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P. R. Cammarata, B. Braun, J. Pack; Characterization and Functional Expression of the Natriuretic Peptide System in Human Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2619.
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The family of natriuretic peptides (NPs) consists of three peptides; atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), as well as three allied receptors (NPRs); natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B) and natriuretic peptide receptor C (NPR-C). We demonstrate both expression and functionality of the natriuretic peptide system (NPS) in the human lens epithelial cell line, HLE-B3, and suggest their potential role in a protection pathway against mitochondrial permeability transition (MPT).
RT-PCR coupled with confirmation by DNA sequencing was used to demonstrate expression of mRNA for the NPS. Protein expression and subcellular localization of the natriuretic peptide receptors was determined by utilizing formaldehyde-fixed, Saponin-permeabilized cells using conventional immunofluorescence techniques. Functionality was illustrated by a cyclic GMP (cGMP) response to NPs using an enzyme-linked immunosorbent assay.
eNOS, a mediator of guanylyl cyclase activation, and a critical player in the PI3K/Akt pathway of protection against MPT, was undetectable in cultured HLE-B3, as confirmed by Western blot analysis and comparison with an eNOS standard. HLE-B3 cells express mRNA for ANP, BNP and CNP along with their associated receptors. Confocal microscopy and immunofluoresence indicated diffuse cytosolic distribution for all three NPRs; immunostaining being greater for NPR-A and NPR-B when compared to NPR-C. All three NPs educe a cGMP response in the rank order CNP>>ANP=BNP.
The natriuretic peptide system is expressed and operative in cultured human lens epithelial cells. Classically, the NPS is involved in the regulation of physiological functions; including blood pressure and fluid homeostasis, and for ocular systems, to influence eye pressure. We suggest a second, novel role for the NPS in HLE-B3 cells. In the event of low or missing eNOS activity, the NPs and NPRs additionally function as local autocrine/paracrine mediators, which under appropriate conditions may substitute for eNOS in the PI3K/Akt pathway of mitochondrial protection, activating guanylyl cyclase and resulting in a cGMP response, stimulating downstream mitoKATP channels, abrogating mitochondrial permeability transition and otherwise ensuing cell death.
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