Premium
INHIBITION OF NITRIC OXIDE GENERATION ENHANCES SUPEROXIDE PRODUCTION IN CULTURED HK2 and M1 CELLS
Author(s) -
Islam Mohammed Toriqul,
Maiti Arpan Kumar,
Sato Ryosuke,
Majid Dewan S.A.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.704.11
Subject(s) - apocynin , nadph oxidase , chemistry , superoxide , nitric oxide , incubation , microbiology and biotechnology , oxidase test , biochemistry , enzyme , biology , organic chemistry
An enhancement in superoxide (O 2 − ) activity due to nitric oxide (NO) deficiency facilitates renal tubular sodium reabsorption leading to the condition of salt‐sensitivity. To examine the interaction between NO and O 2 − activity in the renal epithelial cells, cultured HK2 (proximal tubular) and M1 (distal tubular) cells were incubated with L‐NAME (1 mM) or vehicle for 2, 4 and 6 hrs in different wells (n=6 in each case) separately. O 2 − activity in incubated cells was determined by Lucigenin‐chemiluminescence assay. Compared to vehicle treated cells, L‐NAME treated cells showed increment in O 2 − activity within 2 hr of incubation in HK2 cells (973 ± 99 to 1691 ± 165 RLU/μg protein) and in M‐1 cells (1768 ± 244 to 3548 ± 622 RLU/μg protein). Such increases in O 2 − activity was prevented in cells co‐incubated with NO donor compound, DETA NONOate (1 mM). In separate sets, L‐NAME treated cells were co‐incubated with O 2 − scavenger, tempol (5 mM) or with NADPH oxidase inhibitor, apocynin (5 mM). O 2 − activity was minimal in cells co‐incubated with L‐NAME + tempol (189 ± 13 and 345 ± 24 RLU/μg protein) and L‐NAME + apocynin (242 ± 25 and 333 ± 27 RLU/μg protein) in HK2 and M1 cells respectively indicating that NADPH oxidase is the major source of O 2 − production in these cells. These data demonstrate that a reduction in NO generation leads to increases in O 2 − production induced by NADPH oxidase activity in renal epithelial cells.