Functional expression of constitutive nitric oxide synthases regulated by voltage-gated Na+ and Ca2+ channels in cultured human astrocytes.
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 microM 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 omega-conotoxin GVIA, an N-type Ca(2+) channel blocker, or omega-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 alpha(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.
Department of Biochemical Pharmacology, Kyoto Pharmaceutical University, Kyoto, Japan.
Michiko
M
Oka
Miyuki
M
Wada
Akira
A
Yamamoto
Yoshinori
Y
Itoh
Takuya
T
Fujita
D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology*; D015220:Calcium Channels / Q000502:physiology*; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000096:biosynthesis; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000502:physiology; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D011189:Potassium Chloride / Q000494:pharmacology; D015222:Sodium Channels / Q000502:physiology*; D014701:Veratridine / Q000494:pharmacology
D015220:Calcium Channels; D009419:Nerve Tissue Proteins; D015222:Sodium Channels; D011189:Potassium Chloride; D014701:Veratridine; C496320:NOS1 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP