title: Roles of Ca2+ and the Ca2+-sensing receptor (CASR) in the expression of inducible NOS (nitric oxide synthase)-2 and its BH4 (tetrahydrobiopterin)-dependent activation in cytokine-stimulated adult human astrocytes.
abstract: Since NO production by NOS-2 made by astrocytes activated by proinflammatory cytokines contributes to the killing of neurons in variously damaged human brains, knowing the mechanisms responsible for NOS-2 expression should contribute to developing effective therapeutics. The expression and activation of NOS-2 in normal adult human cerebral cortical astrocytes treated with three proinflammatory cytokines, IL-1beta, TNF-alpha, and IFN-gamma, are driven by two separable mechanisms. NOS-2 expression requires a burst of p38 MAPK activity, while the activation of the resulting enzyme protein requires MEK/ERK-dependent BH4 (tetrahydrobiopterin) synthesis between 24 and 24.5 h after adding the cytokines to the culture medium. Here we show that NOS-2 expression in the activated astrocytes requires that the culture medium contain 1.8 mM Ca2+, but it is unaffected by inhibiting calcium-sensing receptors (CASRs) with NPS 89636. However, NOS-2 activation is inhibited by NPS 89626 during the MEK/ERK-dependent stage between 24 and 24.5 h after adding the cytokines, and this inhibition can be overridden by exogenous BH4. Therefore, NOS-2 expression and the subsequent BH4-dependent NOS-2-activation in human astrocytes need 1.8 mM Ca2+ to be in the culture medium, while NOS-2 activation also needs functional CASRs between 24 and 24.5 h after cytokine addition. These findings raise the possibility that calcilytic drugs prevent NO-induced damage and death of human neurons.
authors.affiliation: Histology & Embryology Unit, Department of Biomedical & Surgical Sciences, University of Verona Medical School, Verona, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Dal Pra
authors.forename: Anna
authors.initials: A
authors.lastname: Chiarini
authors.forename: Edward F
authors.initials: EF
authors.lastname: Nemeth
authors.forename: Ubaldo
authors.initials: U
authors.lastname: Armato
authors.forename: James F
authors.initials: JF
authors.lastname: Whitfield
mesh_terms: D000328:Adult; D000375:Aging / Q000502:physiology; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology*; D002413:Cations, Divalent / Q000737:chemistry; D049109:Cell Proliferation / Q000187:drug effects; D016207:Cytokines / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D016207:Cytokines; D044169:Receptors, Calcium-Sensing; D001708:Biopterins; D009569:Nitric Oxide; D048049:Extracellular Signal-Regulated MAP Kinases; C003402:sapropterin; D002118:Calcium