases in brain excitability and convulsive seizures in rats, Brain Res. 278 (1983) 332–346. [9] C.L. Ehlers, E.K. Killan, The influence of cortisone on EEG and seizure activity in the baboon Papio Papio, Electroneph. Clin. Neurophysiol. 47 (1979) 404–410. [10] F. Foote, K. Gale, Morphine potentiates seizures induced by GABA antagonists and attenuates seizures induced by electroshock in rats, Eur. J. Pharmacol. 95 (1983) 259–264. [11] H.H. Frey, Anticonvulsant effect of morphine and morphine-like analgesics in mongolian gerbils, Pharmacology 23 (1986) 335–339. [12] T.A. Fuller, J.W. Olney, V.T. Conbog, Naloxone blocks morphine enhancement of kainic acid neurotoxicity, Life Sci. 31 (1982) 229–233. [13] T.R. Fuller, J.W. Olney, Effect of morphine or naloxone on kainic acid neurotoxicity, Life Sci. 24 (1979) 1793–1798. [14] A. Gintzler, M. Gershon, S. Spector, A nonpeptide morphine like compound immunocytochemical localization in the mouse brain, Science 199 (1978) 447–448. [15] G.H. Glaser, On the relationship between adrenal cortical activity and the convulsive state, Epilepsia 2 (1953) 7–14. [16] A. Goldstein, R.W. Barrett, I.F. James, C.J. Weitz, L. Kinipnejor, H. Rappoport, Morphine and other opiates from beef brain and adrenal, Proc. Natl. Acad. Sci. USA 82 (1985) 5203–5207. [17] H. Kodaira, S. Spector, Biotransformation of thebaine to oripaviine, codeine and morphine by rat liver, kidney and brain microsomes, Proc. Natl. Acad. Sci. USA 85 (1989) 1267–1271. [18] C.S. Lee, S. Spector, Changes of endogenous morphine and codeine content in fasting rat, J. Pharmacol. Exp. Ther. 257 (1991) 647–651. [19] R.J. Lee, J.G. Bajorek, Similar anticonvulsant but unique behavioral effects of opioid agonists in the seizure sensitive Mongolian gerbil, Neuropharmacology 23 (1984) 517–524. [20] B.W. Meidnen, C. Menini, J.M. Statzman, R. Naquet, Effects of opiate like peptide, morphine and naloxone in the photosensitive baboon, Brain Res. 17 (1979) 330–348. [21] F. Moroni, D.L. Chency, E. Peralta, E. Costa, Opioid receptor agonists and modulators of gamma-amino butyric acid turnover in the nucleus caudatus, globus palludus and substantia nigra of the rat, J. Pharmacol. Exp. Ther. 20 (1978) 870–877. [22] E. Moroni, E. Peralta, D.L. Cheney, E. Costa, On the regulation of
[23]
[24]
[25] [26] [27]
[28] [29] [30]
[31] [32] [33] [34] [35]
[36]
[37] [38]
[39]
gamma-aminobutyric acid neurons on caudate, palludus and nigra effects of opioids and dopamine agonists, J. Pharmacol. Exp. Ther. 208 (1979) 190–194. I.D. Munjal, D.E. Schmidt, S. Spector, Role of endogenous morphine in the attenuation of opiate withdrawal syndrome by Nacetylmuramyl-L-alanine-D-isoglutamine (MDP), Neuropsychopharmacology 15 (1996) 99–103. S. Nakamura, J.M. Tepper, S.J. Young, S. Ling, P.M. Groves, Noradrenergic terminal excitability: effects of opioids, Neurosci. Lett. 30 (1982) 57–62. R.A. North, T.S. Williams, How do opiates inhibit transmitter release, Neuroscience 6 (1983) 337–339. K. Oka, O.J.D. Kantrowitz, S. Spector, Isolation of morphine from skin, Proc. Natl. Acad. Sci. USA 82 (1985) 1852–1854. R.M. Post, S.R.B. Weiss, Endogenous biochemical abnormalities in affective illness therapeutic verses pathogenic, Biol. Psych. 32 (1992) 469–484. R.P. Rose, F. Morell, T.J. Hoeppner, Influence of pituitary Badrenal hormones on kindling, Brain Res. 169 (1979) 303–315. O.C. Snead, Opiate induced seizures. A study of mu and delta specific mechanisms, Exp. Neurol. 