INDUCIBLE EXPRESSION OF N-METHYL-D-ASPARTATE RECEPTOR, AND DELTA-OPIOID AND MU-OPIOID RECEPTOR MESSENGER-RNAS AND PROTEIN IN THE NT2-N HUMAN CELL-LINE

Retinoic acid treatment of NT-era2/cl.D1 (NT2) cells, a human teratoca rcinoma cell line, yields 95% pure cultures of terminally differentiat ed neuronal cells. Concomitant with their terminal differentiation int o neurons, NT2 cells are induced by retinoic acid to express neuronal N-methyl-D-aspartate receptor channels, which are fully functional. We determined the effects of retinoic acid-induced differentiation of NT 2 cells on the levels of N-methyl-D-aspartate, delta opioid and mu opi oid receptor messenger RNAs. RNA levels were measured using quantitati ve solution hybridization assays. The riboprobes were complementary to major portions of the coding regions of the N-methyl-D-aspartate, del ta opioid and mu opioid receptor complementary DNAs. After four weeks of exposure to 10 mu M retinoic acid, followed by four weeks of treatm ent with mitotic inhibitors (1 mu M of cytosine arabinoside, 10 mu M o f fluorodeoxyuridine and 10 mu M of uridine) the levels of N-methyl-D- aspartate receptor messenger RNA in differentiated NT2-N cells increas ed 10-fold, delta opioid receptor messenger RNA increased three-fold, and mu opioid receptor messenger RNA increased four-fold. Northern blo t analysis revealed two transcripts for the N-methyl-D-aspartate recep tor messenger RNA (4.2 and 4.4 kb) and two transcripts for delta opioi d receptor messenger RNA (7.0 and 11.0 kb). To determine whether the i ncreases in messenger RNAs were accompanied by an increased synthesis of the respective proteins, we examined the immunoperoxidase localizat ion of N-methyl-D-aspartate receptor and delta opioid receptor antiser a. N-Methyl-D-aspartate receptor-like immunoreactivity was seen within the cell bodies as well as on the processes of the retinoic acid-diff erentiated cells. Although delta opioid receptor-like immunoreactivity was detected within the soma of isolated cells prior to retinoic acid treatment, the apparent number of these labelled cells and their rami fied processes were markedly enhanced following retinoic acid differen tiation. These results demonstrate parallels between the inducible exp ression of the N-methyl-D-aspartate and opioid receptor messenger RNAs and proteins during the acquisition of the fully differentiated neuro nal phenotype in cultured NT2 cells. Retinoic acid-differentiated NT2 cells express increased levels for the N-methyl-D-aspartate, delta opi oid and mu opioid receptor messenger RNAs, providing the opportunity t o study the interactions among these receptor systems in human termina lly differentiated neuronal cells in culture. (C) 1997 IBRO. Published by Elsevier Science Ltd.