Nox2 is required for amiloride‐sensitive H+ efflux in a rat medullary thick ascending limb cell line

Abstract

We have recently demonstrated that an amiloride (NMA)‐sensitive outward H+ current is present in mTAL of salt‐sensitive (SS) but not salt‐resistant (SS.13BN) rats and that when activated, this stimulates the production of excess superoxide (O2−). The identity of the H+ transporter remains unclear, however it displays many of the characteristics of a nox2 associated H+ current characterized in macrophages. The aim of this study was to determine which subunits of NAD(P)H oxidase are expressed in a rat mTAL cell line and whether a similar H+ transport pathway exists in these cells. mTAL cells were cultured, transfected with nox2, loaded with BCECF dye, imaged on a inverted microscope and intracellular pH (pHi) recovery determined following an NH4Cl prepulse in 0 Na+ solution with BaCl2. pHi recovery following an NH4Cl prepulse was absent in control transfected mTAL (δpHi/sec ‐1.9±2*10−5) however, in mTAL transfected with nox2, amiloride‐sensitive pHi recovery could be detected (δpHi/sec 7.9±3.2*10−5 vs ‐1.6±2*10−5 with NMA; P<0.03). Cellular mRNA expression of endogenous NAD(P)H oxidase subunits are shown in the table below. We conclude that although nox4 is the primary gp91 homolog expressed in mTAL cells, nox2 over‐expression is required to produce the outward NMA‐sensitive H+ current observed in SS mTAL.mTAL copy # per 106 18S RNA (n=4)