Multiple Opiate Receptors

The use of very low concentrations of 1251-[D-Ala2,DLeu5]enkephalin, [3H]naloxone, and [3H]dihydromorphine under similar conditions enables the measurement of different opiate binding sites in a rat brain membrane preparation. In the absence of sodium ions, one such site binds enkephalins and their D-Ala2-substituted analogs with higher affinity than morphine and naloxone (enkephalin receptor), while another site exhibits better affinity for naloxone and morphine (morphine receptor). Morphine, naloxone, and enkephalin do not facilitate the dissociation of bound 12?-[D-Ala2,D-Leu5]enkephalin, suggesting that heterogenous or homogenous cooperativity does not exist for opiate receptors. Sodium ion decreases the high affinity binding of morphine and enkephalins to both receptor sites. Scatchard plots of saturation binding isotherms of ‘251-[D-Ala2,D-Leu5]enkephalin indicate two binding sites with dissociation constants of 0.8 no and 6 11~ and capacities of 43 and 86 fmol/mg of membrane protein. Many neuroblastoma cell lines bear only the enkephalin receptors. So far, cell lines containing only the morphine receptor have not been found. Homogenization or exposure to morphine for 24 h does not affect the binding characteristics of neuroblastoma cells. NCyclopropylmethylnoretorphine, an analgesic about 100 times more potent than morphine with less propensity to induce physical dependence, competes for the binding of ‘251-[D-Ala2,D-Leu5]enkephalin and [3H]naloxone with ICsO values of 2.5 and 1 no, respectively, and shows no “sodium shift” in the C3H]naloxone-binding assay. Partial agonists, pentazocine, butorphanol, and oxilorphan, also have a weak “sodium shift” and a low I&O ratio in competing with [3H]naloxone compared to ‘251-[D-Ala2,D-Leu5]enkephalin binding. Enkephalin and its stable analogs exhibit high affinity for the enkephalin receptors. The analog, Try-GlyGly-Phe, which retains significant intrinsic activity, is still able to bind to the enkephalin receptor better than to the morphine receptor. Tyr-ethyl ester binds to both receptor sites equally well, showing an I& value of about 0.1111~. These data may suggest that the hydrophobic group of the phenylalanine residue of enkephalin could be responsible for the major structural difference between enkephalin and morphine for receptor recognition. This may also explain why N-cyclopropylmethylnoretorphine, which contains a hydrophobic group at the C-19 position of oripavine, binds to the