Abstract
The present study concerns intriguing effects of hydrogen peroxide (H2O2) on cAMP-mediated anion secretion in polarized human airway epithelia. Although H2O2 applied to the apical and basolateral membrane increases short-circuit currents (ISC) with analogous properties, it has opposite effects on subsequent cAMP-activated ISC responses. Namely, forskolin (FK)-induced ISC responses were down-regulated by the apical presence of H2O2, whereas they were up-regulated by its basolateral presence. Despite this contrasting effect, oxidative stimuli from either aspect of the monolayer hindered FK-induced increments in cytosolic cAMP levels and apical membrane Cl- conductance. The site-dependent effects of H2O2 were reproduced in the responses to 8-bromo-cAMP. Estimation of the anionic composition of the ISC revealed that the FK up-regulated both bumetanide [an Na+-K+-2Cl- cotransporter (NKCC1) inhibitor]-sensitive and 4,4′-dinitrostilbene-2,2′-disulfonic acid [an -dependent anion transporter (NBC1/AE2) inhibitor]-sensitive ISC in the control, whereas the up-regulation evidently favored bumetanide-sensitive ISC in the basolateral presence of H2O2. The FK-induced NKCC1 augmentation after exposure to basolateral H2O2 was counteracted by cytochalasin D, an inhibitor of microfilament function, but not by charybdotoxin, a blocker of the intermediate conductance Ca2+-activated K+ channel, whose activation could be related to NKCC1-mediated Cl- secretion. These observations suggest that basolaterally but not apically applied H2O2 potentiates subsequent cAMP-mediated Cl- secretion by an increase in Cl- uptake via basolateral NKCC1, whose sensitivities to cAMP/protein kinase A are up-regulated, overcoming the H2O2-induced inhibition of cAMP-mediated apical anion conductance. The basolateral membrane-specific effects of H2O2 may be relevant to the basolateral cytoskeleton, which is believed to interact with NKCC1.
Footnotes
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This work was supported by Research Grant Fund 14770272 from the Ministry of Education, Culture, Sports, Science, and Technology (to Y.I.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.102541.
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ABBREVIATIONS: ROS, reactive oxygen species; H2O2, hydrogen peroxide; CFTR, cystic fibrosis transmembrane conductance regulator; PSS, physiological saline solution; Rt, transepithelial resistance; ISC, short-circuit current; GCl, apical membrane Cl- conductance; ICl, apical membrane Cl- current; FK, forskolin; 8-Br-cAMP, 8-bromo-cAMP; DNDS, 4,4′-dinitrostilbene-2,2′-disulfonic acid; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate; Cyto-D, cytochalasin D; NS-398, N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide; SC-560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole; ChTx, charybdotoxin; COX, cyclooxygenase; NKCC1, Na+-K+-2Cl- cotransporter; NBC1, cotransporter; AE2, exchanger; KCNN4, human intermediate conductance Ca2+-activated K+ channels; PKA, protein kinase A.
- Received February 7, 2006.
- Accepted March 24, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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