Biochemical Bases for Narcotic Drug Addiction

One primary objective of our research group is the elucidation of the cell biological bases for narcotic addiction (tolerance/dependence). A major complicating factor in the medicinal use of narcotics is their diminished pharmacological effectiveness following persistent administration.

As a result of tolerance formation, it is not uncommon for the antinociceptive effectiveness of opioids to be reduced by an order of magnitude or more. Multiple cellular adaptations are elicited by chronic exposure to opioids. These include diminution of spare opioid receptors, decreased opioid receptor density and G protein content and coupling thereof. All imply that opioid tolerance is a manifestation of a loss of opioid function, i.e., desensitization.

Recent observations in this laboratory challenge the exclusiveness of this formulation and indicate that opioid tolerance also results from qualitative changes in opioid signaling as well. The organizing formulation upon which much of this research is based is that opioid tolerance derives not only from impaired opioid receptor G protein coupling but also from altered consequences of coupling. In other words, opioid tolerance reflects a gain as well as a loss of opioid functionality. Results of recent experiments have indicated that chronic morphine induces fundamental changes in the nature of effectors that are coupled to the opioid receptor-G protein-signaling pathway.

These molecular changes include the up-regulation of adenylyl cyclase (AC) isoforms of the type two family as well as a substantial increase in their phosphorylation state. There are as many as nine isoforms of AC many of which are differentially regulated. We have found that chronic morphine induces phosphorylation of AC isoforms II and/or VII and also increases the synthesis of AC IV and VII. These changes should result in a shift in opioid receptor signaling from predominantly Gia inhibitory to Gbg stimulatory. Indeed, we have demonstrated that Gsa/Gbg stimulation of AC is significantly augmented following chronic morphine. Thus, one consequence of chronic morphine would be the up-regulation of divergent receptor-coupled stimulatory AC signaling.

The figure on the left shows that the ability of Gsa to stimulate AC activity is augmented following chronic morphine treatment. Since this augmented stimulation can be blocked by the Gbg blocking peptide QEHA, it must result from augmented Gbg stimulation of cyclase activity (see Chakrabarti et al., 1998a).

The chronic morphine-induced shift from predominantly Gia AC inhibitory to Gbg AC stimulatory signaling provides a mechanism for the well-documented heterologous consequences of chronic morphine exposure. There are a plethora of G protein (Gi) coupled receptors that signal via AC. Consequently, up-regulation of Gbg-stimulated AC isoforms in combination with covalent modifications thereof (e.g. phosphorylation) that increase Gbg-stimulatory responsiveness should have wide-ranging physiological consequences beyond those directly under the influence of opioids.

The molecular changes that underlie the shift from opioid receptor-coupled inhibitory to stimulatory signaling reflect the plasticity of opioid signal transduction mechanisms and the ability of chronic morphine to augment new signaling strategies.

The schematic diagram shows new signaling strategies after chronic morphine treatment. Chronic morphine augments production of new adenylyl cyclase isoforms of the type II family as well as their phosphorylation. As a result, opioid signaling shifts from predominantly Gsa inhibitory to Gsa/Gbg stimulatory.

Currently, we have broadened our investigations to include the effect of chronic morphine on other protein components of receptor G protein-coupled signaling pathways such as G protein receptor kinases and b-arrestin. Results from this line of study could provide substantial insights into how cells integrate diverse signal inputs.

The above figure illustrates autoradiographs of immunoprecipitates obtained using anti-Gb (A) or anti GRK (B) antibodies. Lane 1, opioid naïve. Lane 2, chronic morphine-treated. Lane 3, immunoprecipitate obtained using pre-absorbed antisera. Note the increased phosphorylation of GRK (~80 kDa), b-arrestin (~45 kDa) and Gb (~3 kDa) following chronic morphine. Phosphorylation of purified Gb (C) decreases its co-immunoprecipitation (association) with GRK (D) (see Chakrabarti et al., 2001).

Selected Publications

1. Wang, L and. Gintzler, A. R. Altered m opiate receptor-G protein signal transduction following chronic morphine exposure, J. Neurochem., 68(1) 248 254, 1997.

2. Rivera, M. and Gintzler, A.R. Differential effect of chronic morphine on mRNA encoding adenylyl cyclase isoforms: relevance to physiological sequela of tolerance/dependence, Mol.Brain Res., 54(1): 165-169, 1998.

3. Chakrabarti, S., Rivera, M., Yan, S.Z., Tang, W.-J. and Gintzler, A. R., Chronic morphine augments Gbg/Gsa stimulation of adenylyl cyclase: relevance to opioid tolerance, Mol. Pharmacol., 54: 655-662, 1998a.

4. Chakrabarti,S., Wang, L., Tang, W.-J., and Gintzler, A.R. Chronic morphine augments adenylyl cyclase phosphorylation: relevance to altered signaling during tolerance/dependence, Mol. Pharmacol., 54: 949-953, 1998b.

5. Chakrabarti,S., Oppermann, M. and Gintzler, A.R. Chakrabarti,S., Oppermann, M. and Gintzler, A.R. Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: a novel mechanism for modulating cell signaling, Proc. Nat'l Acad. Sci. (USA), 98: 4209-4214, 2001.

Laboratory Personnel

Sumita Chakrabarti, PhD, Research Assistant Professor

Daya Gupta, PhD Student

Nai-Jiang Liu, Postdoctoral Fellow

Annette Regec, PhD, Senior Research Associate

Maria Szucs, Visiting Professor