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 (Chang, K.-J., and Cuatrecasas, P. (1979) J. Biol. Chem. 254, 2610-2618) 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 (Knodel, E., and Richelson, E. (1980) Brain Res. 197, 565-570), 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 mu-Bondapak C18 column. Extracted methionine5-enkephalin immunoreactive material behaved similarly to synthetic methionie5-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 for [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.