Regional Differences in Calcium Dynamics in the Intact Lens: The Role of TRPV Channels in Lens Calcium Regulation

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Abstract
To establish an imaging approach capable of mapping Ca²⁺ dynamics across different regions of the intact ocular lens, and to determine the functional contributions of TRPV1 and TRPV4 channels to extracellular Ca²⁺ influx in epithelial and differentiating fiber cells. Rat lenses were mounted in a recording chamber with zonular attachments intact and imaged using confocal laser-scanning microscopy. Epithelial cells were loaded with Cal520 AM, while fiber cells were microinjected with Fluo-8 pentapotassium salt. Region-specific changes in intracellular Ca²⁺ were monitored in response to pharmacological manipulations designed to deplete endoplasmic reticulum (ER) Ca²⁺ stores, activate G-protein coupled receptors, or stimulate extracellular Ca²⁺ influx via mechanosensitive TRPV1 and TRPV4 channels. Central and equatorial epithelial cells exhibited biphasic Ca²⁺ responses to thapsigargin, consistent with ER-dependent release and store-operated Ca²⁺ entry, whereas differentiating fiber cells lacked detectable ER-dependent Ca²⁺ mobilization. In epithelial cells, TRPV4 activation with GSK1016790A (GSK) induced transient Ca²⁺ influx, whereas exposure to the TRPV1 agonist capsaicin (Cap) had no effect. In contrast, fiber cells displayed robust extracellular Ca²⁺ entry in response to TRPV1 and TRPV4 activation by Cap and GSK, respectively. These responses were abolished in Ca²⁺-free conditions or by pre-incubation with TRPV1- or TRPV4-specific antagonists. This study establishes a confocal imaging approach for simultaneous analysis of Ca²⁺ dynamics in epithelial and fiber cells of the intact lens. Our findings reveal region-specific differences in Ca²⁺ regulation and provide insight into how TRPV1 and TRPV4 activate signaling pathways that regulate lens ion homeostasis and water transport to maintain optical properties.

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