The molecular mechanisms of opiate tolerance and dependence remain unknown, and efforts to elucidate them are greatly hampered by the complexity and heterogeneity of the brain. Our approach to this problem has been to use a clonal cell line of neuronal origin (NG108-15). These cells undergo a quantifiable biochemical response-inhibition of adenylate cyclase when administered opiates and a tolerance-like adaptation process during chronic treatment. We have shown that there are actually three distinct adaptation processes triggered in these cells by chronic opiate agonist treatment: 1) receptor desensitization or uncoupling of receptor from adenylate cyclase; 2) receptor down-regulation or disappearance of receptor from the cell surface; and 3) receptor up-regulation or increase in the enzymes' activity following abrupt termination of chronic opiate treatment or addition of narcotic antagonist. We propose to study in detail the molecular mechanisms of the cellular adaptation processes and to determine their relevance to the mechanism of tolerance and dependence in mammalian brain. Specifically, we will 1) attempt to show that opiate receptor desensitization results from a covalent modification of the receptor, possibly dephosphorylation, allowing it to remain physically coupled to the regulatory subunit of adenylate cyclase (Ni), but anable to promote dissociation of GDP; 2) shown that during down-regulation, opiate receptors internalize in NG cells by a pathway similar to that traversed by other down-regulated receptors, and we will try to establish the exact nature of this pathway. We will also determine the kinetics of internalization and the signal initiating it; 3) study the relationship of intracellular Ca++ to the up-regulation of adenylate cyclase activity by altering Ca++, by determining whether a cyclase-stimulatory material from up-regulated cells is calmodulin, and by investigating the role of polyphosphoinositide turnover in up-regulation; 4) differentiate NG108-15 cells, and compare chronic opiate effects in them to undifferentiated cells; and 5) investigate the recently discovered phenomenon of down-regulation in mammalian brain to determine which opiate agonists can initiate it, and whether receptors are internalized in the process.
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