Effect of cocaine and morphine on neutral endopeptidase activity of human peripheral blood mononuclear cells cultured with lectins

We have tested the effect of alkaloids (cocaine, morphine) and enkephalins on neutral endopeptidase of peripheral blood mononuclear cells activated by lectins. When treated with concanavalin A and cocaine, peripheral blood mononuclear cells showed an enhanced activity (+110 per cent) of the membrane neutral endopeptidase, which was not related to the expression of the common acute lymphoblastic leukemia antigen at the cell surface, although both molecules have the identical amino acid sequence. Phytohemagglutinin-P, morphine and synthetic enkephalins did not induce the activity of neutral endopeptidase nor the expression of common acute lymphoblastic leukemia antigen. Our findings suggested that the drugs of abuse, cocaine and morphine, affected specific membrane constituents without altering proliferation, subcellular localization of membrane enzymes or the surface immune phenotype of peripheral blood mononuclear cells.     

Effect of a cholecystokinin tetrapeptide analogue on opioid reception under acute and chronic morphine administration

Effects of a modified CCK-4, a tetrapeptide fragment of cholecystokinin, on opioid reception and cAMP level were studied. The modified CCK-4 changed the ligand binding of the opioid receptors of μ-and δ-types in vitro. In vivo, it prevented changes in opioid reception caused by an acute morphine administration or by morphine withdrawal after its long-term administration. The CCK-4 analogue did not exert any effect in the state of intoxication after a long-term administration of morphine or even promoted the morphine effect. The injection of the CCK-4 analogue alone or together with morphine changed the forskolin-stimulated level of cAMP. These changes depended on the brain structure and the duration of the administration of morphine and the CCK-4 analogue.     

Gβγ that interacts with adenylyl cyclase in opioid tolerance originates from a Gs protein

We previously demonstrated that chronic morphine induces a change in G protein coupling by the mu opioid receptor (MOR) from Gi/o to Gs, concurrent with the instatement of an interaction between Gβγ and adenylyl cyclase types II and IV. These two signaling changes confer excitatory effects on the cell in place of the typical inhibition by opioids and are associated with morphine tolerance and dependence. Both signaling changes and these behavioral manifestations of chronic morphine are attenuated by cotreatment with ultra‐low‐dose naloxone. In the present work, using striatum from chronic morphine‐treated rats, we isotyped the Gβ within Gs and Go heterotrimers that coupled to MOR and compared these to the Gβ isotype of the Gβγ that interacted with adenylyl cyclase II or IV after chronic morphine treatment. Isotyping results show that chronic morphine causes a Gs heterotrimer associated with MOR to release its Gβγ to interact with adenylyl cyclase. These data suggest that the switch to Gs coupling by MOR in response to chronic morphine, which is attenuated by ultra‐low‐dose opioid antagonist cotreatment, leads to a two‐pronged stimulation of adenylyl cyclase utilizing both Gα and Gβγ subunits of the Gs protein novel to this receptor. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Decreased mitochondrial DNA copy number in the hippocampus and peripheral blood during opiate addiction is mediated by autophagy and can be salvaged by melatonin

Drug addiction is a chronic brain disease that is a serious social problem and causes enormous financial burden. Because mitochondrial abnormalities have been associated with opiate addiction, we examined the effect of morphine on mtDNA levels in rat and mouse models of addiction and in cultured cells. We found that mtDNA copy number was significantly reduced in the hippocampus and peripheral blood of morphine-addicted rats and mice compared with control animals. Concordantly, decreased mtDNA copy number and elevated mtDNA damage were observed in the peripheral blood from opiate-addicted patients, indicating detrimental effects of drug abuse and stress. In cultured rat pheochromocytoma (PC12) cells and mouse neurons, morphine treatment caused many mitochondrial defects, including a reduction in mtDNA copy number that was mediated by autophagy. Knockdown of the Atg7 gene was able to counteract the loss of mtDNA copy number induced by morphine. The mitochondria-targeted antioxidant melatonin restored mtDNA content and neuronal outgrowth and prevented the increase in autophagy upon morphine treatment. In mice, coadministration of melatonin with morphine ameliorated morphine-induced behavioral sensitization, analgesic tolerance and mtDNA content reduction. During drug withdrawal in opiate-addicted patients and improvement of protracted abstinence syndrome, we observed an increase of serum melatonin level. Taken together, our study indicates that opioid addiction is associated with mtDNA copy number reduction and neurostructural remodeling. These effects appear to be mediated by autophagy and can be salvaged by melatonin.     

