NADPH-diaphorase activity and nitric oxide synthase activity in the kidney of the clawed frog, Xenopus laevis.
Nitric oxide (NO) may play a central role in controlling renal hemodynamics and renal salt excretion. Thus, several investigations focused on localization and function of nitric oxide synthase (NOS) isoforms in the mammalian kidney. Although studies of amphibians have contributed significantly to the elucidation of renal physiology, NOS has not been investigated in the amphibian kidney. Therefore, we characterized NOS and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase biochemically and, furthermore, visualized putative NO-producing cells in the kidney of the clawed frog, Xenopus laevis. Our results indicate that NADPH-diaphorase activity correlates with NOS activity. Both enzyme activities eluted at 225 mM NaCl on a diethylaminoethanol anion exchange column and had an apparent molecular weight of 235 kDa, as estimated on an S-300 Sephacryl column. In addition, these enzymes were sensitive to Ca2+ and NADPH, but insensitive to calmodulin antagonists (trifluoperazine, W-13) or omission of calmodulin from the reaction medium. The molecular identity of NOS in Xenopus kidney extract was estimated using polymerase chain reaction. Primers to Xenopus neuronal NOS hybridized to a transcript in Xenopus kidney homogenate. NADPH-diaphorase histochemistry revealed staining in the neck segment, distal tubules, collecting segment, and peritoneal funnels. NOS-immunoreactive material was visualized in distal tubules. These results indicate that Xenopus kidney contains at least neuronal NOS, but may contain an additional NOS isoform, which is less calmodulin sensitive.
Kerschbaum
H H
HH
Department of Molecular Neurobiology and Cell Physiology, Institute of Zoology, University of Salzburg, Austria. Hubert.Kerschbaum@sbg.ac.at
Huang
S
S
Xie
M
M
Hermann
A
A
D000818:Animals; D015342:DNA Probes; D018076:DNA, Complementary; D015870:Gene Expression / Q000502:physiology; D007685:Kidney Tubules, Collecting / Q000201:enzymology*; D007686:Kidney Tubules, Distal / Q000201:enzymology*; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014982:Xenopus laevis
D015342:DNA Probes; D018076:DNA, Complementary; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009252:NADPH Dehydrogenase