Nitric oxide reduces the molecular activity of Na+,K+-ATPase in opossum kidney cells.
BACKGROUND
Nitric oxide (NO) directly inhibits fluid and solute reabsorption in the proximal tubule. In the present study, we investigated the effect of NO on the Na+, K+-ATPase of opossum kidney (OK) cells, a proximal tubule cell line, and its mechanisms.


METHODS
Na+,K+-ATPase activity in the membrane fraction of OK cells was measured as the ouabain-sensitive ATP hydrolytic activity. The enzyme unit number on intact cells was measured by ouabain-binding assay.


RESULTS
Incubation with 0.5 mM sodium nitroprusside (SNP), a NO donor, for two hours inhibited the catalytic activity of the membrane-associated Na+,K+-ATPase in OK cells to 65.5 +/- 9.7% of control (N = 6, P < 0.05 vs. control). This effect of SNP was concentration- and time-dependent. The NO scavenger hemoglobin blunted, while another NO donor spermine NONOate (5 microM) mimicked this effect of SNP. At all concentrations and time points tested, SNP did not alter the molecular number of Na+,K+-ATPase on intact OK cells, indicating that NO inhibited the molecular activity of Na+,K+-ATPase. The soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ), blunted the inhibitory effect of SNP on the Na+,K+-ATPase activity. An exogenous cGMP analog similarly inhibited the Na+,K+-ATPase activity. Neither lipid soluble antioxidants vitamin E/probucol or thiol group compound DL-dithiothreitol (DTT) altered the inhibitory effect of SNP on the Na+,K+-ATPase activity.


CONCLUSIONS
NO inhibited the molecular activity of the Na+,K+-ATPase of the OK proximal tubule cell line probably via cGMP-dependent mechanisms.
Liang
M
M
Department of Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Knox
F G
FG
D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D000959:Antihypertensive Agents / Q000494:pharmacology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D015227:Lipid Peroxidation / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D009893:Opossums; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology; D011341:Probucol / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014810:Vitamin E / Q000494:pharmacology
D000924:Anticholesteremic Agents; D000959:Antihypertensive Agents; D004791:Enzyme Inhibitors; D014810:Vitamin E; D009599:Nitroprusside; D009569:Nitric Oxide; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D006152:Cyclic GMP; D011341:Probucol