NEUROCHEMICAL MECHANISM OF NARCOTIC ACTION

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In this proposal, two independent but related projects will be combined which describe some main research activities in my laboratory. 1) Molecular characterization of opioid receptors and 2) determination of the neurochemical mechanisms of tolerance and dependence. The first project will utilize a mu type opioid receptor which we recently purified to homogeneity, and will include a) reconstitution of opioid binding and opioid-mediated function into a membrane environment; b) raising monoclonal and polyclonal antibodies to the receptor, and using them to map the receptor's distribution in brain, determine the role of different portions of the receptor in binding and function, and for cloning the receptor; c) molecular characterization of opioid binding to the purified receptor, including tests of negative cooperativity, thermodynamic analysis of agonist and antagonist binding, effects of ions, guanine nucleotides and lipids on binding, and tests for interconversion of mu receptors to other types; and d) cloning the receptor by synthesis of oligodeoxynucleotide probes, isolation of receptor mRNA, and insertion into a cloning vector. Studies of opioid tolerance/dependence will employ NG108-15 hybrid cells model which exhibit opioid binding, opioid-mediated function (inhibition of adenylate cyclase), and a tolerance-like adaptation process during chronic treatment. In previous work, our lab has shown that chronic opioid agonist treatment induces three distinct adaptation processes in these cells: 1) receptor desensitization, or uncoupling from adenylate cyclase; 2) receptor down-regulation, or disappearance from cell surface; and 3) an increase in adenylate cyclase activity following withdrawal or antagonism of chronic of chronic agonist. We propose to study each of these processes in detail and determine their relevance to tolerance/dependence in mammalian brain. We will try to show that a) desensitization results from a covalent change in the receptor; b) during down-regulation, receptors move along a pathway similar to that traversed by other down-regulated receptors; we will also determine the kinetics of internalization, and the signal initiating it; and c) study the involvement of Ca++ and Ca++-binding proteins in the increase in adenylate cyclase activity. To make the NG cells model more similar to these in brain, we will induce differentiation in them and compare chronic effects of these cells with those in undifferentiated cells. Finally, we will characterize opioid receptor down-regulation in brain, which we have recently reported.