Modeling of Ion and Water Transport on the Human Ocular Surface

Background Introduction

The ocular surface plays a crucial role in human physiology and diseases, especially the stability and composition of the tear film, which directly impact ocular surface health. The tear film consists of three layers: an outer lipid layer (secreted by the meibomian glands), a middle aqueous layer (produced by the lacrimal glands), and an inner mucin layer (secreted by conjunctival epithelial cells). Functions of the tear film include providing a smooth optical surface, clearing debris, and defending against microbial invasion. Abnormalities or defects in the ocular surface can lead to conditions such as dry eye disease. However, despite its importance, research on the expression, characteristics, and regulation mechanisms of ion channels in ocular surface epithelial cells is relatively scarce. Most studies have relied on animal models such as rats, mice, or rabbits, while research on human ocular surface epithelial cells remains limited.

To address this gap, a research team from the University of Poitiers, Poitiers University Hospital, ImageUP platform, and H4 Orphan Pharma conducted a study to explore the mechanisms of ion and water transport in human conjunctival epithelial cells (hconec) and meibomian gland epithelial cells (hmgec). This study not only generated human epithelial cells capable of secreting mucins and lipids but also described in detail the expression and function of ion and water channels in these cells using techniques such as Ussing chambers and quantitative phase microscopy.

Source of the Paper

The study was conducted by Chloë Radji, Christine Barrault, Roxane Flausse, Nicolas Leveziel, Anne Cantereau, Catherine Bur, Gaëtan Terrasse, and Frédéric Becq, and was published in American Journal of Physiology-Cell Physiology on January 28, 2025. The research was funded by the French National Research Agency (ANR) and the New Aquitaine Region.

Research Workflow and Results

1. Generation and Characterization of Human Conjunctival and Meibomian Gland Epithelial Cells

The research team first isolated and cultured primary conjunctival epithelial cells (hconec) and meibomian gland epithelial cells (hmgec) from human ocular tissue. Through immunofluorescence staining, they confirmed the epithelial nature of these cells, ruling out contamination by endothelial cells, fibroblasts, and myofibroblasts. Subsequently, ELISA assays and Oil Red O staining were used to verify that hconec could secrete the mucin protein Muc5AC, while hmgec could secrete lipids. These results indicate that the generated epithelial cell models can simulate the physiological functions of human ocular surface epithelial cells.

2. Functional Study of Ion Channels

To investigate the ion transport mechanisms in these cells, researchers cultured the cells under air-liquid interface conditions and recorded transepithelial short-circuit currents (isc) using Ussing chambers. They discovered multiple ion channels in the apical membranes of both hconec and hmgec, including amiloride-sensitive epithelial sodium channels (ENaC), cAMP-dependent cystic fibrosis transmembrane conductance regulator (CFTR), UTP-dependent TMEM16A, and Chromanol 293B-sensitive KCNQ1 potassium channels. In the basolateral membrane, they also identified bumetanide-sensitive Na-K-Cl cotransporters (NKCC) and barium-sensitive potassium channels.

Through pharmacological experiments, the researchers further validated the functions of these channels. For instance, amiloride inhibited ENaC-mediated sodium current, while Forskolin (a cAMP activator) and VX-770 (a CFTR potentiator) activated CFTR-mediated chloride secretion. Additionally, UTP triggered transient calcium-dependent chloride secretion via activation of P2Y2 receptors and TMEM16A channels.

3. Study of Water Transport

The researchers also measured water transport in hconec and hmgec using quantitative phase microscopy. They found that Forskolin could stimulate water influx through the cAMP pathway, while mercuric chloride (HgCl2) inhibited this process. Western blot experiments further confirmed the expression of aquaporins AQP3 and AQP5 in hconec and hmgec, indicating that cAMP-dependent water transport is primarily mediated by these aquaporins.

4. Regulation of Ion Transport by VIP

The study also revealed that vasoactive intestinal peptide (VIP) could activate CFTR via the cAMP pathway, thereby stimulating chloride secretion. The effect of VIP was concentration-dependent, with EC50 values of 1 nM for hconec and 8 nM for hmgec. This finding suggests that VIP may play an important physiological role in ion and water transport in ocular surface epithelial cells.

Conclusions and Significance

This study provides the first systematic description of the molecular mechanisms of ion and water transport in human conjunctival and meibomian gland epithelial cells, proposing a detailed transport model. It not only fills the research gap on ion channels in human ocular surface epithelial cells but also offers important experimental evidence for understanding the molecular mechanisms of tear secretion and related diseases. These findings may provide new targets and strategies for the treatment of ocular surface diseases such as dry eye syndrome.

Highlights of the Study

1. Generation of Human Primary Cells: The study successfully cultured and characterized human conjunctival and meibomian gland epithelial cells capable of secreting mucins and lipids, providing a reliable model for subsequent research.
2. Comprehensive Analysis of Ion Channels: The study details the expression and function of various ion channels, including ENaC, CFTR, TMEM16A, and KCNQ1, in ocular surface epithelial cells.
3. Exploration of Water Transport Mechanisms: Using quantitative phase microscopy, the study revealed the mechanism of cAMP-dependent water transport and confirmed the key roles of AQP3 and AQP5.
4. Regulatory Role of VIP: The study found that VIP can activate CFTR via the cAMP pathway, offering new insights into the regulation of secretion in ocular surface epithelial cells.

Other Valuable Information

The research team also provided access to experimental data, further enhancing the transparency and reproducibility of the study. Additionally, the use of quantitative phase microscopy in this study offers a new method for real-time monitoring of water transport in living cells, with broad application prospects.

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Through this study, we gain a deeper understanding of the molecular mechanisms of ion and water transport in human ocular surface epithelial cells, providing new ideas for the treatment of related diseases. Future research based on these findings is expected to bring breakthroughs in the treatment of ocular surface diseases such as dry eye syndrome.