A Genetic Approach to Reveal the Action of the Opiate Receptor in Selected Neuroblastoma‐Glioma Cells

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Abstract
Three clones of neuroblastoma-glioma cells that contain low amounts of calmodulin were selected from mouse and rat NG108-15 cells after several treatments with high concentrations of chlorpromazine. Purified membranes of the 3 clones had decreased numbers of both .alpha.-adrenergic and opiate receptors, monitored with [3H]yohimbine and [3H,D-Ala2]methionine enkephalinamide, respectively. No changes were observed in the affinity of these radioactive ligands to the receptors of the selected cells as compared to the parent cells. Addition of bovine brain calmodulin did not affect the binding of [3H,D-Ala2]methionine enkephalinamide to the membranes of the selected cells and they had the same number of acetylcholine receptors, determined with 1-quinuclidinyl-[phenyl-4-3H]-benzilate, as the parent NG108-15 cells. The basal ATPase activity in the membranes of the selected cells was 35-50% of the parent cells, with a decreased V [reaction rate] value and no significant change in the affinity constant Ka to ATP. Addition of Ca2+ to the purified membranes increased the V of the ATPase in the selected as well as the parent cells but the V of the selected cells remained lower than that of the parent cells. Ca2+ had no effect on the Ka to ATP in either cell type. The Ca2+-dependent ATPase activity of both the parent and the selected cells was also calmodulin-dependent since it was blocked in vitro by chlorpromazine. The co-regulation of opiate and adrenergic receptors and their interaction with calmodulin and Ca2+-ATPase is discussed in view of recent observations indicating biochemical and physiological association between opiates, Ca2+ and adrenergic compounds.