Methods and Findings in Experimental and Clinical Pharmacology
Vol. 25, Suppl. A, 2003
ISSN 0379-0355
Copyright 2003 Prous Science, S.A.
CCC: 0379-0355/2003
http://www.prous.com

Regulation and Signaling of Opioid Receptors in the Brains of Opiate Addicts

J.A. García Sevilla

Laboratory of Neuropharmacology, University of the Balearic Islands, Palma de Mallorca, Spain; Clinical Research Unit, Department of Psychiatry, University of Geneva, Geneva, Switzerland

The modulation of m-opioid receptors and a compensatory upregulation of the cAMP signaling pathway are established molecular adaptations of morphine addiction in the brains of laboratory animals. Since little is known regarding similar or different adaptive changes in the brains of human opiate addicts, the regulation of m-opioid receptors and various signal transduction mechanisms related to these receptors were investigated in postmortem brains (n = 25) of well-defined chronic opiate abusers (tolerant/dependent state). Drug screening in blood samples showed the presence of fatal concentrations of morphine (range: 0.3-2.0 mg/ml) or methadone (range: 0.3-1.5 mg/ml) in all opiate addicts in whom an opiate overdose was the cause of death. The mode of death was rapid, as revealed by high concentrations of 6-monoacetylmorphine and free morphine or methadone in the blood. Moreover, the detection of 6-monoacetylmorphine (range: 0.2-5.2 ng/mg) or methadone (range: 0.4-14.2 ng/mg) in hair samples indicated a long-term abuse of opiates in these subjects (at least 6-12 months before death). For comparison, control brain specimens were obtained at autopsy from healthy subjects dying in the same period of time and who met specific criteria for matching with the opiate addicts. The target proteins were quantitated by immunoblot assays with specific antibodies.

In the brains of opioid addicts (frontal cortex), modest but significant decreases in the immunodensities of m-opioid receptors (22%), G protein-coupled receptor kinases 2 (18%) and 6 (23%), and b-arrestin-2 (21 %), involved in the phosphorylation and desensitization of agonist-occupied receptors, were found compared with matched controls. The content of GRK 6, but not GRK 2, correlated significantly with that of b-arrestin-2 (r = 0.64) in the same brains of opiate addicts. In contrast to findings in the brains of laboratory animals, these modest changes and previous negative data (normal density of m-agonist binding sites and unaltered stimulation of 35S-GTPgammaS by DAMGO) indicate an apparent normal activity of brain Jl-opioid receptors and some adaptation in receptor regulation during the process of opioid dependence in humans.

The components of the cAMP signaling pathway (adenylate cyclase, type I; protein kinase A, catalytic subunit Ca; total and phosphorylated CREB, cAMP response element-binding protein) were found unchanged in the brains of chronic opiate addicts. In spite of these negative results, there was a good correlation (r = 0.72) between the contents of PKA and phosphorylated CREB in the same brains, suggesting a concert modulation of this kinase and its downstream nuclear target by opiate drugs. In contrast to the compensatory upregulation of the cAMP system induced by chronic morphine in the rat brain (mainly in the locus caeruleus), the components of this signaling pathway were not altered in the cerebral cortex of chronic opiate addicts. The lack of upregulation of the cAMP pathway in the human brain after chronic opiate exposure might be related to species sensitivity and/or differences in brain regions (greater cellular heterogeneity in cortex). Alternatively, the apparent normalization of cAMP signaling in the brains of chronic opiate addicts suggest that this system (perhaps after an initial upregulation) has achieved a new steady-state in which the opioid receptor signaling through this pathway is normosensitive.

In the brains of chronic opiate addicts, the active forms of the mitogen-activated protein kinase (MAPK) signaling cascade (Ras, Raf, MEK, ERK) were markedly downregulated (phosphorylated MEK: 80%; phosphorylated ERK1/2: 75%). Similarly, decreased immunodensities of cdk5 (neuronal cyclin-dependent kinase-5) and its neuron-specific activator p35 (20-44%) were also found in the brains of opiate addicts. In the same brains, the densities of p25 (a truncated neurotoxic form of p35), protein phosphatase PP2Ac and m-calpain were found unaltered. Because ERK and cdk5 playa major role in regulating cytoskeleton dynamics, the relation of cdk5/p35 with neurofilament (NF) phosphorylation was also investigated. In the brains of opiate addicts, the density of phosphorylated NF-H was increased (43%) as well as the ratio of phosphorylated to nonphosphorylated NF-H forms (two-fold). In these brains, phosphorylated NF-H significantly correlated with p35 (r = 0.58) but not with cdk5 (r = 0.03). It therefore appears that, in marked contrast to the cAMP pathway, the neuronal MAPK signaling cascade and the cdk5/p35 complex are readly modulated in the cerebral cortex of chronic opiate addicts. The sustained downregulation of cdk5/p35 and MAPK, and the aberrant hyperphosphorylation of NF-H proteins, might have important consequences in the development of neural plasticity associated with opiate addiction in humans.

REFERENCES

1. Ferrer-Alcón, M., García-Sevilla, J.A., Jaquet, P.H. et al. Regulation of nonphosphorylated and phosphorylated forms of neurofilament proteins in theprefrontal cortex of human opioid addicts. J Neurosci Res 2000, 61: 338-49.

2. Ferrer-Alcón, M., La Harpe, R., Guimón, J., García-Sevilla, J.A. Downregulation of neuronal cdk5/p35 in opioid addicts and opiate-treated rats: Relation to neurofilament phosphorylation. Neuropsychopharmacology 2003 (In press).

3. Meana, J.J., González-Maeso, J., García-Sevilla, J.A, Guimón J. m-Opioid receptor and a₂-adrenoceptor agonist stimulation of [35S]GTPgS binding to G-proteins in postmortem brains of opioid addicts. Mol Psychiatry 2000, 5: 308-15.

Methods and Findings in Experimental and Clinical Pharmacology Vol. 25, Suppl. A, 2003
ISSN 0379-0355 Copyright 2003 Prous Science, S.A. CCC: 0379-0355/2003 http://www.prous.com