Opiates exert actions by binding to specific receptors, whose interactions are translated across the cell membrane into specific biological responses by second messenger systems. A variety of data suggest that opiate tolerance may be associated with a step distal from the receptor binding site. Therefore, a logical candidate for the molecular mechanism of tolerance is coupling of opiate receptors with second messenger systems. The principal second messenger system associated with opiate receptors thus far has been opiate-mediated reductions in cellular cyclic AMP levels through inhibition of adenylate cyclase. However, other second messenger systems are also possibly coupled to opiate receptors. Preliminary data in cultures of C-6 glioma cells has revealed the existence of another such system: opiate-stimulated adenylate cyclase. In these cells, opiate agonists both inhibit and stimulate adenylate cyclase, and both activities are blocked by naloxone. Each activity can be studied separately by selective blockade of the other activity: pertussis toxin to block inhibited activity, and pretreatment of membranes at pH 4.5 to block stimulated activity. This proposal will explore the specificity of opiate-stimulated and -inhibited adenylate cyclase in C-6 glioma cells, and determine whether different receptor subtypes are responsible for these two activities. Because these two activities are opposite, they may mask each other in many different cell types. Therefore, other cell types in addition to C-6 glioma cells will be screened for either opiate-stimulated or -inhibited adenylate cyclase with the same techniques. The control of the balance between stimulated and inhibited activities will be studied by fusion of membranes from different cell types with different activities. Finally, the effects of chronic opiate exposure on these systems will be examined by treatment of cells in culture with different agonists. The goal of these experiments is to determine whether these model cell systems can reveal important information about the role of opiate receptor-coupled second messenger systems in the development of opiate tolerance.
