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

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.