Semester

Summer

Date of Graduation

2014

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Jeffrey S. Fedan

Committee Co-Chair

James M. Antonini

Committee Member

Patrick S. Callery

Committee Member

Richard D. Dey

Committee Member

Gregory M. Dick

Abstract

Nerve growth factor (NGF) was discovered for its ability to enhance nerve growth, but recent evidence suggests there is a correlation between elevated NGF levels in the lung and airway diseases, including lung inflammatory diseases and respiratory virus infections. NGF can be produced and act upon both structural and non-structural cells of the airways, and overexpression of NGF causes morphological and physiological changes in the airways, such as an increased innervation resulting in a neuronal remodeling of the lung, airway smooth muscle thickening, increased vascularization, airway hyperreactivity to capsaicin, and subepithelial thickening. Although work has been conducted to investigate NGFs effects on ion transport in non-airway cells, such as PC12, MTAL, and HEK-293 cells, no information is available regarding the effect NGF has on ion transport of airway epithelial cells. To investigate whether NGF can affect epithelial ion transport, a well-differentiated human primary cultured epithelial cell model was developed. The ability for these cells to differentiate into epithelial cells, which represent in situ epithelial morphology, was confirmed using several imaging techniques. Cells were placed in Ussing chambers to obtain transepithelial voltage (Vt, -7.1 +/- 3.4 mV), short-circuit current ( Isc, 5.9 +/- 1.0 muA), and transepithelial resistance (Rt, 750 Ohm x cm2), and to measure responses to ion transport inhibitors. Apical and basolateral NGF concentration-response curves were generated, but NGF only evoked bioelectric responses apically with the maximum response occurring at 1 ng/ml. To investigate the ionic basis for the bioelectric responses to NGF, responses to known ion transport inhibitors were generated in the absence or presence of 1 ng/ml NGF. The addition of 1 ng/ml of NGF to the apical membrane decreased Isc by 5.3 %. Amiloride (apical, 3.5x10-5 M), which inhibits Na+ transport, decreased Isc by 55.3 % in the absence of NGF, but this response was reduced (41.6 %; p = 0.0127) in the presence of 1 ng/ml NGF, which indicated NGF was affecting amiloride-sensitive Na + transport. There were no differences in response to NPPB or ouabain, indicating NGF did not have an affect on Cl- transport or the Na+/K+-ATPase. To investigate if the trkA receptor was responsible for mediating the NGF-induced reduction in Na+ transport, the non-specific tyrosine kinase inhibitor, K-252a (10 nM, apical), was used. K-252a reduced the NGF bioelectric response as well as attenuated the NGF-induced reduction in Na+ transport. The trkA receptor activates the Erk 1/2 signaling pathway, which has been shown to phosphorylate ENaC and reduce Na+ transport by channel degradation through a NEDD4-mediated ubiquitin pathway. To investigate if NGF is activating the Erk 1/2 signaling pathway downstream of trkA, the specific Erk 1/2 inhibitor, PD-98059 (30 microM, apical and basolateral), was used. PD-98059 reduced the NGF-induced bioelectric response as well as attenuated the NGF-induced reduction in Na+ transport. Protein analysis using western blot techniques confirmed NGF-mediated reduction in Na+ transport was a result of Erk 1/2 activation and ENaC phosphorylation.;To investigate if incubation with NGF can elicit changes in ion transport, cells were incubated with NGF for 24 or 48 h prior to placing to cells into Ussing chambers. Cells exposed to NGF for either 24 or 48 h did not demonstrate changes in ion transport as compared to control cells, indicating NGF did not have a genomic effect on ion transporter subunit expression. These results also suggest that the rapid reduction in amiloride-sensitive Na+ transport is a transient reduction. To determine if this lack of response was a result of a decreased concentration of NGF during the incubation period, a NGF-specific ELISA assay was used. Cells internalized or metabolized 94% of initial concentration of NGF applied within 5 min, as inserts without cells did not demonstrate a reduction in NGF concentration.;The findings discussed in this dissertation indicate that NGF causes a transient and non-genomic reduction in Na+ transport in epithelium through a trkA-Erk1/2-mediated signaling pathway, resulting in the internalization and degradation of ENaC. This reduction in Na+ transport would result in the hydration of the airway surface liquid.

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