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

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 (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 (Յ80 mosM) increased V t and elicited dilation of the smooth muscle with a similar concentrationdependence; higher concentrations decreased V t . In tracheas exposed to 30 mosM D-mannitol (ϳEC 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.

Activation and Clinical Significance of p38 MAPK Signaling Pathway in Patients With Severe Trauma

“…p38 MAPK can be activated in response to a variety of extracellular stimuli including UV [10], osmolarity [11], inflammatory cytokines, growth factor, and shock. p38 MAPK is transduced through highly conserved 3-level kinase cascade [12,13].…”

Tracheal epithelium cell volume responses to hyperosmolar, isosmolar and hypoosmolar solutions: relation to epithelium-derived relaxing factor (EpDRF) effects

“…Amiloride, DIDS and NPPB, but not bumetanide, inhibited hyperosmolar solution-induced relaxation of IPT and bioelectric responses (Fedan et al, 1999; Wu et al, 2004; Jing et al, 2008a). In unstimulated, dispersed cells, amiloride and bumetanide had little or no effect on cell volume, while DIDS (but not NPPB) reduced cell volume.…”

“…The IPT (Munakata et al, 1988; Fedan and Frazer, 1992; Jing et al, 2008a) is a novel preparation that permits agents to be applied separately to the mucosal (intraluminal or IL) or serosal (extraluminal or EL) surfaces of the trachea while monitoring contractile or relaxant responses of the airway smooth muscle from changes in diameter. It allows assessment of the role of the epithelium in integrated responses of the organ (Jing et al, 2008b) and has been used to demonstrate that both the apical and basolateral membranes of airway epithelial cells respond to hyperosmolar challenge (Fedan et al, 2004a).…”

“…A corollary question, “what are the effects of raised osmolarity of the ASL on airway function?,” has not been addressed and, therefore, has been investigated in our laboratory. In the guinea-pig isolated, perfused trachea (IPT) preparation, hyperosmolar challenge of the epithelium induces relaxation of the airway smooth muscle (Munakata et al, 1988; Fedan et al, 1999, 2004a,b; Johnston et al, 2004; Wu et al, 2004; Jing et al, 2008a) that is inhibited by the Na + channel blocker, amiloride, and the Cl − channel blockers, 4, 4′-diisothiocyano-2, 2′-stilbenedisulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Ionic and non-ionic, permeant and impermeant osmolytes have similar relaxant potencies (~9–25 mOsM).…”

“…EpDRF resembles, in part, carbon monoxide; it is not nitric oxide or a prostanoid. p38 is involved in EpDRF-mediated relaxation (Jing et al, 2008a). Relaxation responses are not inhibited by cytoskeleton/microtubule-interfering agents.…”