NEUROFILAMENT (NF) proteins, the major components of the neuronal cytoskeleton, have been shown to represent previously unknown targets for the chronic effects of morphine in rats. This study was designed to evaluate the abundance of immunoreactive NF-L (68 kDa) proteins in post-mortem brains of chronic opiate addicts who had died of a heroin or methadone overdose. Levels of NF-L proteins were assessed by immunoblotting techniques. Levels of immunoreactive NF-L proteins were markedly decreased (47%, n = 17) in the frontal cortex. The reduced abundance of brain NF-L proteins was not related to the post-mortem delay or to the plasma concentrations of opiates, suggesting that the observed changes represent a specific long-term effect of opiate drugs. Because of the functions associated with NF proteins (e.g. axonal transport), this finding suggests that opiate drugs may induce neuronal damage after chronic abuse in humans.

The abundance of protein kinase C-α and β isoforms (PKC-αβ), PKC-α messenger (m) RNA and guanine nucleotide-binding G protein subunits (Gα i1/2 , Gα o and Gβ) were quantitated in the rat cerebral cortex after acute and chronic treatments with various opiate drugs. Acute (100 mg/kg for 2 h) and chronic (10 to 100 mg/kg for 5 days) treatment with morphine decreased similarly the immunoreactivity of PKC-αβ (28% and 32%, respectively). Acute (2 h) and chronic treatment (5 days) with other µ-agonists heroin (30 mg/kg and 10 to 30 mg/kg) and methadone (30 mg/kg and 5 to 30 mg/kg) also induced similar decreases of PKC-αβ (acute: 25% and 23%; chronic: 28% and 18%). After the chronic treatments, spontaneous (48 h) or naloxone (2 mg/kg)-precipitated opiate withdrawal (2 h) resulted in up-regulation of PKC-αβ above control levels (30-38%), and in the case of morphine withdrawal in a concomitant marked increase in the expression of PKC-α mRNA levels (2.3-fold). Acute (2 h) treatments with pentazocine (80 mg/kg, mixed κ/δ-agonist and µ-antagonist), spiradoline (30 mg/kg, selective κ-agonist) and [D-Pen 2 , D-Pen 5 ] enkephalin (14 nmol i.c.v., selective δ-agonist) induced significant decreases of PKC-αβ (19-33%). Chronic (5 days) treatment with pentazocine (10 to 80 mg/kg), but not spiradoline (2 to 30 mg/kg), also induced a similar decrease of PKC-αβ (35%). In pentazocine-or spiradoline-dependent rats, naloxone (2 mg/kg) did not induce up-regulation of brain PKC-αβ. Acute (10 mg/kg for 2 h) and chronic (2×10 mg/kg for 5 and 14 days) treatment with naloxone did not alter PKC-αβ immunoreactivity.Chronic, but not acute, treatment with µ-agonists (morphine, heroin and methadone) increased the immunoreactivities of Gα i1/2 (33-37%), Gα o (25-41%) and Gβ (10-33%) protein subunits. In heroin-and methadone-dependent rats naloxone (2 mg/kg)-precipitated withdrawal (2 h) did not modify the up-regulation of these G proteins induced by chronic µ-opiate treatment. In marked contrast to µ-agonists, chronic treatment with high doses of pentazocine and spiradoline or acute treatment with [D-Pen 2 , D-Pen 5 ] enkephalin did not result in up-regulation of these G protein subunits.After chronic treatment with µ-agonists, significant negative correlations were found when the percentage changes in immunoreactivity of PKC-αβ were related to the percentage changes in immunoreactivity of Gα i1/2 (r = -0.53, n = 29) and Gβ (r = -0.41, n = 24) in the same brains. PKC-αβ abundance did not correlate significantly with the density of Gα o (r = -0.21, n = 28). Together the results indicate that the brain PKC-αβ system may play a major regulatory role in opiate tolerance and dependence. Moreover, the possible in vivo cross-communication between this regulatory enzyme and specific inhibitory G proteins may also be of relevance in the cellular and molecular processes of opiate addiction.

The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein‐coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate‐treated rats as well as in opiate‐dependent rats after spontaneous or naloxone‐precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane‐associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose‐ and time‐dependent increases (8–22%) in total GRK2 concentrations [higher increases were observed for the membrane‐associated enzyme (46%)]. Spontaneous and naloxone‐precipitated withdrawal after chronic morphine or methadone induced a marked up‐regulation in the levels of total GRK2 in the rat frontal cortex (18–25%). These results suggest that GRK2 is involved in the short‐term regulation of μ‐opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.

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