NADPH-diaphorase activity and nitric oxide synthase activity in the kidneyof 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 localiza tion and function of nitric oxide synthase (NOS) isoforms in the mammalian kidney. Although studies of amphibians have contributed significantly to th e elucidation of renal physiology, NOS has not been investigated in the amp hibian kidney. Therefore, we characterized NOS and reduced nicotinamide ade nine dinucleotide phosphate (NADPH) diaphorase biochemically and, furthermo re, visualized putative NO-producing cells in the kidney of the clawed frog , Xenopus laevis. Our results indicate that NADPH-diaphorase activity corre lates with NOS activity. Both enzyme activities eluted at 225 mM NaCl on a diethylaminoethanol anion exchange column and had an apparent molecular wei ght of 235 kDa, as estimated on an S-300 Sephacryl column. In addition, the se enzymes were sensitive to Ca2+ and NADPH, but insensitive to calmodulin antagonists (trifluoperazine, W-13) or omission of calmodulin from the reac tion medium. The molecular identity of NOS in Xenopus kidney extract was es timated using polymerase chain reaction. Primers to Xenopus neuronal NOS hy bridized to a transcript in Xenopus kidney homogenate. NADPH-diaphorase his tochemistry revealed staining in the neck segment, distal tubules, collecti ng segment, and peritoneal funnels. NOS-immunoreactive material was visuali zed 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.