Characterization and functional expression of the natriuretic peptide system in human lens epithelial cells

Abstract

Purpose: The family of natriuretic peptides (NPs); atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) as well as three associated receptors (NPRs); natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B), and natriuretic peptide receptor C (NPR-C) has never been documented in human lens epithelial cells. The study described herein was designed to demonstrate both expression and functionality of components of the natriuretic peptides and natriuretic peptide receptors in the human lens epithelial cell line, HLE-B3 and in normal human lens epithelial cell cultures (nHLE).

Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) along with confirmation by DNA sequencing and real-time quantitative RT-PCR was used to identify and demonstrate expression of mRNA for the natriuretic peptide family. Authentication of protein expression of the natriuretic peptide receptors was determined by using formaldehyde-fixed, Saponin-permeabilized cells (HLE-B3) or methanol:acetone-fixed and permeabilized cells (nHLE) using conventional immunofluorescence techniques. Enzyme-linked immunosorbent assay was used to determine cyclic GMP (cGMP) activity as stimulated by exogenous addition of natriuretic peptides.

Results: Using RT-PCR with confirmation by DNA sequencing and real-time quantitative RT-PCR, HLE-B3 cells were shown to express mRNA for ANP, BNP, and CNP along with their associated receptors. Conventional immunofluorescence on the permeabilized cells confirmed positive diffuse staining indicating the presence of the three natriuretic peptide receptors in both HLE-B3 and nHLE cells. All three natriuretic peptides educe a cGMP response in the rank order CNP>>ANP approximately BNP indicating that the natriuretic peptide family is functional in HLE-B3 cells.

Conclusions: The data indicates that ANP, BNP, and CNP and natriuretic peptide receptor transcripts are expressed and are functional in human lens epithelial cells. The cellular expression of NPs and NPRs, as well as the demonstration that all three NPs activate guanylyl cyclase suggests a potential role in maintaining lens epithelial cell homeostasis.

Figure 1

Reverse Transcriptase-PCR analysis of the natriuretic peptides (ANP, BNP, and CNP) and receptors (NPR-A, NPR-B, and NPR-C) in HLE-B3 cells. Total RNA was isolated from HLE-B3 cells and subjected to RT–PCR for cDNA synthesis. Polymerase chain reactions were run with the synthesized cDNA with the primer pairs for each of the three natriuretic peptides and natriuretic peptide receptors. PCR products were run on 2.5% agarose gel alongside a 100 bp step ladder (Promega Corporation, Madison, WI). All products resolved in the expected location.

Figure 2

Immunoflourescent analysis of natriuretic peptide receptor expression in HLE-B3 cells. Immunoflourescence and confocal microscopy was used to determine expression of natriuretic peptide receptors on HLE-B3 cells. Control images included HLE-B3 cells incubated with no primary antibody but with a goat-anti-rabbit secondary and cells incubated with rabbit IgG as the primary antibody. The data are typical of images representative of eight random fields of view per receptor taken from two independent populations of cells. The scale bar equals 50 microns.

Figure 3

Immunoflourescent analysis of natriuretic peptide receptor expression in normal human lens epithelial cells (nHLE). Immunoflourescence and confocal microscopy was used to determine expression of natriuretic peptide receptors in cultured normal human lens epithelial cells. Control image included nHLE cells incubated with no primary antibody but with a goat-anti-rabbit secondary. The data are typical of images representative of three or four random fields of view per receptor taken from a population of cells derived from whole globes (passage 2). The scale bar equals 50 microns.

Figure 4

Analysis of natriuretic peptide induced cyclic GMP production in HLE-B3 cells. Cyclic GMP activity was measured to determine functionality for the natriuretic peptide system in HLE-B3 cells. Values shown are representative of means of two independent populations of cells examined in triplicate samples (n=6). Each point on the graph represents the mean±standard error.

Figure 5

eNOS production in human lens epithelial cell lines HLE-B3 and SRA 01/04. Protein expression for eNOS was measured by western blot analysis with HLE-B3 and SRA 01/04 cells subjected to hypoxia followed by reintroduction of oxygen. Western blots were run with an actin control and the lanes were equally loaded at a concentration of 10 µg protein per lane. Note that authentic eNOS standard was detected with the commercial antibody.