Characterisation and Functional Activity of Thrombin Receptors in the Human Lens

Abstract

Abstract: : Purpose: Activation of thrombin receptors affects many diverse cellular functions including inhibition of active sodium-potassium transport in the pig lens. This study was undertaken to elucidate which of the 4 PAR subtype(s) are present in the human lens and to investigate the functional effects of receptor activation on calcium (Ca²⁺) signalling, ERK phosphorylation and lens cell growth. Methods: Changes in cytosolic Ca²⁺ were measured by fluorometric imaging techniques in the intact human lens and in the human lens cell line FHL 124, following Fura-2 incorporation. ERK phosphorylation (pERK) was measured in FHL 124 cells by the Western blot technique using the pERK E10 monoclonal antibody (Cell Signalling Technology). Cell growth in FHL 124 cells was quantified using a patch assay where 5-10K cells were seeded, drop-wise onto a 35mm culture dish. The cells were serum-starved for 24 hours prior to exposure to experimental conditions and the growth of the patch determined by image analysis techniques. Pharmacological characterisation of the four PAR sub-types was carried out using subtype-specific activating peptides (PAR-AP, all at 10µM). Results: Thrombin (0.1 - 1 unit/ml) induced an elevation in intracellular Ca²⁺ in both the equatorial and anterior epithelial cells of the intact human lens while only the PAR1-AP induced a Ca²⁺ increase. Thrombin and the PAR1-AP also increased cytosolic Ca²⁺ in FHL 124 cells. Prior application of thapsigargin (1µM) abolished the response to thrombin and the PAR1-AP in both lens cell systems. A 20 minutes exposure to thrombin (0.1 unit/ml) or PAR1-AP also induced a significant increase in pERK levels in serum-starved (24hours) FHL 124 cells. The patch areas of FHL-124 cells increased in serum-free medium and growth was further stimulated following 4 days continuous exposure to thrombin or PAR1-AP. Conclusions: PAR-1 receptors coupled to Ca²⁺ mobilisation in the human lens are located in both the equatorial and anterior epithelial cells. FHL-124 lens cells respond to thrombin and PAR1-AP with an increased phosphorylation of ERK proteins and stimulated growth.

Keywords: calcium • growth factors/growth factor receptors • receptors: pharmacology/physiology