Semester

Summer

Date of Graduation

2010

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Jeffrey S. Fedan.

Abstract

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 Vt. LPS increased the Vt 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 Vt 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 alphaENaC, alpha1 Na+,K+-pump, COX-2, eNOS, iNOS, IL-1beta, and TNF-alpha were measured at 3 and 18 h. In the epithelium, LPS increased the transcription of COX-2, IL-1beta, and, to nonsignificant extent, TNF-alpha at 3 h, but not at 18 h. In alveolar macrophages, TNF-alpha, and, to a nonsignificant extent, COX-2 and IL-1beta were up-regulated at 3 h, but not at 18 h. Even though LPS stimulated the transcription of some genes, alphaENaC and alpha1 Na +,K+-ATPase transcription were not affected. The expression of alpha-, beta-, and gamma-ENaC and alpha1 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 alpha1 subunit and the alpha 1-, alpha2-, and alpha3-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.

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