Hyperosmolarity-Induced Dilation and Epithelial Bioelectric Responses of Guinea Pig Trachea in Vitro: Role of Kinase Signaling

  1. Yi Jing,
  2. Janet A. Dowdy,
  3. Michael R. Van Scott and
  4. Jeffrey S. Fedan
  1. Department of Biochemistry and Molecular Pharmacology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia (Y.J., J.S.F.); Department of Physiology, The Brody School of Medicine, East Carolina University, Greenville, North Carolina (M.R.V.S.); and Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia (J.A.D., J.S.F.)
  1. Address correspondence to:
    Dr. Jeffrey S. Fedan, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Rd., Morgantown, WV 26505-2888. E-mail: jsf2{at}cdc.gov

Abstract

Exercise-induced airway obstruction is thought to involve evaporative water loss and hyperosmolarity of the airway surface liquid. Hyperosmolar challenge of the epithelium of isolated, perfused guinea pig trachea rapidly alters transepithelial potential difference (Vt), and it elicits smooth muscle relaxation mediated by epithelium-derived relaxing factor (EpDRF). In many cell types, protein kinases mediate responses to hyperosmolarity and regulatory volume increase. In this study, inhibitors were used to investigate the involvement of kinases and phosphatases in bioelectric responses of epithelium to hyperosmolarity and their possible relationship to EpDRF-mediated relaxation. After contraction of the perfused trachea with extraluminal methacholine, d-mannitol applied intraluminally (≤80 mosM) increased Vt and elicited dilation of the smooth muscle with a similar concentration-dependence; higher concentrations decreased Vt. In tracheas exposed to 30 mosM d-mannitol (∼EC50), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580) and SKF 86002 [6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridyl)imidazo[2,1-b]thiazole] (p38 inhibitors) potentiated the dilation, whereas SP 600125 [anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone] and dicumarol [c-Jun NH2-terminal kinase (JNK) inhibitors], chelerythrine [nonselective protein kinase C (PKC) inhibitor], and NaAsO2 (mitogen-activated protein kinase stress inducer) and Na3VO4 (protein tyrosine phosphatase inhibitor) inhibited the hyperpolarization. Large increases in the phosphorylation of p38 and JNK occurred at concentrations higher than those needed to elicit functional responses. The phosphatidylinositol 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002) and Na3VO4 did not affect the Vt responses, but they inhibited methacholine-induced constriction; SP 600125 and dicumarol potentiated, and chelerythrine inhibited, methacholine-induced epithelial hyperpolarization. These results suggest that JNK, PKC, and phosphatase(s) are involved in hyperosmolarity-induced hyperpolarization of the tracheal epithelium but that p38 is involved in EpDRF-mediated relaxation.

Footnotes

  • This work was supported, in part, by a predoctoral fellowship from the Ohio Valley Affiliate of the American Heart Association (to Y.J.).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

  • doi:10.1124/jpet.107.135871.

  • ABBREVIATIONS: ASL, airway surface liquid; D-M, d-mannitol; EpDRF, epithelium-derived relaxing factor; RVI, regulatory volume increase; MAP, mitogen-activated protein; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; CaM-K-II, calmodulin kinase-II, MLCK, myosin light chain kinase; PI-3-K, phosphatidylinositol 3-kinase; MCh, methacholine; MKH, modified Krebs-Henseleit; SB 203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; PD 98059, 2′-amino-3′-methoxyflavone; U 0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; LY 294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine; DMSO, dimethyl sulfoxide; Vt, transepithelial potential difference; ΔP, inlet minus outlet perfusion pressure difference; Rt, transepithelial resistance; SKF 86002, 6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridyl)imidazo[2,1-b]thiazole; SP 600125, anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone; ML-7, 1-(5-iodonaphthalene-1-sulfonyl)homopiperazine.

    • Received December 21, 2007.
    • Accepted April 14, 2008.
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  1. Published online before print April 15, 2008, doi: 10.1124/jpet.107.135871 JPET July 2008 vol. 326 no. 1 186-195
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