93 (1986) 348–358. S. Spector, I.D. Munjal, D.E. Schmidt, Effect of the immune stimulant levamisole on opiate withdrawal and levels of endogenous opiate alkaloids and monoamine neurotransmitters in rat brain, Neuropsychopharmacology 19 (1998) 417–427. S. Spector, Quantitative determination of morphine in serum by radioimmunoassay, J. Pharmacol. Expt. Ther. 178 (1971) 253–258. R.M. Post, T.W. Uhde, Carbamazepine in bipolar illness, Psychopharmacol. Bull. 21 (1985) 10–17. S. Spector, C. Parker, Morphine: radioimmunassay, Science 168 (1970) 1347. C.L. Swarts, Propoxyphene poisoning, Am. J. Dis. Child 107 (1984) 177–179. C. Torda, H.G. Wolff, Effect of various concentrations of adrenocorticotropin hormone on electrical activity of brain and on sensitivity in convulsive inducing agent, Am. J. Physiol. 168 (1952) 406–415. F.C. Tortella, J.B. Long, Characterization of opioid peptide like anticonvulsant activity in rat cerebrospinal fluid, Brain Res. 456 (1988) 139–146. G. Urca, H. Franck, Pro and anticonvulsant action of morphine in rats, Pharmacol. Biochem. Behav. 13 (1980) 343–348. A.P. Zis, R.T. Haskett, A.A. Albate, B.J. Caroll, Morphine inhibits cortisol and stimulates prolactin secretion, Psychoendocrinology 9 (1984) 423–427. A.P. Zis, R.A. Remick, M. Campbell, E. Clark, R. Goldner, B.F. Kelly, G.M. Brown, Effect of morphine on cortisol and prolactin secretion in anorexia nervosa and depression, Clin. Endocrinol. 30 (1980) 421–427.
Research report
Endogenous morphine and codeine Possible role as endogenous anticonvulsants S. Spector*, I. Munjal, D.E. Schmidt Department of Psychiatry, Vanderbilt University Medical Center, Nashville, TN 37232, USA Accepted 12 July 2001
Abstract Exogenously administered morphine can have both convulsive or anticonvulsive effects, depending on the dose and species. The levels of the endogenous opiate alkaloids morphine and codeine were significantly elevated in specific rat brain regions by the convulsive drug, pentylenetetrazole, as well as by the anticonvulsant drugs, carbamazepine and phenytoin. Morphine and codeine levels in peripheral tissues (heart, lung, spleen and adrenal) were unaffected by these drugs. Maximal increases in morphine levels were seen in the hypothalamus and striatum (2–10-fold), while lesser increases occurred in the midbrain and brain stem (2–4-fold). Codeine levels were also markedly increased in hypothalamus (5–10 fold), In contrast to morphine, codeine levels were also increased in the hippocampus (2–10-fold), but were unchanged in the striatum. These studies suggest that the endogenous alkaloids morphine and codeine are involved in the modulation of convulsions and that morphine and / or codeine may act as an endogenous anticonvulsant. 2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Opioids: anatomy, physiology and behavior Keywords: Endogenous morphine and codeine; Pentylenetetrazole; Carbamazepine; Phenytoin; Convulsion; Anticonvulsant
1. Introduction The opiate alkaloids morphine and codeine have been identified in mammalian tissues [6,16,26] and mammalian tissues have the capacity to synthesize these alkaloids both in vivo [6,16] and in vitro [17]. It has also been reported that morphine is located in neurons [3,14] and can be released under conditions known to release other neurotransmitters [3]. The endogenous opiate alkaloids exhibit plasticity: their levels are altered by immuno-stimulants, and pain and fasting have been shown to modify the levels of morphine and codeine in brain [18,23,30]. Thus, it appears that a morphinergic neuronal pathway is present in mammalian brain. The effect of morphine administration is controversial. Exogenous morphine results in either a pro-convulsant or anticonvulsant effect in different animal models, depend*Corresponding author. Tel.: 11-615-343-7676; fax: 11-615-3438639. E-mail address: [email protected] (S. Spector).
