It has previously been reported that H+ efflux via Na+/H+ exchange stimulates NAD(P)H oxidase dependent O2 − production in medullary thick ascending limb. We have recently demonstrated that N-methyl-amiloride sensitive O2 − production is enhanced in thick ascending limb of salt-sensitive SS rats suggesting that H+ efflux through Na+/H+ exchangers may promote renal oxidative stress and the development of hypertension in these animals. In the current study we demonstrate, using selective and potent inhibitors, that inhibition of Na+/H+ exchange does not mediate the ability of N-methyl-amiloride to inhibit thick ascending limb O2 − production. To determine the mechanism of action of N-methyl-amiloride, we examined H+ efflux and O2 − production in SS and SS.13BN thick ascending limb of pre-hypertensive, 0.4% NaCl fed rats. Tissue strips containing medullary thick ascending limb were isolated from male SS and salt-resistant consomic SS.13BN rats, loaded with either DHE or BCECF, and imaged in a heated tissue bath. In Na+ replete media, activation of Na+/H+ exchange using an NH4Cl prepulse did not stimulate thick ascending limb O2 −production. In Na+ free media containing BaCl2 in which Na+/H+ activity was inhibited, a NH4Cl pre-pulse stimulated mTAL O2 −. This response was enhanced in mTAL of SS rats (slope ΔEth/ΔDHE=0.029±0.004) compared to SS.13BN rats (slope=0.010±0.004; p<0.04) and could be inhibited by N-methyl-amiloride (slope=0.005±0.002 & 0.006±0.002 for SS and SS.13BN, respectively). We conclude that only H+ efflux through a specific, as yet unidentified, amiloride-sensitive H+ channel promotes O2 − production in medullary thick ascending limb, and that this channel is up-regulated in SS rats.
Keywords: amiloride, blood pressure, free radicals, H+ transport, kidney, NAD(P)H oxidase, pH