Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physiology

College

College of Graduate Studies

First Advisor

Bradley A. Schulte

Second Advisor

James A. Cook

Third Advisor

Jerome Ondo

Fourth Advisor

Samuel Spicer

Fifth Advisor

George Tempel

Abstract

Mongolian gerbils were used in an investigation into the expression of cochlear sodium/hydrogen (NHE) exchanger isoforms 1,2,3 and 4. NHE 1-4 gene products were identified in the gerbil inner ear by reverse-transcription polymerase chain reaction (RT-PCR). The distribution of NHE-1 and 3 subsequently was mapped in the adult gerbil inner ear using isoform-specific polyclonal antibodies generated against rat antigens. Changes in the cellular expression level of NHE-1 protein in response to treatment with dexamethasone or NH4 CI-induced acidification were then assessed by semiquantitative immunohistochemical analysis. Cochlear cDNA was amplified by the polymerase chain reaction (PCR) using NHE isoform-specific primers based on rat sequences. PCR products spanning selected segments of NHE mRNA were cloned and sequenced. NHE-1, 2, 3 and 4 shared 98.7, 100, 99.4 and 88.9% amino acid homology, respectively, with their rat counterparts. The cellular distribution of NHE isoforms 1 and 3 was mapped in the adult gerbil inner ear by immunostaining with isoform-specific polyclonal antibodies generated against rat antigens. In the cochlea, NHE-1 antiserum reacted strongly with the basolateral membrane of strial marginal epithelial cells as well as with inner and outer hair cells and spiral ganglion neurons. Less intense staining for NHE-1 was present in subpopulations of fibrocytes in the spiral limbus and in inferior and superior areas of the spiral ligament. In the vestibular system, dark cells and transitional cells expressed abundant basolateral NHE-1 as did hair cells in the neurosensory epithelium and neurons in the vestibular ganglia. Immunostaining with anti-NHE-3 was limited to the apical surface of marginal cells in the stria vascularis. Changes in the expression of NHE-1 in response to chronic metabolic acidosis (CMA) and dexamethasone treatment were examined immunohistochemically. In the cochlea, CMA resulted in 127±58% and 221±84% increases in immunostaining intensity for NHE-1 in the stria vascularis and outer hair cells, respectively. Spiral ganglion neurons also appeared to upregulate their NHE-1 protein expression whereas fibrocytes and inner hair cells showed little change in immunostaining intensity in response to CMA. In contrast, dexamethasone treatment increased NHE-1 immunostaining intensity at all sites throughout the cochlea including strial marginal epithelial cells, inner and outer hair cells, spiral ganglion neurons and certain populations of fibrocytes.

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