Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney

Citation
F. Wu et al., Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney, AM J P-REN, 45(6), 1999, pp. F874-F881
Citations number
35
Categorie Soggetti
da verificare
Journal title
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
ISSN journal
03636127 → ACNP
Volume
45
Issue
6
Year of publication
1999
Pages
F874 - F881
Database
ISI
SICI code
0363-6127(199906)45:6<F874:QONOSA>2.0.ZU;2-V
Abstract
This study was designed to quantify nitric oxide synthase (NOS) activity in microdissected glomeruli (Glm), pars convoluta, pars recta, cortical colle cting duct, cortical thick ascending limb, outer medullary collecting duct, medullary thick ascending limb and thin limb, inner medullary collecting d uct (IMCD) and thin limb, and vasa recta (VR). Total protein from microdiss ected segments was incubated with L-[H-3]arginine and appropriate cofactors , and the L-arginine and converted L-citrulline were separated by reverse-p hase HPLC and radiochemically quantitated. NOS activity was found to be gre atest in IMCD (11.5 +/- 1.0 fmol citrulline mm(-1) h(-1)) and moderate in G lm (1.9 +/- 3 fmol.glomerulus(-1)h(-1)) and VR (3.2 +/- 0.8 fmol mm-l h-l). All other renal structures studied exhibited significantly less NOS activi ty. The mRNA for NOS isoforms in the NOS activity-positive segments was the n identified by RT-PCR. The IMCD contained mRNA for neuronal (nNOS), endoth elial (eNOS), and inducible NOS (iNOS), but Glm and VR only expressed the m RNA for nNOS and eNOS. These experiments demonstrate that the greatest enzy matic activity for NO production in the kidney is in the IMCD, three- to si xfold less activity is present in the Glm and VR, and minimal NOS activity is found in other segments studied.