Date of Award

Winter 12-2009

Degree Type

Restricted Access Thesis

Degree Name

Master of Science

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Dr. Patrick Cammarata

Abstract

Purpose: The natriuretic peptide (NP) family consists of three peptides; atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) as well as three receptors; natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B) and natriuretic peptide receptor C (NPR-C). The natriuretic peptide family, although well examined in some systems, has not been extensively studied in ocular systems. The purpose of this study was to demonstrate both existence and functionality of components of the natriuretic peptides and natriuretic peptide receptors in a human lens epithelial cell line, HLE-B3. , Master of Science (Biomedical Sciences), December, 2009, 49 pp, 2 tables, 7 illustrations, bibliography, 38 titles.

Methods: HLE-B3, a virally transformed human lens epithelial cell line, was used for all experiments. The effects of stress on the expression of natriuretic peptide and receptor mRNA and protein was determined with the use of a step down procedure. The cells were grown to confluence in Eagle’s minimal essential medium (MEM) containing 20% fetal bovine serum (FBS) and subsequently, stepped down into 2% FBS MEM, followed by serum free MEM. After 24hr of serum free MEM the cells were placed into hypoxic conditions for 24 hours and then subjected to re-oxygenation. In order to determine the presence and expression profile of messenger RNA (mRNA) for the natriuretic peptides and their associated receptors reverse transcriptase PCR (RT-PCR) and quantitative real time PCR (QRT-PCR) were used. Protein expression for the natriuretic peptide receptors was observed using Western Blotting. Immunohistochemistry along with confocal imaging was employed in order to illustrate subcellular localization of the natriuretic peptide receptors. Enzyme-linked immunosorbent assay was used to determine cyclic GMP (cGMP) activity in HLE-B3 cells.

Results: Messenger RNA expression was detected for all three natriuretic peptides and their associated receptors. Protein expression was observed for all three natriuretic peptide receptors. Protein expression remained at a relatively constant level, regardless of the cells being subjected to a variety of stressors, the exception being NPR-B. Cellular localization was observed for the three natriuretic peptide receptors with the image density being higher for NPR-A and NPR-B when compared to NPR-C. Localization appeared to be diffuse throughout the cytosol with no indication of nuclear staining. Functionality of all three natriuretic peptides was demonstrated by a cGMP activity assay with the rank order of potency of activation being CNP»ANP≥BNP.

Conclusions: This article is the first demonstration that all members of the natriuretic peptide family, including both peptides and receptors, are expressed and are functional in the human lens epithelial cell line, HLE-B3.

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