ing on the dose. Low, sub-analgesic doses of morphine elevates the electroconvulsive threshold in rats and this anticonvulsive effect can be reversed by naloxone [1,4,12,13,29,31,34,35,37]. Ahmed and Pleuvry [2] reported that when seizures were induced with either bicuculline or kainic acid morphine exerted an anticonvulsant action. They also showed that morphine enhanced the anticonvulsant effect of phenytoin in the mouse electroshock model of seizures. Foote and Gale [10] also demonstrated that morphine exerted an anticonvulsant effect on electroshock seizures. Frey [11] reported that morphine suppressed blast-induced seizure. Using another animal model, the seizure-sensitive Mongolian gerbil, Lee and Bajorek [19] showed that morphine exerted an anticonvulsant effect. The brain levels of endogenous morphine or codeine are consistent with their possible role as an anticonvulsant [20]. Post and Weiss [27] have hypothesized the existence of compensatory and adaptive mechanisms that come into play to counterbalance pathological processes. They have reported that repeated amygdala-kindled seizures evoked
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02837-2
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the release of an endogenous substance that acts to enhance the efficacy of the anticonvulsant drug carbamazepine. Tortella and Long [36] have suggested that the endogenous anticonvulsant substance is an opioid peptide. However, since carbamazepine also exhibits antinocioceptive, anti-manic and antidepressant effects, the effects of carbamazepine and other anticonvulsant agents (phenytoin and phenobarbital) and the convulsant drug pentylenetetrazole on the endogenous levels of morphine and codeine were measured in brain and selected peripheral tissues.
(95.5%), 1-propanol (3%), pyridine (0.8%) and acetic acid (0.3%). The eluate was evaporated to dryness and the sample was resuspended in 0.250 ml of mobile phase (pH 5.2). The sample was then filtered and injected into a Lichrosorb RP-18 column at a flow-rate of 1.5 ml / min. The fractions corresponding to the elution times of morphine and codeine were collected and evaporated to dryness. The recovery, which was about 70–80%, was determined by parallel analysis of replicate samples spiked with authentic morphine and codeine and the values corrected accordingly.
2. Materials and methods
2.1. Radioimmunoassay for morphine and codeine
All research was conducted accordance with the NIH guide for the care and use of laboratory animals and was approved by the Vanderbilt Animal Care Committee. Male 225–250-g Sprague–Dawley rats were injected with either pentylenetetrazole (60 mg / kg, i.p.) or carbamazepine (25 mg / kg in 20% DMSO–saline, i.p.), phenytoin (25 mg / kg, i.p.) or phenobarbital (25 mg / kg, i.p.) dissolved in saline and the response and time course on the morphine and codeine content in specific brain regions and selected peripheral tissues was measured. Control rats received an equivalent volume of the appropriate vehicle and were sacrificed at the indicated times. At various times following drug administration, the rats were anesthetized by 50% carbon dioxide and decapitated. The brains were quickly removed, chilled in ice cold saline and dissected on ice. The peripheral tissues, lung, heart, spleen and adrenals were also removed and frozen at 2708C until assayed. Morphine and codeine were extracted from the tissues and assayed by RIA as described by Donnerer et al. [6]. The morphine antibody has been previously shown to be highly selective for morphine and does not cross react with opioid peptides, narcotic antagonists or the morphine precursors salutaridine, reticuline or thebaine [7,32]. Briefly, the tissues were washed free of blood or faeces, blotted and frozen immediately. At the time of assay, tissues were weighed and then homogenized in 10 mM HCl to yield 100 mg tissue / ml. The opiate conjugates were hydrolyzed by adding 6 ml of 12 N HCl to 10 ml of tissue homogenate and heating in a water bath (95–1008) for 30 min. The hydrolyzed samples were centrifuged 30 min at 10 0003g at 48C and filtered through glass wool. The pH was adjusted to 8.5–9.0. Five volumes of a 1:9 mixture of n-butanol in chloroform was added and the mixture was shaken for 5 min. The organic phase was collected and back-extracted into 10 mM HCl. The aqueous phase was evaporated to dryness in a Savant vacuum centrifuge. The dried samples were reconstituted in 5 ml of 10 mM HCl plus phosphate-buffered saline, pH 7.4, and the pH was adjusted to 8.5–9.0. The samples were passed through C 18 -Sep Pak cartridge which had been activated with methanol and then flushed with water. The samples were eluted with a mobile phase, containing water
The samples were resuspended in 200 ml of phosphate buffered saline (pH 7.4), 50 ml of the morphine antibody [33] and [ 125 I]iodo-morphine were added and the samples were incubated for 90 min at 48C. A standard curve of authentic morphine and codeine were similarly treated. Following equilibration, 250 ml of saturated ammonium sulfate was added and the samples are incubated for 45 min and filtered through GF / G glass fiber filter. The filters were counted in a gamma counter and concentrations of morphine and codeine established using Riacole Software. Significance was determined by one-way ANOVA followed by Bonferroni’s post-hoc testing of individual values.