The effect of morphine upon DNA methylation in ten regions of the rat brain

Morphine is one of the most effective analgesics in medicine. However, its use is associated with the development of tolerance and dependence. Recent studies demonstrating epigenetic changes in the brain after exposure to opiates have provided insight into mechanisms possibly underlying addiction. In this study, we sought to identify epigenetic changes in ten regions of the rat brain following acute and chronic morphine exposure. We analyzed DNA methylation of six nuclear-encoded genes implicated in brain function (Bdnf, Comt, Il1b, Il6, Nr3c1, and Tnf) and three mitochondrially-encoded genes (Mtco1, Mtco2, and Mtco3), and measured global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5?hmC) levels. We observed differential methylation of Bdnf and Il6 in the pons, Nr3c1 in the cerebellum, and Il1b in the hippocampus in response to acute morphine exposure (all P value < 0.05). Chronic exposure was associated with differential methylation of Bdnf and Comt in the pons, Nr3c1 in the hippocampus and Il1b in the medulla oblongata (all P value < 0.05). Global 5mC levels significantly decreased in the superior colliculus following both acute and chronic morphine exposure, and increased in the hypothalamus following chronic exposure. Chronic exposure was also associated with significantly increased global 5hmC levels in the cerebral cortex, hippocampus, and hypothalamus, but significantly decreased in the midbrain. Our results demonstrate, for the first time, highly localized epigenetic changes in the rat brain following acute and chronic morphine exposure. Further work is required to elucidate the potential role of these changes in the formation of tolerance and dependence.     

Inverse agonists and neutral antagonists at µ opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence

The mu opioid receptor, MOR, displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgammaS binding to cell membranes and cAMP levels in intact cells. beta-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6alpha- and 6beta-naltrexol and -naloxol, and 6beta-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6beta-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.     

Presynaptic Dopaminergic Function in the Nucleus Accumbens Following Chronic Opiate Treatment and Precipitated Withdrawal

Naloxone treatment at three days following implantation of pellets containing morphine base increased uptake of tritiated dopamine by the nucleus accumbens but did not alter efflux of tritiated dopamine by the nucleus accumbens or tritiated norepinephrine by the hippocampus. At six days following placement of pellets containing morphine base, withdrawal score was increased after treatment with either saline or naloxone, indicating that animals were undergoing spontaneous opiate withdrawal. Fractional efflux of tritiated dopamine was decreased at this time point following intermittent stimulation with 317 and 1000 M 4-aminopyridine, for striatal slices obtained from animals pretreated with either saline or naloxone. For the nucleus accumbens at six days after placement of morphine pellets, similar decreases in the efflux of tritiated dopamine were only observed in slices obtained from naloxone treated animals. Fractional dopamine efflux was also diminished after in vitro exposure to rising concentrations of 4-aminopyridine, amphetamine, or cocaine for tissue obtained from the nucleus accumbens, but not for slices from the striatum at six days following morphine pellet implantation. In conclusion, deficits in dopamine efflux by the nucleus accumbens occur at a time when animals are undergoing spontaneous opiate withdrawal at six days following morphine pellet implantation, but do not occur at an earlier time point when withdrawal is precipitated by naloxone treatment. These deficits are apparent for brain slices obtained from the striatum or nucleus accumbens after exposure to rising concentrations of different in vitro treatments, with tissue obtained from the nucleus accumbens being more sensitive than the striatum to dopamine efflux produced by a wider range of treatments.