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Published ahead of print on November 7, 2003, doi:10.1165/rcmb.2003-0325OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 720-728, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0325OC

Regulation of Amiloride-Sensitive Na+ Transport by Basal Nitric Oxide

Karin M. Hardiman, Carmel M. McNicholas-Bevensee, James Fortenberry, Carpantato T. Myles, Bela Malik, Douglas C. Eaton and Sadis Matalon

Departments of Physiology and Biophysics, and Department of Anesthesiology, Gregory Fleming James Cystic Fibrosis Research Center, and the Medical Scientists Training Program, University of Alabama at Birmingham, Birmingham, Alabama; and Department of Physiology and Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia

Address correspondence to: Sadis Matalon, Ph.D., Department of Anesthesiology, University of Alabama at Birmingham, 901 19th Street S, BMR2, Room 224, Birmingham, AL 35205–3703. E-mail: Sadis{at}uab.edu

We investigated the mechanisms of endogenous nitric oxide (NO) modulation of lung sodium (Na+) transport. C57BL/6 mice injected intraperitoneally with the specific inducible NO synthase (iNOS) inhibitor 1400W (10 mg/kg every 8 h for 72 h) exhibited decreased alveolar nitrite levels and Na+-dependent amiloride-sensitive alveolar fluid clearance as compared with mice injected with vehicle. Similarly, pretreatment of mouse tracheal epithelial cells with 1400W abolished the inhibitory effects of amiloride on their Na+ short circuit currents. On the other hand, mouse tracheal epithelial cells pretreated with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of guanylate cyclase, had lower levels of cGMP, but normal values of amiloride-sensitive Na+ currents. Amiloride also inhibited whole-cell Na+ currents across A549 cells treated with vehicle (Ki = 249 nM), but had no effect in A549 cells treated with 1400W. Western blotting studies showed significantly lower levels of {alpha} and {gamma}ENaC in lung tissues and alveolar type II (ATII) cells from iNOS–/– as well as iNOS+/+ mice treated with 1400W, as compared with the corresponding values from vehicle-treated iNOS+/+ mice. Similar values for ratios of {alpha}, ß, and {gamma}enac to gapdh were obtained by real-time polymerase chain reaction for iNOS+/+ mice and iNOS–/– mice. We concluded that NO derived from iNOS under basal conditions is necessary for amiloride-sensitive Na+ transport across lung epithelial cells and modulates the amount of {alpha} and {gamma}ENaC via post-transcriptional, cGMP-independent mechanisms.

Abbreviations: alveolar fluid clearance, AFC • alveolar type II cells, ATII • bronchoalveolar lavage, BAL • bovine serum albumin, BSA • 2,3-diaminonaphthalene, DAN • Dulbecco's modified Eagle's medium, DMEM • dimethyl sulfoxide, DMSO • enhanced chemiluminescence, ECL • horseradish peroxidase, HRP • equivalent short circuit current, Ieq • short circuit current, Isc • inducible nitric oxide synthase, iNOS • mouse tracheal epithelial cells, MTE • sodium, Na+ • nitric oxide, NO • nasal potential difference, NPD • 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, ODQ • polymerase chain reaction, PCR • prostaglandin E2, PGE2




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