Prostasin-dependent activation of epithelial [Na.sup.+] channels by low plasmin concentrations

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Publisher: American Physiological Society
Document Type: Author abstract
Length: 299 words

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Svenningsen P, Uhrenholt TR, Palarasah Y, Skjodt K, Jensen BL, Skott O. Prostasin-dependent activation of epithelial [Na.sup.+] channels by low plasmin concentrations. Am J Physiol Regul Integr Comp Physiol 297: RI733-R1741, 2009. First published September 30, 2009; doi: I0.1152/ajpregu.00321.2009.--Several pathophysiological conditions, including nephrotic syndrome, are characterized by increased renal activity of the epithelial [Na.sup.+] channel (ENaC). We recently identified plasmin in nephrotic urine as a stimulator of ENaC activity and undertook this study to investigate the mechanism by which plasmin stimulates ENaC activity. Cy3-1abeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a glycosylphosphatidylinositol (GPI)anchored protein. Biotin-label transfer showed that plasmin interacted with the GPI-anchored protein prostasin on M-1 cells and that plasmin cleaved prostasin. Prostasin activates ENaC by cleavage of the "y-subunit, which releases an inhibitory peptide from the extracellular domain. Removal of GPI-anchored proteins from the M-1 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 [micro]g/ml). At a high plasmin concentration of 30 [micro]g/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked plasmin-stimulated ENaC current in single M-1 cells, as measured by whole-cell patch clamp. In M-1 cells expressing heterologous FLAG-tagged prostasin, "[gamma]ENaC and prostasin were colocalized. A monoclonal antibody directed against the inhibitory peptide of [gamma]ENaC produced specific immunofluorescence labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. We conclude that, at low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the [gamma]-subunit and activation of ENaC, while at higher concentrations, plasmin directly activates ENaC. serine protease; sodium retention; kidney; M-1 cells; nephrotic syndrome doi: 10.1152/ajpregu.00321.2009

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Gale Document Number: GALE|A215115996