Function and regulation of methionine⁵-enkephalin and its receptors in murine neuroblastoma cells

A homogeneous class of enkephalin receptors found in murine neuroblastoma clone N1E-115 has been confirmed using a centrifugation assay employing cellular membranes. In intact N1E-115 cells, synthetic methionine⁵-enkephalin inhibited prostaglandin E₁-induced intracellular cyclic AMP formation in a naloxone-sensitive manner. Upon demonstrating intracellular methionine⁵-enkephalin immunocytochemically, analyses of crude N1E-115 extract were made by radioimmunoassay or opiate receptor binding assay following fractionation by molecular sieve chromatography and high pressure liquid chromatography on a μ-Bondapak C₁₈ column. Extracted methionine⁵-enkephalin immunoreactive material behaved similarly to synthetic methionine⁵-enkephalin in these analyses. Growth curve studies of the N1E-115 cells indicated that the quantity of methionine⁵-enkephalin immunoreactive material synthesized per milligram of cellular protein and the maximum number of enkephalin receptor sites per milligram of membrane protein increased as the cells progressed from logarithmic to stationary phase, with no change in the apparent affinity of the enkephalin receptors of [³H]methionine⁵-enkephalin. These data suggest that adrenergic clone N1E-115 has functional methionine⁵-enkephalin membrane receptors, that this clone synthesizes methionine⁵-enkephalin, and that both the enkephalin receptor number and the content of stored methionine⁵-enkephalin are regulated with respect to cell division.