Cammarata Patrick R, Braun Brittany, Dimitrijevich Slobodan D, Pack Jessica
Department of Cell Biology and Anatomy, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, USA.
Mol Vis. 2010 Apr 9;16:630-8.
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).
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.
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.
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.
利钠肽家族，包括心房利钠肽（ANP）、脑利钠肽（BNP）和C型利钠肽（CNP），以及三种相关受体，即利钠肽受体A（NPR-A）、利钠肽受体B（NPR-B）和利钠肽受体C（NPR-C），在人晶状体上皮细胞中从未有过记录。本文所述的研究旨在证明利钠肽及其受体的成分在人晶状体上皮细胞系HLE-B3和正常人晶状体上皮细胞培养物（nHLE）中的表达和功能。
采用逆转录聚合酶链反应（RT-PCR），并通过DNA测序和实时定量RT-PCR进行确认，以鉴定和证明利钠肽家族mRNA的表达。利钠肽受体蛋白表达的鉴定是通过使用甲醛固定、皂素通透的细胞（HLE-B3）或甲醇：丙酮固定并通透的细胞（nHLE），采用传统免疫荧光技术进行的。酶联免疫吸附测定用于确定外源性添加利钠肽刺激后的环磷酸鸟苷（cGMP）活性。
通过RT-PCR并经DNA测序和实时定量RT-PCR确认，HLE-B3细胞显示出ANP、BNP和CNP及其相关受体的mRNA表达。对通透细胞进行的传统免疫荧光证实了阳性弥漫性染色，表明HLE-B3和nHLE细胞中均存在三种利钠肽受体。所有三种利钠肽均能引起cGMP反应，其顺序为CNP>>ANP≈BNP，表明利钠肽家族在HLE-B3细胞中具有功能。
数据表明，ANP、BNP和CNP以及利钠肽受体转录本在人晶状体上皮细胞中表达且具有功能。NPs和NPRs的细胞表达，以及所有三种NPs均能激活鸟苷酸环化酶的证明，提示其在维持晶状体上皮细胞稳态中可能发挥作用。
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