Expression of Nox1 in 3T3 cells increases cellular acid production but not proton conductance

Abstract

The role of the NADPH oxidase homolog 1 (Nox1) in plasma membrane H + conductance and cellular H + production was investigated in 3T3 cells stably expressing Nox1 (Nox1 3T3) compared to vector-expressing control cells (mock 3T3). In whole cell patch clamp experiments both Nox1 and mock 3T3 expressed a similar H + conductance (Nox1 3T3, 13.2 ± 8.6 pS/pF; mock 3T3, 16.6 ± 13.4 pS/pF) with a number of similar characteristics (e.g., current–voltage relations, current activation kinetics, Zn 2+-sensitivity). When the intracellular pH of cells was alkalinized with NH 4Cl, rates of intracellular acidification were significantly higher in Nox1 3T3 compared to mock 3T3. Nox1 3T3 showed a time course of acidification that followed a double-exponential function with a fast and a slow component of, on average, τ = 165 s and 1780 s, whereas mock 3T3 showed only a single slow τ of 1560 s. Expression of Nox1 also caused cells to acidify the extracellular medium at higher rates than control cells; Nox1 3T3 released 96 ± 19 fmol h −1 cell −1 of acid equivalents compared to 19 ± 12 fmol h −1 cell −1 in mock 3T3. These data show that expression of Nox1 results in a mechanism that has the capacity to rapidly acidify the cytosol and generate significant amounts of acid. No significant effect of Nox1 expression on the plasma membrane H + conductance was found.