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 (V(t)), 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 (< or = 80 mosM) increased V(t) and elicited dilation of the smooth muscle with a similar concentration-dependence; higher concentrations decreased V(t). In tracheas exposed to 30 mosM D-mannitol (approximately EC(50)), 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 NH(2)-terminal kinase (JNK) inhibitors], chelerythrine [nonselective protein kinase C (PKC) inhibitor], and NaAsO(2) (mitogen-activated protein kinase stress inducer) and Na(3)VO(4) (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 Na(3)VO(4) did not affect the V(t) 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.