title: Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells.
abstract: The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.
authors.affiliation: Natural Product Sciences, Inc., Salt Lake City, UT 84108.
authors.forename: T N
authors.initials: TN
authors.lastname: Parks
authors.forename: L D
authors.initials: LD
authors.lastname: Artman
authors.forename: N
authors.initials: N
authors.lastname: Alasti
authors.forename: E F
authors.initials: EF
authors.lastname: Nemeth
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D016257:Fura-2; D005998:Glycine / Q000494:pharmacology; D007700:Kinetics; D016202:N-Methylaspartate / Q000494:pharmacology*; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000502:physiology*; D013050:Spectrometry, Fluorescence
chemical_list: D002121:Calcium Channel Blockers; D016194:Receptors, N-Methyl-D-Aspartate; D004533:Egtazic Acid; D016202:N-Methylaspartate; D011189:Potassium Chloride; D002118:Calcium; D005998:Glycine; D016257:Fura-2