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.
Highlights
Defective Nitric oxide (NO) signaling in thick ascending limbs contributes to several forms of salt-sensitive hypertension [1,2,3]
We found that 100 mmol/l resveratrol increased DAF-FM signal by 1.360.2 arbitrary fluorescence units (AFU)/mg/min as compared to vehicle (p,0.03; Figure 1) while lower concentrations had no significant effect
We found that L-NAME blunted resveratrol-stimulated NO by 96611% (p, 0.03; Figure 2)
Summary
Defective NO signaling in thick ascending limbs contributes to several forms of salt-sensitive hypertension [1,2,3]. Binding of Ca2+/calmodulin to NOS3 facilitates the electron flux through its domains and increases NO production [7,8]. Several stimuli such as luminal flow, ATP, endothelin and angiotensin II stimulate NOS3 by enhancing phosphatidylinositol 3 kinase and Akt activity. Akt phosphorylates of NOS3 at the serine 1177 (S1177) which reduces dissociation of calmodulin at low Cai levels, such that normal basal Cai is sufficient to increase NO production. Other kinases such as AMP kinase (AMPK) phosphorylate NOS3 at S1177, activating it independently of increases in Cai [8]. Deacetylation of the lysines 496 and 506 within the calmodulin binding domain by NAD-dependent deacetylase sirtuin (SIRT1) is thought to favor calmodulin binding and NOS3 activation [9]
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