TY - JOUR
T1 - Role of local production of endothelium-derived nitric oxide on cGMP signaling and S-nitrosylation
AU - Qian, Jin
AU - Zhang, Qian
AU - Church, Jarrod E.
AU - Stepp, David W.
AU - Rudic, Radu D.
AU - Fulton, David J.R.
PY - 2010/1
Y1 - 2010/1
N2 - Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), exerts control over vascular function via two distinct mechanisms, the activation of soluble guanylate cyclase (sGC)/cGMP-dependent signaling or through S-nitrosylation of proteins with reactive thiols (S-nitrosylation). Previous studies in cultured endothelial cells revealed that eNOS targeted to the plasma membrane (PM) releases greater amounts of NO compared with Golgi tethered eNOS. However, the significance of eNOS localization to sGC-dependent or -independent signaling is not known. Here we show that PM-targeted eNOS, when expressed in human aortic endothelial cells (HAEC) and isolated blood vessels, increases sGC/cGMP signaling to a greater extent than Golgi-localized eNOS. The ability of local NO production to influence sGC-independent mechanisms was also tested by monitoring the secretion of Von Willebrand factor (vWF), which is tonically inhibited by the S-nitrosylation of N-ethylmaleimide sensitive factor (NSF). In eNOS "knockdown" HAECs, vWF secretion was attenuated to a greater degree by PM eNOS compared with a Golgi-restricted eNOS. Moreover, the PM-targeted eNOS induced greater S-nitrosylation of NSF vs. Golgi eNOS. To distinguish between the amount of NO generated and the intracellular location of synthesis, we expressed Golgi and PM-targeted calcium-insensitive forms of eNOS in HAEC. These constructs, which generate equal amounts of NO regardless of location, produced equivalent increases in cGMP in bioassays and equal inhibition of vWF secretion. We conclude that the greater functional effects of PM eNOS are due to the increased amount of NO produced rather than effects derived from the local synthesis of NO.
AB - Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), exerts control over vascular function via two distinct mechanisms, the activation of soluble guanylate cyclase (sGC)/cGMP-dependent signaling or through S-nitrosylation of proteins with reactive thiols (S-nitrosylation). Previous studies in cultured endothelial cells revealed that eNOS targeted to the plasma membrane (PM) releases greater amounts of NO compared with Golgi tethered eNOS. However, the significance of eNOS localization to sGC-dependent or -independent signaling is not known. Here we show that PM-targeted eNOS, when expressed in human aortic endothelial cells (HAEC) and isolated blood vessels, increases sGC/cGMP signaling to a greater extent than Golgi-localized eNOS. The ability of local NO production to influence sGC-independent mechanisms was also tested by monitoring the secretion of Von Willebrand factor (vWF), which is tonically inhibited by the S-nitrosylation of N-ethylmaleimide sensitive factor (NSF). In eNOS "knockdown" HAECs, vWF secretion was attenuated to a greater degree by PM eNOS compared with a Golgi-restricted eNOS. Moreover, the PM-targeted eNOS induced greater S-nitrosylation of NSF vs. Golgi eNOS. To distinguish between the amount of NO generated and the intracellular location of synthesis, we expressed Golgi and PM-targeted calcium-insensitive forms of eNOS in HAEC. These constructs, which generate equal amounts of NO regardless of location, produced equivalent increases in cGMP in bioassays and equal inhibition of vWF secretion. We conclude that the greater functional effects of PM eNOS are due to the increased amount of NO produced rather than effects derived from the local synthesis of NO.
KW - Endothelial nitric oxide synthase
KW - Endothelium
KW - Intracellular location
KW - Nitric oxide
KW - Nitrosylation
KW - Von willebrand factor
UR - http://www.scopus.com/inward/record.url?scp=73549093794&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=73549093794&partnerID=8YFLogxK
U2 - 10.1152/ajpheart.00614.2009
DO - 10.1152/ajpheart.00614.2009
M3 - Article
C2 - 19855060
AN - SCOPUS:73549093794
SN - 0363-6135
VL - 298
SP - H112-H118
JO - American Journal of Physiology - Heart and Circulatory Physiology
JF - American Journal of Physiology - Heart and Circulatory Physiology
IS - 1
ER -