Isoforms and functions of NAD(P)H oxidase at the macula densa.
ABSTRACT Macula densa cells produce superoxide (O2-) during tubuloglomerular feedback primarily via NAD(P)H oxidase (NOX). The purpose of the present study was to determine NOXs expressed by the macula densa and the role of each one in NaCl-induced O2- production. To identify which isoforms are expressed, we applied single-cell RT-PCR to macula densa cells isolated by laser capture microdissection and to MMDD1 cells (a macula densa-like cell line). The captured cells expressed neuronal NOS (marker of macula densa), NOX2, and NOX4 but not NOX1. Expression of the NOXs and neuronal NOS was essentially identical in the MMDD1 cells. Thus, we used MMDD1 cells to investigate which isoform is responsible for NaCl-induced O2- production. We used small-interfering RNA to knock down NOX2 or NOX4 in MMDD1 cells and measured O2- exposed to low-salt solution (LS; 70 mmol/L of NaCl) or high-salt solution (HS; 140 mmol/L of NaCl). Exposing control cells (scrambled small-interfering RNA) to HS increased O2- concentrations from 0.75+/-0.28 to 1.48+/-0.46 U/min per 10(5) cells in LS and HS, respectively (P<0.001). Inhibiting NOX2 blocked the HS-induced increase in O2- (0.62+/-0.39 versus 0.76+/-0.31 U/min per 10(5) cells in LS and HS groups, respectively). Blocking NOX4 did not affect HS-induced O2- levels. O2- levels in the control cells during LS and HS were 0.80+/-0.30 and 1.56+/-0.49 U/min per 10(5) cells, respectively (P<0.001); whereas O2- levels in NOX4-small-interfering RNA-treated cells during LS and HS were 0.40+/-0.25 and 1.26+/-0.51 U/min per 10(5) cells, respectively (P<0.001). We conclude that, whereas macula densa cells express the NOX2 and NOX4 isoforms, NOX2 is primarily responsible for NaCl-induced O2- generation.
Article: Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox.[show abstract] [hide abstract]
ABSTRACT: High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for "dual oxidase" because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.The Journal of Cell Biology 09/2001; 154(4):879-91. · 10.26 Impact Factor