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Luminal flow induces eNOS activation and translocation in the rat thick ascending limb. II. Role of PI3-kinase and Hsp90

Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202

Submitted 30 October 2003 ; accepted in final form 13 April 2004

Endothelial nitric oxide synthase (eNOS) regulates NaCl absorption by the thick ascending limb of the loop of Henle (THAL). We found that augmenting luminal flow induces eNOS activation and translocation to the apical membrane of THALs (Ortiz PA, Hong NJ, and Garvin JL. Am J Physiol Renal Physiol 287: F274–F280, 2004). In other cells, eNOS activation by shear stress is mediated by phosphatidylinositol 3-OH kinase (PI3)-kinase. We hypothesized that luminal flow induces eNOS activation via PI3-kinase. Pretreatment of THALs with wortmannin, a PI3-kinase inhibitor, significantly reduced flow-induced nitric oxide (NO) release by 75% (from 53.6 ± 6 to 13.2 ± 5.7 pA/mm). Increasing luminal flow from 0 to 20 nl/min induced eNOS translocation to the apical membrane, whereas in the presence of wortmannin eNOS translocation was prevented (basolateral = 32 ± 2%, middle = 38 ± 1%, apical = 30 ± 1%, n = 5, not significant vs. no flow). We next studied which PI3-kinase product mediates eNOS translocation. Addition of PI(3,4,5)P₃ (5 µM) in the absence of flow increased NO levels (P < 0.05) and induced eNOS translocation to the apical membrane (from 40 ± 4 to 60 ± 2% of total eNOS, n = 6, P < 0.05). Incubation with PI(3,4)P₂ or PI(4,5)P₂ did not change eNOS localization. We next tested whether heat shock protein (Hsp)90 is involved in eNOS translocation. The Hsp90 inhibitor geldanamycin blocked flow-induced eNOS translocation to the apical membrane (n = 6). Flow also induced translocation of Hsp90 to the apical membrane (from 35 ± 2 to 57 ± 2%; P < 0.05) in a PI3-kinase-dependent manner. We conclude that luminal flow induces eNOS translocation and activation in the THAL via PI3-kinase and that Hsp90 is involved in eNOS translocation to the apical membrane.

nitric oxide; renal tubules; trafficking; phosphatidylinositol; heat shock protein; endothelial nitric oxide synthase

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