Systemic lipopolysaccharide stimulates airway transepithelial Na+ transport by increasing ENaC and Na+,K+ -pump activity

Abstract

Systemic lipopolysaccharide stimulates airway transepithelial Na + transport by increasing ENaC and Na + ,K +-pump activity Michael W. Dodrill Our laboratory found that systemic administration of lipopolysaccharide (LPS; 4 mg/kg) hyperpolarized the transepithelial potential difference (V t) of tracheal epithelium in the isolated, perfused trachea (IPT) of the guinea pig 18 h after injection. We hypothesized that LPS stimulates the transepithelial movement of Na + via the epithelial sodium channel (ENaC)/Na + ,K +-pump axis, leading to hyperpolarization of V t. LPS increased the V t response to amiloride, i.e., offset the effect of LPS, indicating that Na + transport was increased. The functional activity of ENaC was measured in the IPT after short-circuiting the Na + ,K +-pump with basolateral amphotericin B. LPS had no effect on the hyperpolarization response to apical trypsin in the Ussing chamber, indicating that channel activating proteases are not involved in the LPS-induced activation of ENaC. To assess Na + ,K +-pump activity in the IPT, ENaC was short-circuited with apical amphotericin B. The greater V t in the presence of amphotericin B in tracheas from LPS-treated animals compared to controls revealed that LPS increased Na + ,K +-pump activity. This finding was confirmed in the Ussing chamber by inhibiting the Na + ,K +-pump via extracellular K + removal, loading the epithelium with Na + , and observing a greater hyperpolarization response to K + restoration. Using qPCR, the effects of LPS on the transcription of αENaC, α 1 Na + ,K +pump, COX-2, eNOS, iNOS, IL-1β, and TNF-α were measured at 3 and 18 h. In the epithelium, LPS increased the transcription of COX-2, IL-1β, and, to nonsignificant extent, TNF-α at 3 h, but not at 18 h. In alveolar macrophages, TNF-α, and, to a nonsignificant extent, COX-2 and IL-1β were up-regulated at 3 h, but not at 18 h. Even though LPS stimulated the transcription of some genes, αENaC and α 1 Na + ,K +-ATPase transcription were not affected. The expression of α-, β-, and γ-ENaC and α 1 Na + ,K +-pump from tracheal epithelium and kidney cortex/medulla were investigated by western blotting. All three ENaC subunits were detected as cleavage fragments, yet LPS had no effect on their expression. LPS increased the expression of the α 1 subunit and the α 1-, α 2-, and α 3-subunits, collectively, of the Na + ,K +-pump. Taken together, the findings of this study reveal that LPS hyperpolarizes the airway epithelium by increasing the activities of ENaC and the Na + ,K +-pump. ENaC activation by LPS is not accomplished via a change in ENaC regulation involving proteolytic cleavage. LPS increases Na + transport downstream of the genetic level, in part, by stimulating the expression of the Na + ,K +-pump.