Submitted on November 25, 2008
Revised on February 19, 2009
Accepted on February 21, 2009
Nitric oxide reduces flow-induced superoxide production via cGMP-dependent protein kinase in thick ascending limbs
Nancy J Hong1 and Jeffrey L. Garvin1*
1 Henry Ford Hospital
* To whom correspondence should be addressed. E-mail: jgarvin1{at}hfhs.org.
We have shown that increased renal tubular flow induces O2- and NO production in thick ascending limbs (TAL). However, the interaction of flow-stimulated NO and O2- in TALs is unclear. We hypothesized that NO inhibits flow-induced O2- production in TALs via cGMP-dependent protein kinase (PKG). We measured flow-stimulated O2- production in rat TALs using dihydroethidium in the absence and presence of L-arginine (0.3 mM), the substrate for NO synthase. Addition of L-arginine reduced flow-induced net O2- production from 68±to 17±4 AU/s (p<0.002). Addition of the NO synthase inhibitor L-NAME (5 mM) in the presence of L-arginine stimulated production (L-arginine: 15±4 AU/s vs. L-arginine + L-NAME: 63±7 AU/s; p<0.002). The guanylate cyclase inhibitor LY-83583 (10 µM) also enhanced flow-induced net O2- production in the presence of L-arginine (L-arginine: 7±4 AU/s vs. L-arginine + LY-83583: 53±7 AU/s; p<0.01). In the presence of LY-83583, L-arginine only reduced flow-induced net O2- by 36% (LY-83583: 80±7 AU/s vs. LY-83583 + L-arginine: 51±3 AU/s; p<0.006). The cGMP analog dibutyryl (db)-cGMP reduced flow-induced net O2- from 39±9 to 7±3 AU/s (p<0.03). The PKG inhibitor KT-5823 (5 µM) partially restored flow-induced net O2- in the presence of L-arginine (L-arginine: 4±4 AU/s vs. L-arginine + KT-5823: 32±9 AU/s; p<0.03) and db-cGMP (db-cGMP: 9±7 AU/s vs. db-cGMP + KT-5823: 54±5 AU/s; p<0.01). Phosphodiesterase II inhibition had no effect on arginine-inhibited O2- production. We conclude that: 1) NO reduces flow-stimulated O2- production; 2) this occurs primarily via the cGMP/PKG pathway; and 3) O2- scavenging by NO plays a minor role.