3. Results The effect of pentyleneterazole (60 mg / kg, i.p.) on regional morphine and codeine levels in brain are presented in Fig. 1a and b. TonicBclonic convulsions occurred within 2–3 min of drug administration and the rats were sacrificed 15 and 30 min after the drug was given. There was about a three-fold increase in morphine content in the hypothalamus, striatum and midbrain within 15 min. The elevated levels persisted for more than 30 min in the hypothalamus, while morphine concentrations in the striatum and midbrain returned to control values by 30 min. A smaller, non-significant increase in morphine levels at 15 min was observed in the hippocampus.The morphine content in the brain stem, cortex and cerebellum was unaffected (Fig. 1a). Similar to morphine, codeine levels in the hypothalamus, striatum and midbrain were significantly elevated, while levels in the cortex and cerebellum were unchanged (Fig. 1b). In contrast to morphine, codeine concentrations were also increased in the hippocampus and brainstem. These regional differences and the increases observed persisted longer than seen with morphine. Peripheral tissues content of morphine and codeine in the heart, lung, spleen and adrenal levels were not changed (data not shown). Blocking the mu opiate receptor with naltrexone (5
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Fig. 1. (A) and (B) The effect of pentylenetetrazole on morphine and codeine levels in rat brain. Groups of rats (N56) were sacrificed 15 and 30 min following administration of pentylenetetrazole (60 mg / kg, i.p.) or vehicle only. In an additional group, naltrexone (5 mg / kg, i.p.) was administered 10 min prior to the pentylenetetrazole. Mean values are stated as pmol / g tissue6s.e.m. There was no differences between the 15- and 30-min vehicle groups and the values were combined. Significance (*P.0.05) was determined by one-way ANOVA followed by Bonferroni’s post-hoc testing of individual values.
mg / kg, i.p.) administered 10 min prior to the convulsant agent did not cause a feedback-induced increase in either morphine or codeine (Fig. 1a and b). Naltrexone alone did not alter the levels of morphine or codeine. Fig. 2a shows that the anticonvulsant agent carbamazepine markedly elevated the morphine content in the hypothalamus, striatum and midbrain within 15 min. There was also a significant increase in the cerebellum. Again, there was a different effect of carbamazepine on codeine content in the brain (Fig. 2b). The hypothalamus and hippocampus showed the greatest effect following carbamazepine administration, while the striatum, cerebellum and cortex were unchanged. The midbrain had an elevated codeine content at 30 min and the brainstem had a significant elevation at 15 min. The peripheral tissue content of morphine and codeine were unchanged (data not shown). Fig. 3 shows that phenytoin also increased the morphine levels in specific brain regions. The morphine levels in the hypothalamus was increased seven-fold within 15 min and returned to control levels by 30 min. The striatal levels of
morphine increased ten-fold within 15 min and the elevated levels persisted for the 30-min period. Midbrain, brainstem and cerebellum all showed significant increases in morphine content. The administration of vehicle alone did not change the morphine or codeine levels at any time point and the control values were combined from all time points. There also were no changes in morphine or codeine levels in the peripheral tissues (data not shown).
4. Discussion The data presented here demonstrates that the brain concentrations of both morphine and codeine are elevated following pentylenetetrazole-induced convulsions. The levels also increased following administration of the anticonvulsant drugs carbamazepine and phenytoin, drugs with different chemical structures. It has been previously reported that pain, stress, starvation and immunostimulants [18,23,30] also alter the levels of endogenous morphine and codeine in the brain. This plasticity in response to
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Fig. 2. (A) and (B) The effect of carbamazepine on morphine and codeine levels in rat brain. Groups of rats (N56) were sacrificed 15 and 30 min following administration of carbamazepine (25 mg / kg, i.p.) or vehicle only. Mean values are stated as pmol / g tissue6s.e.m. There was no differences between the 15- and 30-min vehicle groups and the values were combined. Significance (*P.0.05) was determined by one-way ANOVA followed by Bonferroni’s post-hoc testing of individual values.
