Evidence for multiple mechanisms of kappa opioid tolerance in mesencephalic cultures.

Opioid tolerance limits the effectiveness of chronic opioid therapy. Kappa opioid tolerance in mesencephalic cultures has been established as a model system for evaluating neuronal mechanisms of tolerance. Our studies were carried out in primary cultures derived from the mesencephalon of rat embryos. [3H]Dopamine release assays were used to evaluate the kinetics of onset and recovery from tolerance to the kappa opioid receptor agonist (U69,593) which were measured with and without (1) modulators of cAMP-dependent kinase or (2) cycloheximide, an inhibitor of protein synthesis. Results were compared to the kinetics of recovery from kappa receptor alkylation by beta-chlornaltrexamine. The results showed that 8-bromo-cAMP and forskolin significantly accelerated the development of tolerance to U69,593. Rp-cAMP significantly slowed the onset of tolerance. Rates of recovery of cultures after 18 h of U69,593 were not significantly different regardless of whether cultures were incubated with U69,593 alone or in combination with 8-bromo-cAMP, forskolin or Rp-cAMP. Cycloheximide significantly slowed the recovery of cultures after 7 days of exposure to U69,593 but not after 18 h of exposure to U69,593. Recovery of cultures after alkylation of kappa receptors by beta-chlornaltrexamine was significantly slower than the recovery of cultures after 7 days of U69,593 exposure indicating that receptor synthesis is unlikely to be the major mechanism of recovery after prolonged opioid exposure. It is suggested that multiple mechanisms are responsible for tolerance to U69,593 in primary neuronal cultures. There are long lasting mechanisms of tolerance that do not involve destruction or permanent inactivation of cellular components.