Characterization of [3H]-diprenorphine binding in Rana pipiens: observations of filter binding enhanced by naltrexone.
Initial studies were undertaken to examine the properties of [3H]-diprenorphine binding to Rana pipiens whole brain tissue using naltrexone for the definition of nonspecific binding. Saturation analysis demonstrated the binding of [3H]-diprenorphine to be saturable with a K(D) value of 0.65 nM and a Bmax value of 287.7 fmol/mg protein. Unlabeled diprenorphine dose-dependently displaced [3H]-diprenorphine from a single noninteractive site in competition studies which yielded a Ki of 0.22 nM. However, control studies in the absence of tissue revealed significant binding of [3H]-diprenorphine to the filter alone. Interestingly, [3H]-diprenorphine in the presence of unlabeled naltrexone as well as with unlabeled naloxone showed significantly greater binding to the filter than did [3H]-diprenorphine alone. Given this observation of increased nonspecific binding, an artificially low Bmax value would be expected. It is our hypothesis that the unlabeled nonspecific drug forms a complex with [3H]-diprenorphine preventing it from being effectively washed through the filter or the unlabeled drug itself is blocking the flow of [3H]-diprenorphine through the filter. The latter is unlikely however as other binding studies done in our lab using the radioligand [3H]-naloxone with unlabeled naltrexone do not show significant binding to the filter.
Newman
L C
LC
Department of Pharmacology and Physiology, Oklahoma State University, College of Osteopathic Medicine, Tulsa 74107-1898, USA.
Wallace
D R
DR
Stevens
C W
CW
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects*; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D004174:Diprenorphine / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005374:Filtration / Q000295:instrumentation; D009271:Naltrexone / Q000378:metabolism / Q000494:pharmacology*; D009292:Narcotic Antagonists / Q000378:metabolism*; D011894:Rana pipiens
D009292:Narcotic Antagonists; D004174:Diprenorphine; D009271:Naltrexone