various pharmacological and physiological stimuli strongly suggests that the endogenous opiate alkaloids are functionally significant within the brain. However, not all anticonvulsive agents are able to modify the CNS morphine content, since phenobarbital
had no effect (data not presented). Blocking the mu opiate receptor with naltrexone failed to produce further feedback increases in the opiate alkaloid content following convulsions. This could be interpreted that either the opiate levels achieved during convulsions were already maximal or that
Fig. 3. The effect of phenytoin on morphine levels in rat brain. Groups of rats (N56) were sacrificed 15 and 30 following phenytoin (25 mg / kg, i.p.) administration. Mean values are stated as pmol / g tissue6s.e.m. Significance (*P.0.05) was determined by one-way ANOVA followed by Bonferroni’s post-hoc testing of individual values.
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15 min was too short a time for feedback-induced increases to occur. Since the evidence would indicate that the turnover of the opiate alkaloids is quite rapid, one would expect an effect by 15 min. Carbamazepine, although an effective anticonvulsant, also exerts antinocioceptive, antimanic and antidepressive actions [32]. Carbamazepine caused a robust elevation of morphine in the hypothalamus, striatum and midbrain within 15 min. Phenytoin, having a hydantoin structure in contrast to the iminostilbine structure of carbamazepine, showed an effect on CNS morphine levels that was similar to that seen with carbamazepine. Both phenytoin and carbamazepine affected the specific brain region without altering the levels of opiate alkaloids in peripheral tissues. Both anticonvulsant drugs elevated the morphine content in the hypothalamus. The hypothalamus is a multi-functional center for the control of vasomotor and endocrine activity and regulates endocrine function of the adrenohypophysis through a vascular portal system via the action of various releasing factors. Corticotropin releasing factor (CRF) has been reported to elicit, in a dose-related fashion, a spectrum of effects ranging from arousal to signs of convulsions [8,9]. Exogenously administered morphine inhibits the release of CRF [38,39]. We hypothesize that one of the mechanisms for the anticonvulsant actions of both carbamazepine and phenytoin is to produce an elevation of the endogenous morphine content in the hypothalamus, thereby inhibiting CRF secretion which, in turn, alters ACTH and corticosteroid secretion. Studies also have shown that ACTH and corticosteroids can increase susceptibility to seizures [5,9,15,28,35,39]. We have demonstrated that both pentylenetetrazole convulsions and the anticonvulsant drugs causes an increase in striatal morphine levels. Exogenous morphine has been shown to decrease GABAergic turnover in the caudate [22], while increasing activity in the GAGAergic pathway from the caudate to the globus pallidus [21]. GABA turnover in the substantia nigra is also increased [22]. The efferent neurons from the substantia nigra inhibit thalamic neurons that project to the cortex and thereby inhibit excitatory fibers to the cortex. The effects of carbamazepine on codeine levels are very interesting. Codeine content in the hypothalamus and hippocampus was elevated 7–10-fold, while levels in the striatum remained unchanged. The midbrain showed smaller, but statistically significant increases. Codeine is a precursor for morphine synthesis and normally a precursor would not be expected both to accumulate and also to show a different brain distribution than its product. Based on these findings, we suggest that codeine has an independent role in the CNS, as well as acting as a precursor to morphine. We have seen a similar relationship between dopamine and norepinephrine. The other interesting finding is that both the convulsant and the anticonvulsant drugs altered hippocampal codeine levels without altering striatal levels. This is markedly different than the effect of these
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drugs on morphine levels. The hippocampal formation is integral to many functions. One such function is the regulation of corticosteroid release from the adrenal gland that accompany changes in mood and behavioral states. Under conditions of stress the paraventricular neurosecretory cells are driven by signals from the hippocampus. To date, there is no evidence that codeine has a role as a neurotransmitter. The results of this study raises that possibility. Elevated levels of brain morphine, and possibly codeine, following both convulsions and the administration of anticonvulsant drugs suggest a new role for the endogenous opiate alkaloids, i.e., they play a modulatory role in promoting seizure arrest and refractoriness. We suggest that during a convulsion morphine levels are elevated as part of the homeostaic mechanisms present in the brain to counteract and / or terminate the convulsion. We also propose that the endogenous anticonvulsant morphine is involved in the mechanism of action of some anticonvulsant drugs and, thus, the brain levels of morphine increase in response to their administration. North and Williams [25] have proposed that potassium conductance is increased by opiates and some anticonvulsant drugs, which results in a reduction of the action potential duration. Nakamura et al. [24] reported that morphine reduced excitability due to membrane hyperpolarization and / or conductance increases. We recognize that carbamazepine and phenytoin exert an action on sodium channels and consequently we are not proposing that the two anticonvulsant drugs studied act only through the opiate alkaloid system, particularly since both have long half lives and exert their anticonvulsant action for many hours. However, the ability of both convulsant and anticonvulsant drugs to increase the levels of the endogenous opiate alkaloids only in specific brain regions suggests a new approach to the pathophysiology of seizures by exerting an effect of the propagation of action potentials in nerve trunks. We propose that the opiate alkaloids be considered as neurotransmitters that play a role in modulation of convulsions and introduce the possibility that drugs that raise brain opiate alkaloid concentrations offer a new therapeutic approach to certain forms of epilepsy.
