Autocrine effects of nitric oxide on HCO3- transport by rat thick ascending limb

Abstract

Background. In vivo and in vitro studies have shown that nitric oxide (NO) is an important modulator of transport processes along the nephron. The thick ascending limb (TAL) plays a significant role in the urine-concentrating mechanism and in the maintenance of acid/base balance. Methods. TALs from male Sprague-Dawley rats were isolated and perfused, and net bicarbonate flux (J(HCO3)-) was determined. Results. In perfused TALs, 0.5 mmol/L L-arginine (L-Arg), the substrate for NO synthase, significantly lowered J(HCO3)- from 35.4 ± 4.6 to 23.2 ± 2.9 pmol · mm-1 · min-1, a decrease of 36.9 ± 11.6% (P < 0.025). D-Arg (0.5 mmol/L) had no effect on J(HCO3)- (N = 7). In the presence of 5 mmol/L L-NAME, an NO synthase (NOS) inhibitor, the addition of L-Arg did not affect TAL J(HCO3)- (43.4 ± 4.4 vs. 44.6 ± 5.0 pmol · mm-1 · min-1). L-NAME alone (5 mmol/L) did not affect TAL J(HCO3)-. After removing L-Arg from the bath, J(HCO3)- increased from 26.2 ± 3.9 to 34.8 ± 3.2 pmol · mm-1 · min-1 (P < 0.01), indicating no cytotoxicity of NO. We next investigated the effect of cGMP analogues on TAL J(HCO3)-. 8-Br-cGMP (50 μmol/L) and db-cGMP (50 μmol/L) significantly decreased J(HCO3)- by 26.3 ± 9.1% and 35.1 ± 11.6%, respectively. In the presence of cGMP (50 μmol/L), the addition of L-Arg had no effect on J(HCO3)-. In the presence of KT-5823 (2 μmol/L), a protein kinase G inhibitor, the addition of L-Arg did not change TAL J(HCO3)- (N = 5). Conclusions. We conclude that (1) endogenously produced NO inhibits TAL J(HCO3)- in an autocrine manner, (2) cGMP mediates all of the effects of NO, and (3) this effect is mediated by protein kinase G activation.