Inducible expression of N-methyl-D-aspartate receptor, and delta 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 teratocarcinoma cell line, yields 95% pure cultures of terminally differentiated neuronal cells. Concomitant with their terminal differentiation into 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 NT2 cells on the levels of N-methyl-D-aspartate, delta opioid and mu opioid receptor messenger RNAs. RNA levels were measured using quantitative solution hybridization assays. The riboprobes were complementary to major portions of the coding regions of the N-methyl-D-aspartate, delta opioid and mu opioid receptor complementary DNAs. After four weeks of exposure to 10 microM retinoic acid, followed by four weeks of treatment with mitotic inhibitors (1 microM of cytosine arabinoside, 10 microM of fluorodeoxyuridine and 10 microM of uridine) the levels of N-methyl-D-aspartate receptor messenger RNA in differentiated NT2-N cells increased 10-fold, delta opioid receptor messenger RNA increased three-fold, and mu opioid receptor messenger RNA increased four-fold. Northern blot analysis revealed two transcripts for the N-methyl-D-aspartate receptor messenger RNA (4.2 and 4.4 kb) and two transcripts for delta opioid receptor messenger RNA (7.0 and 11.0 kb). To determine whether the increases in messenger RNAs were accompanied by an increased synthesis of the respective proteins, we examined the immunoperoxidase localization of N-methyl-D-aspartate receptor and delta opioid receptor antisera. N-Methyl-D-aspartate receptor-like immunoreactivity was seen within the cell bodies as well as on the processes of the retinoic acid-differentiated 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 ramified processes were markedly enhanced following retinoic acid differentiation. These results demonstrate parallels between the inducible expression of the N-methyl-D-aspartate and opioid receptor messenger RNAs and proteins during the acquisition of the fully differentiated neuronal phenotype in cultured NT2 cells. Retinoic acid-differentiated NT2 cells express increased levels for the N-methyl-D-aspartate, delta opioid and mu opioid receptor messenger RNAs, providing the opportunity to study the interactions among these receptor systems in human terminally differentiated neuronal cells in culture.
Beczkowska
I W
IW
Department of Pharmacology, Cornell University Medical College, New York, NY 10021, U.S.A.
Gracy
K N
KN
Pickel
V M
VM
Inturrisi
C E
CE
D015152:Blotting, Northern; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000378:metabolism*
D011506:Proteins; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu