Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells

Citation
K. Schmidt et al., Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells, AM J P-HEAR, 281(5), 2001, pp. H2053-H2061
Citations number
36
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cardiovascular & Hematology Research
Journal title
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
ISSN journal
0363-6135 → ACNP
Volume
281
Issue
5
Year of publication
2001
Pages
H2053 - H2061
Database
ISI
SICI code
0363-6135(200111)281:5<H2053:CONAEI>2.0.ZU;2-N
Abstract
The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS a nd eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their ac tivity in a common cellular environment. NOS activity was determined by mea suring L-[H-3] citrulline production in homogenates and intact cells, the c onversion of oxyhemoglobin to methemoglobin, and the production of cGMP. Th e results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble m utant of eNOS appeared to be slightly more active than wild-type eNOS in te rms of NO and cGMP formation, suggesting that membrane association may be c rucial for inhibition of basal NO release but is not required for stimulati on by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyc lase was significantly reduced by transfection with wild-type eNOS due to d ownregulation of mRNA expression. These results demonstrate that nNOS and e NOS behave differently even in an identical cellular environment.