Characterization of a Labile Naloxone Binding Site (λ Site) in Rat Brain

A high‐affinity binding site selective for naloxone and other 4,5‐epoxymorphinans (λ site) has been previously described in rat brain. Following homogenization of freshly dissected brain, the λ sites convert from a high‐affinity to a low‐affinity state. When measured with [ 3 H]naloxone, the decay is very rapid at 20°C (t 1/2 < 2 min), whereas it is progressively slowed at lower temperatures. Proteinase inhibitors, antoxidants, and sulfhydryl group‐protecting agents failed to prevent this conversion. Kinetic measurements of μ and λ binding at varying temperatures demonstrated that the decrease in λ binding does not coincide with the concurrent increase in μ binding and that the loss of high‐affinity λ binding at 20°C can be partially restored when the temperature is lowered to 0°C. The low‐affinity state of the λ site is rather stable in the Tris buffer homogenates and is susceptible to digestion by a protease. The (–)‐isomer of WIN 44,441 , a benzomorphan drug, binds to λ sites with moderate affinity (dissociation constant, K D = 63 n M ), whereas the (+)‐isomer does not ( K D > 10,000 n M ), thus establishing stereoselectivity of the binding process. Neither the high‐affinity nor the low‐affinity state of λ binding is significantly affected by the presence of 100 m M sodium chloride or 50 μ M Gpp(NH)p, (a GTP analog), which is in contrast to the dramatic effect of these agents on the established opioid receptor system. Naltrexone, naloxone, nalorphine, and morphine (in this order of decreasing potency) bind to the λ site in vivo in intact rat brain over dosage ranges that are commonly employed in pharmacological studies.