A method for the rapid development of opiate tolerance in the guinea pig ileum

The rate and degree of in vitro tolerance development to morphine, normorphine and d,1-methadone were assessed on the excised guinea pig ileum. Agonists in fixed concentrations at 1/4, 1/2, 1 and 2 x IC50 were incubated with the tissue for 1, 2 or 4 hours. The degree of tolerance development was expressed as a ratio of the IC50 after and before incubation. A high degree of tolerance developed to all three agonists and the effect could be prevented by co-incubation with naloxone. Tolerance development was stereo-specific; levorphanol and 1-methadone developed much higher degrees of tolerance than their respective d-isomers. Furthermore, under the same conditions, subsensitivity to acetylcholine, norepinephrine, and adenosine monophosphate did not develop. The in vitro tolerance was accompanied by physical dependence development as evidenced by the fact that naloxone elicited muscular contracture in the tolerant ileum. cAMP enhanced the development of tolerance to normorphine and cycloheximide could reduce this phenomenon. It is concluded that the procedure may facilitate studies on the mechanisms involved in the development of opiate tolerance and physical dependence.