title: Resveratrol increases nitric oxide production in the rat thick ascending limb via Ca2+/calmodulin.
abstract: The thick ascending limb of the loop of Henle reabsorbs 30% of the NaCl filtered through the glomerulus. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl absorption by this segment. Resveratrol, a polyphenol, has beneficial cardiovascular and renal effects, many of which are mediated by NO. Resveratrol increases intracellular Ca2+ (Cai) and AMP kinase (AMPK) and NAD-dependent deacetylase sirtuin1 (SIRT1) activities, all of which could activate NO production. We hypothesized that resveratrol stimulates NO production by thick ascending limbs via a Ca2+/calmodulin-dependent mechanism. To test this, the effect of resveratrol on NO bioavailability was measured in thick ascending limb suspensions. Cai was measured in single perfused thick ascending limbs. SIRT1 activity and expression were measured in thick ascending limb lysates. Resveratrol (100 µM) increased NO bioavailability in thick ascending limb suspensions by 1.3±0.2 AFU/mg/min (p<0.03). The NOS inhibitor L-NAME blunted resveratrol-stimulated NO bioavailability by 96±11% (p<0.03). The superoxide scavenger tempol had no effect. Resveratrol elevated Cai from 48±7 to 135±24 nM (p<0.01) in single tubules. In Ca2+-free media, the resveratrol-induced increase in NO was blunted by 60±20% (p<0.05) and the rise in Cai reduced by 80%. Calmodulin inhibition prevented the resveratrol-induced increase in NO (p<0.002). AMPK inhibition had no effect. Resveratrol did not increase SIRT1 activity. We conclude that resveratrol increases NO production in thick ascending limbs via a Ca2+/calmodulin dependent mechanism, and SIRT1 and AMPK do not participate. Resveratrol-stimulated NO production in thick ascending limbs may account for part of its beneficial effects.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, Ohio, United States of America; Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Agustin
authors.initials: A
authors.lastname: Gonzalez-Vicente
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, Ohio, United States of America; Universidad de Buenos Aires, Facultad de Medicina, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Pablo D
authors.initials: PD
authors.lastname: Cabral
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, Ohio, United States of America; Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan, United States of America.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Garvin
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015870:Gene Expression; D008138:Loop of Henle / Q000187:drug effects* / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D000077185:Resveratrol; D015398:Signal Transduction / Q000187:drug effects; D056564:Sirtuin 1 / Q000235:genetics / Q000378:metabolism; D013267:Stilbenes / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D047428:Protein Kinase Inhibitors; D013267:Stilbenes; D013449:Sulfonamides; D009569:Nitric Oxide; C017967:W 7; D055372:AMP-Activated Protein Kinases; C488368:Sirt1 protein, rat; D056564:Sirtuin 1; D000077185:Resveratrol; D002118:Calcium