Luminal flow induces eNOS activation and translocation in the rat thick ascending limb.
Nitric oxide (NO) produced by endothelial NO synthase (eNOS) acts as an autacoid to inhibit NaCl absorption in the thick ascending limb of the loop of Henle (THAL). In the vasculature, shear stress activates eNOS. We hypothesized that increasing luminal flow activates eNOS and enhances NO production in the THAL. We measured NO production by isolated, perfused THALs using a NO-sensitive microelectrode. Increasing luminal flow from 0 to 20 nl/min increased NO production by 43.1 +/- 4.1 pA/mm of tubule (n = 10, P < 0.05), and this response was blunted (92%) by the NOS inhibitor L-(omega)nitro-methylarginine (P < 0.05). We studied the effect of flow on eNOS subcellular localization. In the absence of flow, eNOS was diffusely localized throughout the cell (basolateral = 33 +/- 4%; middle = 27 +/- 3%; apical = 40 +/- 4% of total eNOS). Increasing luminal flow induced eNOS translocation to the apical membrane, as evidenced by a 60% increase in eNOS immunoreactivity in the apical membrane (from 40 +/- 4 to 65 +/- 2%; n = 6; P < 0.05). Disrupting the actin cytoskeleton with cytochalasin D (10 microM) reduced flow-induced NO production by 62% (from 37.1 +/- 3.4 to 14.0 +/- 2.4 pA/mm tubule, n = 7, P < 0.04) and blocked flow-induced eNOS translocation. Flow also increased the amount of phosphorylated eNOS (Ser1179) at the apical membrane (from 25 +/- 2 to 56 +/- 2%; P < 0.05). We conclude that increasing luminal flow induces eNOS activation and translocation to the apical membrane in THALs. These are the first data showing that flow regulates eNOS in epithelial cells. This may be an important mechanism for regulation of NO levels in the renal medulla.
Ortiz
Pablo A
PA
Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202, USA. portiz1@hfhs.org
Hong
Nancy J
NJ
Garvin
Jeffrey L
JL
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008138:Loop of Henle / Q000378:metabolism / Q000502:physiology*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019335:Nitroarginine / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D014563:Urodynamics
D004791:Enzyme Inhibitors; D019335:Nitroarginine; D015638:Cytochalasin D; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat