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1 Department of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, MI, USA
* To whom correspondence should be addressed. E-mail: portiz1{at}hfhs.org.
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). Little is known about regulation of NO production and eNOS activity in this nephron segment. In the vasculature, shear stress increases NO production via activation of eNOS. We hypothesized that increasing luminal flow activates eNOS and enhances NO production in the THAL. We first measured the effect of flow on NO production by isolated perfused THALs using a NO-sensitive microelectrode. We found that increasing luminal flow from 0 to 20-25 nl/min increased NO production by 43.1 ± 4.1 pA/mm of tubule length (n = 10, p < 0.05). The NOS inhibitor L-NAME (5 mM), blunted flow-induced NO production by 92% (n = 6; p < 0.05). Since eNOS changes its localization when activated in endothelial cells, we studied the effect of flow in eNOS subcellular localization. In the absence of luminal flow, eNOS was diffusely localized throughout the cell (basolateral = 33±4%; middle = 27±3%; apical = 40±4% of total eNOS immunofluorescence; n = 6). Increasing luminal flow to 20-25 nl/min induced eNOS translocation to the apical membrane, as evidenced by a 60% increase in eNOS immunoreactive protein in the apical membrane (from 40±4% to 65±2%; n = 6; p < 0.05). We next tested whether disruption of the cytoskeleton could block flow-induced NO production and eNOS translocation. We found that pretreatment of THALs with cytochalasin D (10 µM) reduced flow-induced NO production by 62% (from 37.1 ± 3.4 to 14.0 ± 2.4 pA/mm tubule length, n = 7, p<0.04) and blocked flow-induced eNOS translocation to the apical membrane (basolateral = 49±3%; middle = 25±1%; apical = 26±2%; n = 6). Finally, we tested whether flow induces phosphorylation of eNOS at the apical membrane using a phospho-specific antibody. Flow increased the amount of phosphorylated eNOS (ser 1179) in the apical membrane (from 25 ± 2% to 56 ± 2%; p<0.05). We concluded that increasing luminal flow activates eNOS and induces its translocation to the apical membrane in the THAL. We believe these are the first data showing that flow regulates eNOS in epithelial cells. Because luminal flow in the THAL may be affected by various conditions, this may be an important mechanism for overall regulation of NO le vels in the renal medulla.
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