References [1] M.W. Adler, C.H. Lin, S.H. Keimath, S. Braverman, E.B. Geller, Anticonvulsant action of acute morphine administration in rats, J. Pharmacol. Exp. Ther. 195 (1976) 655–660. [2] I. Ahmad, B.J. Pleuvry, Interactions between opioid drugs and propofol in laboratory models of seizures, Brit. J. Aneth. 74 (1995) 311–314. [3] E. Bianchi, M. Guana, A. Tagliamont, Immunocytochemical localization of endogenous codeine and morphine, Adv. Neuroimmunol. 4 (1999) 83–92. [4] J.E. Chapman, E.J. Walesek, Antagonism of some toxic effects of dextro-propoxyphene by morphine, Toxicol. Appl. Pharmacol. 4 (1962) 752–758.
160
S. Spector et al. / Brain Research 915 (2001) 155 – 160
[5] N. Conforti, S. Feldman, Effect of cortisol on the excitability of limbic structure of the brain in freely moving rats, J. Neurol. Sci. 26 (1975) 29–38. [6] J. Donnerer, G. Cardinale, J. Coffey, C.A. Lisek, I. Tardine, S. Spector, Chemical characterization and regulation of endogenous morphine and codeine in rat, J. Pharmacol. Exp.Ther. 212 (1989) 583–587. [7] J. Donnerer, K. Oka, A. Brossi, K.C. Rice, S. Spector, Presence and formation of codeine and morphine in the rat, Proc. Natl. Acad. Sci. USA 83 (1986) 4566–4567. [8] C.L. Ehlers, S.J. Henriksen, M. Wang, J. Riviera, W. Vale, F.E. Bloom, Corticotropin releasing factor increases in brain excitability and convulsive seizures in rats, Brain Res. 278 (1983) 332–346. [9] C.L. Ehlers, E.K. Killan, The influence of cortisone on EEG and seizure activity in the baboon Papio Papio, Electroneph. Clin. Neurophysiol. 47 (1979) 404–410. [10] F. Foote, K. Gale, Morphine potentiates seizures induced by GABA antagonists and attenuates seizures induced by electroshock in rats, Eur. J. Pharmacol. 95 (1983) 259–264. [11] H.H. Frey, Anticonvulsant effect of morphine and morphine-like analgesics in mongolian gerbils, Pharmacology 23 (1986) 335–339. [12] T.A. Fuller, J.W. Olney, V.T. Conbog, Naloxone blocks morphine enhancement of kainic acid neurotoxicity, Life Sci. 31 (1982) 229–233. [13] T.R. Fuller, J.W. Olney, Effect of morphine or naloxone on kainic acid neurotoxicity, Life Sci. 24 (1979) 1793–1798. [14] A. Gintzler, M. Gershon, S. Spector, A nonpeptide morphine like compound immunocytochemical localization in the mouse brain, Science 199 (1978) 447–448. [15] G.H. Glaser, On the relationship between adrenal cortical activity and the convulsive state, Epilepsia 2 (1953) 7–14. [16] A. Goldstein, R.W. Barrett, I.F. James, C.J. Weitz, L. Kinipnejor, H. Rappoport, Morphine and other opiates from beef brain and adrenal, Proc. Natl. Acad. Sci. USA 82 (1985) 5203–5207. [17] H. Kodaira, S. Spector, Biotransformation of thebaine to oripaviine, codeine and morphine by rat liver, kidney and brain microsomes, Proc. Natl. Acad. Sci. USA 85 (1989) 1267–1271. [18] C.S. Lee, S. Spector, Changes of endogenous morphine and codeine content in fasting rat, J. Pharmacol. Exp. Ther. 257 (1991) 647–651. [19] R.J. Lee, J.G. Bajorek, Similar anticonvulsant but unique behavioral effects of opioid agonists in the seizure sensitive Mongolian gerbil, Neuropharmacology 23 (1984) 