Morphine and other opioids are widely used in clinical management of severe pain. One problem of prolonged use of morphine is that tolerance develops. Morphine exerts its effects mainly at the mu opioid receptors. Development of morphine tolerance is a complex process, involving changes at the cellular level and in neural circuitry. Despite numerous studies, mechanisms for morphine tolerance are still not well-understood, partly because there are no cellular models in which the mu receptor can be manipulated to address specific questions. Recently a mu opioid receptor has been molecularly cloned, thus making it possible for the first time to generate such cellular models. This is the goal of the present proposal. The underlying hypothesis for this investigation is that the development of morphine tolerance at the cellular level results from specific molecular events that are mediated through the mu opioid receptor. Two mammalian cell lines, PC12 pheochromocytoma cells and Chinese hamster ovary cells, will be used to stably express the mu receptor. Coupling of the mu receptor to the cAMP pathway and ion channels will be determined with acute opioid treatment. Then, development of morphine tolerance will be studied by chronically exposing the cells to morphine and determining the molecular events that underlie the changes during tolerance development. Furthermore, the role of phosphorylation in regulating receptor function will be studied by systematically mutating the potential phosphorylation sites of protein kinases A and C, generating cell lines that express the mutant receptors, and examining the effect of these mutations on receptor function. These cells will also be chronically exposed to morphine to determine the potential role of receptor phosphorylation in tolerance development. These studies will provide important information as to the molecular mechanisms of tolerance development through the mu opioid receptor. Such information will contribute to our knowledge on how opioid drugs may act at receptors and will provide a basis in designing strategies to circumvent the problem of opioid tolerance in the clinical management of pain.
