Abstract
Macula densa (MD) modulates tubuloglomerular feedback system by generating nitric oxide (NO) in response to changes of luminal NaCl concentration. A newly established mouse cell line of MD (NE-MD) shows up-regulation of nNOS protein when NE-MD cells were incubated with either low [NaCl] or 12 μM furosemide for 5 hrs. Molecular weights of furosemide-induced nNOS proteins were 65 kDa and 150 kDa (Western blotting), although a faint single band of 150 kDa was only recognized under the control condition. This suggests that truncated nNOS protein may play a key role for regulation of NO generation. To further investigate nNOS, we examined the control and furosemide-induced proteomes of NE-MD by two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MALDI-TOF-MS) as well as a study of nNOS mRNA by RT-PCR technique. We found that nNOS mRNA was about 4.0 kb corresponding to a nearly full length of nNOS protein (150 kDa). More over, we found that 6 protein spots were increased by 1.5 times in NE-MD cells treated with furosemide (2-DE), and that only one unique protein spot, determined as nNOS, increased by more than 5 times. By using MALDI-TOF-MS, although N-terminal heme binding domain was conserved, a C-terminal reductase domain was missing. In conclusion, N-terminal oxidative domain of nNOS protein may explain a unique regulation of NO generation in the mouse kidney macula densa. [J Physiol Sci. 2008;58 Suppl:S21]
