This project is directed toward understanding the molecular mechanisms for the development of opiate addiction and tolerance with the expectation that such an understanding could suggest new methods for treatment of opiate abuse. Since a drug's potential for abuse is a function of its ability to reward and since opiate tolerance, physical dependence, and reward potential are separable, the mechanism for separation of these aspects of response to chronic opiate treatment is a particular focus. The signal transduction pathways underlying each of these responses will be characterized in respect to the changes that occur during the development of tolerance and dependence. A model system, the SK-N-SH human neuroblastoma cell line will be used. Since the abuse potential of opiates is thought to primarily involve the mu-type opiate receptor, and in SK-N-SH cells, 80% of the opiate receptors are mu-type receptors, this is a particularly valuable system to analyze. There are two subtypes of the mu-opiate receptor. Each subtype of the mu- opiate receptor, will be characterized with respect to the G-protein(s) with which it couples and the effector systems thus regulated. DAMGO, a mu-specific agonist and naloxonazine, an irreversible inhibitor of mu1 receptors and reversible inhibitor of mu2 receptors will be used in competitive binding studies to distinguish mu subtypes. A combination of receptor-dependent [35S]-GTPgammaS binding with immunoprecipitation of specific G-proteins will be used to examine receptor/G-protein coupling. Adenylyl cyclase activity and K+ and Ca++ conductance will be assayed to detect effector system coupling as well as tolerance and dependence. Changes in the amounts and interactions of the signal transduction components with chronic mu-opiate stimulation will be correlated with the development of tolerance and dependence. In addition, to confirm connection between changes involving signal transduction system components and responses characteristic of tolerance and dependence, gene transfection of specific components and reconstitution will be used to construct systems having pre-defined response characteristics.
| Carty, D J; Iyengar, R (1994) Guanosine 5'-(gamma-thio)triphosphate binding assay for solubilized G proteins. Methods Enzymol 237:38-44 |
