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1 Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, MI, USA
* To whom correspondence should be addressed. E-mail: portiz1{at}hfhs.org.
We have identified endothelial nitric oxide synthase (eNOS) as the NOS isoform that regulates NaCl absorption by the thick ascending limb of the loop of Henle (THAL). Recently, we found that augmenting luminal flow induces eNOS activation and translocation to the apical membrane of THALs (companion study, Ortiz PA, Hong NJ, and Garvin J.L.). However, it is not known how flow regulates eNOS activity and translocation in these cells. In endothelial cells eNOS activation by shear stress, endothelial growth factor and estradiol is mediated by the phosphatidylinositol 3-OH kinase (PI3 kinase) pathway. We hypothesized that increasing luminal flow induces eNOS activation and translocation via activation of the PI3 kinase cascade. Pretreatment of THALs with wortmannin, a PI3 kinase inhibitor, reduced flow-induced NO release by 75% (from 53.6 ± 6 to 13.2 ± 5.7 pA/mm of tubule, n = 7; p<0.05). We then studied whether wortmannin could prevent flow-induced translocation of eNOS. Increasing luminal flow from 0 to 20-25 nl/min induced eNOS translocation to the apical membrane (from 40±4% to 65±2% of total eNOS, n = 6, p < 0.05) whereas in the presence of wortmannin eNOS translocation was prevented (basolateral = 32±2%, middle = 38±1%, apical = 30±1%, n = 5, n.s vs no flow). We next tested whether the PI3 kinase product phosphatidylinositol(3,4,5)3P could induce eNOS activation and translocation in the absence of luminal flow. Addition of PI(3,4,5)3P (5 µM) in the absence of flow increased intracellular NO levels as evidenced by a 12% increase in fluorescence of the NO-sensitive dye DAF-2DA (n = 6, p<0.05). Incubation of THALs with PI(3,4,5)3P (5 µM) induced eNOS translocation to the apical membrane (from 40±4% to 60±2% of total eNOS, n = 6,p < 0.05). Incubation with the PI3 kinase product PI(3,4)2P or the PI(3,4,5)3P precursor PI(4,5)2P, did not change eNOS localization (n = 5). Finally, we tested whether Hsp90, a protein that interacts with eNOS, is involved in eNOS translocation and whether its localization is affected by luminal flow. We found that the Hsp90 inhibitor geldanamycin, blocked flow-induced eNOS translocation to the apical membrane (n = 6). Flow also induced the translocation of Hsp90 to the apical membrane (from 35 ± 2% to 57 ± 2%; n = 6, p < 0.05) in a PI3 kinase-dependent manner. We concluded that luminal flow induces eNOS translocation and activation via the PI3 kinase cascade in the THAL, and that Hsp90 is involved in eNOS translocation to the apical membrane.
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