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Functional expression of constitutive nitric oxide synthases regulated by voltage‐gated Na + and Ca 2+ channels in cultured human astrocytes
Author(s) -
Oka Michiko,
Wada Miyuki,
Yamamoto Akira,
Itoh Yoshinori,
Fujita Takuya
Publication year - 2004
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/glia.10359
Subject(s) - veratridine , channel blocker , biology , astrocyte , nitric oxide synthase , nitric oxide , microbiology and biotechnology , voltage dependent calcium channel , verapamil , neuroglia , calmodulin , endocrinology , medicine , sodium channel , biochemistry , calcium , chemistry , sodium , enzyme , organic chemistry , central nervous system
We report the functional characterization of constitutive nitric oxide synthase(s) (NOS) such as neuronal and endothelial NOS in cultured human astrocytes. Exposure of cultured human astrocytes to 1 μM veratridine or 50 mM KCl produced a pronounced increase in a calmodulin‐dependent NOS activity estimated from cGMP formation. The functional expression of voltage‐gated Na + channel, which is estimated by the response to veratridine, appeared to be earlier (at second day in culture) than that of voltage‐gated Ca 2+ channels, which are estimated by the response to the KCl stimulation (at fourth day in culture). The KCl‐evoked NO synthesis was totally reversed by L‐type Ca 2+ channel blockers such as nifedipine and verapamil, but not by ω‐conotoxin GVIA, an N‐type Ca 2+ channel blocker, or ω‐agatoxin IVA, a P/Q‐type Ca 2+ channel blocker. In addition, verapamil abolished the KCl‐induced increase in the intracellular free Ca 2+ concentration. RT‐PCR analysis revealed that mRNA for neuronal and endothelial NOS was expressed in human astrocytes. In addition, Western blot analysis and double labeling of NOS and glial fibrillary acidic protein (GFAP) showed that cultured human astrocytes expressed neuronal NOS and endothelial NOS as well as the α 1 subunit of Ca 2+ channel. These results suggest that human astrocytes express constitutive NOS that are regulated by voltage‐gated L‐type Ca 2+ channel as well as Na + channel. © 2004 Wiley‐Liss, Inc.