Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intracellular pH in a mouse macula densa cell line (NE-MD)

Abstract

Changes in the luminal NaCl concentration ([NaCl]) at the macula densa (MD) modulate the tubuloglomerular feedback (TGF) responses via an affect on the release of nitric oxide (NO). This study was performed in a newly established mouse macula densa cell line (NE-MD) to investigate the effects of lowering [NaCl] on the neuronal NO synthase (nNOS) protein expression and l-arginine (Arg)-induced NO release. Expression of nNOS protein and release of NO were evaluated by Western blot analysis and an NO-sensitive electrode, respectively. Intracellular pH (pHi) was monitored by the BCECF assay. Although there was weak staining of the nNOS protein expression, l-Arg-induced NO generation was negligible in normal (140 mM NaCl) solution. Both were significantly (P < 0.05) increased either in the presence of furosemide (12 μM), an inhibitor of the Na+-K+-2Cl- cotransporter, or in a low (23 mM) Cl- solution. Furosemide- and low Cl--induced NO generation was completely inhibited by 50 μM 7-nitroindasole (7-NI), a nNOS inhibitor. Moreover, these increases were significantly (P < 0.05) inhibited by the addition of 100 μM amiloride, an inhibitor of the Na+/H+ exchanger, or by its analogue 5-(N)-ethyl-N-isopropyl amiloride (EIPA), and also at a lower pH of 7.1. Furthermore, nNOS expression and NO release were not stimulated in as low as 19 mM Na+ solution. In conclusion, low [Cl-], but not low [Na+] in the lumen at the MD, increased nNOS protein expression and NO generation. Changes in the luminal [NaCl] may modulate the TGF system via an effect on the NO generation from the MD. © 2009 The Physiological Society of Japan and Springer.