517–524. [20] B.W. Meidnen, C. Menini, J.M. Statzman, R. Naquet, Effects of opiate like peptide, morphine and naloxone in the photosensitive baboon, Brain Res. 17 (1979) 330–348. [21] F. Moroni, D.L. Chency, E. Peralta, E. Costa, Opioid receptor agonists and modulators of gamma-amino butyric acid turnover in the nucleus caudatus, globus palludus and substantia nigra of the rat, J. Pharmacol. Exp. Ther. 20 (1978) 870–877. [22] E. Moroni, E. Peralta, D.L. Cheney, E. Costa, On the regulation of
[23]
[24]
[25] [26] [27]
[28] [29] [30]
[31] [32] [33] [34] [35]
[36]
[37] [38]
[39]
gamma-aminobutyric acid neurons on caudate, palludus and nigra effects of opioids and dopamine agonists, J. Pharmacol. Exp. Ther. 208 (1979) 190–194. I.D. Munjal, D.E. Schmidt, S. Spector, Role of endogenous morphine in the attenuation of opiate withdrawal syndrome by Nacetylmuramyl-L-alanine-D-isoglutamine (MDP), Neuropsychopharmacology 15 (1996) 99–103. S. Nakamura, J.M. Tepper, S.J. Young, S. Ling, P.M. Groves, Noradrenergic terminal excitability: effects of opioids, Neurosci. Lett. 30 (1982) 57–62. R.A. North, T.S. Williams, How do opiates inhibit transmitter release, Neuroscience 6 (1983) 337–339. K. Oka, O.J.D. Kantrowitz, S. Spector, Isolation of morphine from skin, Proc. Natl. Acad. Sci. USA 82 (1985) 1852–1854. R.M. Post, S.R.B. Weiss, Endogenous biochemical abnormalities in affective illness therapeutic verses pathogenic, Biol. Psych. 32 (1992) 469–484. R.P. Rose, F. Morell, T.J. Hoeppner, Influence of pituitary Badrenal hormones on kindling, Brain Res. 169 (1979) 303–315. O.C. Snead, Opiate induced seizures. A study of mu and delta specific mechanisms, Exp. Neurol. 93 (1986) 348–358. S. Spector, I.D. Munjal, D.E. Schmidt, Effect of the immune stimulant levamisole on opiate withdrawal and levels of endogenous opiate alkaloids and monoamine neurotransmitters in rat brain, Neuropsychopharmacology 19 (1998) 417–427. S. Spector, Quantitative determination of morphine in serum by radioimmunoassay, J. Pharmacol. Expt. Ther. 178 (1971) 253–258. R.M. Post, T.W. Uhde, Carbamazepine in bipolar illness, Psychopharmacol. Bull. 21 (1985) 10–17. S. Spector, C. Parker, Morphine: radioimmunassay, Science 168 (1970) 1347. C.L. Swarts, Propoxyphene poisoning, Am. J. Dis. Child 107 (1984) 177–179. C. Torda, H.G. Wolff, Effect of various concentrations of adrenocorticotropin hormone on electrical activity of brain and on sensitivity in convulsive inducing agent, Am. J. Physiol. 168 (1952) 406–415. F.C. Tortella, J.B. Long, Characterization of opioid peptide like anticonvulsant activity in rat cerebrospinal fluid, Brain Res. 456 (1988) 139–146. G. Urca, H. Franck, Pro and anticonvulsant action of morphine in rats, Pharmacol. Biochem. Behav. 13 (1980) 343–348. A.P. Zis, R.T. Haskett, A.A. Albate, B.J. Caroll, Morphine inhibits cortisol and stimulates prolactin secretion, Psychoendocrinology 9 (1984) 423–427. A.P. Zis, R.A. Remick, M. Campbell, E. Clark, R. Goldner, B.F. Kelly, G.M. Brown, Effect of morphine on cortisol and prolactin secretion in anorexia nervosa and depression, Clin. Endocrinol. 30 (1980) 421–427.