title: Expression of functional NR1/NR2B-type NMDA receptors in neuronally differentiated SK-N-SH human cell line.
abstract: The present study demonstrates that human SK-N-SH neuroblastoma cells, differentiated by retinoic acid (RA), express functional NMDA receptors and become vulnerable to glutamate toxicity. During exposure to RA, SK-N-SH cells switched from non-neuronal to neuronal phenotype by showing antigenic changes typical of postmitotic neurons together with markers specific for cholinergic cells. Neuronally differentiated cells displayed positive immunoreactivity to the vesicular acetylcholine transporter and active acetylcholine release in response to depolarizing stimuli. The differentiation correlated with the expression of NMDA receptors. RT-PCR and immunoblotting analysis identified NMDA receptor subunits NR1 and NR2B, in RA-differentiated cultures. The NR1 protein immunolocalized to the neuronal cell population and assembled with the NR2B subunit to form functional N-methyl-D-aspartate (NMDA) receptors. Glutamate or NMDA application, concentration-dependently increased the intracellular Ca2+ levels and acetylcholine release in differentiated cultures, but not in undifferentiated SK-N-SH cells. Moreover, differentiated cultures became vulnerable to NMDA receptor-mediated excitotoxicity. The glutamate effects were enhanced by glycine application and were prevented by the NMDA receptor blocker MK 801, as well as by the NR2B selective antagonist ifenprodil. These data suggest that SK-N-SH cells differentiated by brief treatment with RA may represent an unlimited source of neuron-like cells suitable for studying molecular events associated with activation of human NR1/NR2B receptors.
authors.affiliation: Division of Pharmacology, Department of Biomedical Sciences and Biotechnologies, School of Medicine, University of Brescia, 25123 Brescia, Italy. pizzi@med.unibs.it
authors.forename: Marina
authors.initials: M
authors.lastname: Pizzi
authors.forename: Flora
authors.initials: F
authors.lastname: Boroni
authors.forename: Andrea
authors.initials: A
authors.lastname: Bianchetti
authors.forename: Costantinos
authors.initials: C
authors.lastname: Moraitis
authors.forename: Ilenia
authors.initials: I
authors.lastname: Sarnico
authors.forename: Marina
authors.initials: M
authors.lastname: Benarese
authors.forename: Francesca
authors.initials: F
authors.lastname: Goffi
authors.forename: Alessandra
authors.initials: A
authors.lastname: Valerio
authors.forename: PierFranco
authors.initials: P
authors.lastname: Spano
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D005998:Glycine / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D008275:Magnesium Deficiency / Q000378:metabolism; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
chemical_list: D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; C409466:NR1 NMDA receptor; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D014212:Tretinoin; D000109:Acetylcholine; D005998:Glycine