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Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Published on: July 16, 2013
Thrombin inhibits intercellular calcium wave propagation in corneal endothelial cells by modulation of hemichannels
Catheleyne D'hondt1, Raf Ponsaerts, Sangly P Srinivas
1Laboratory of Physiology, KULeuven, Leuven, Belgium.
Purpose:
Thrombin, a serine protease, breaks down the barrier integrity of corneal endothelial cells by phosphorylation of the regulatory light chain of myosin II (myosin light chain; MLC), which induces contractility of the actin cytoskeleton. This study was undertaken to investigate the effect of thrombin on gap junctional (GJIC) and paracrine (PIC) intercellular communication in cultured bovine corneal endothelial cells (BCECs).
Methods:
An intercellular Ca(2+) wave, a form of cell-cell communication, was elicited by applying a mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca(2+)](i) were imaged by fluorescence microscopy with a fluorescent calcium indicator, and the images were used to calculate the area reached by the Ca(2+) wave (active area). GJIC was assessed by fluorescence recovery after photobleaching (FRAP). Activity of hemichannels was assayed by lucifer yellow (LY) uptake and also by adenosine triphosphate (ATP) release by using the luciferin-luciferase technique.
Results:
RT-PCR showed transcripts for PAR-1 and -2 receptors, but not for PAR-4 receptors. Immunocytochemistry showed thrombin-sensitive PAR receptors as well as trypsin-sensitive PAR-2 receptors. Both thrombin and the selective PAR-1 agonist TRAP-6 reduced the active area of the Ca(2+) wave. These agents also reduced the fluorescence recovery in FRAP experiments. The effect of thrombin on the Ca(2+) wave was inhibited by a peptide antagonist of PAR-1, but not by a PAR-4 antagonist. Pretreatment with ML-7 (an MLCK inhibitor), Y-27632 (a Rho kinase inhibitor) or chelerythrine (a PKC inhibitor) prevented the effect of thrombin on the Ca(2+) wave. Activation of PAR-1 did not affect the Ca(2+) wave propagation in cells pretreated with Gap26, which blocks hemichannels. However, PAR-1 activation decreased the active area in cells pretreated with Gap27, which inhibits gap junctions. Thrombin abolished enhancement of the Ca(2+) wave propagation by ARL-67156 (inhibitor of ecto-ATPases). The effect of the PAR-1 agonists on the Ca(2+) wave was not detectable in cells pretreated with exogenous apyrases.
Conclusions:
Thrombin inhibits intercellular Ca(2+) wave propagation in BCECs. This effect is due to activation of PAR-1 receptors and involves MLC phosphorylation by MLCK-, PKC- and Rho kinase-sensitive pathways. Thrombin mainly inhibits the ATP-mediated PIC pathway, and also reduces GJIC to a lesser extent.
Insights
Thrombin significantly impairs intercellular communication in bovine corneal endothelial cells by activating PAR-1 receptors. This disruption affects both gap junctional and paracrine signaling pathways, impacting cell barrier integrity.
Area of Science:
- Cell Biology
- Ophthalmology
- Physiology
Background:
- Thrombin, a serine protease, disrupts corneal endothelial cell barrier integrity via myosin light chain (MLC) phosphorylation.
- This process induces actin cytoskeleton contractility, affecting cell-cell communication.
Purpose of the Study:
- To investigate thrombin's effects on gap junctional intercellular communication (GJIC) and paracrine intercellular communication (PIC).
- To elucidate the mechanisms underlying thrombin-induced alterations in communication in cultured bovine corneal endothelial cells (BCECs).
Main Methods:
- Assessed intercellular Ca(2+) wave propagation using fluorescence microscopy after mechanical stimulation.
- Measured GJIC via fluorescence recovery after photobleaching (FRAP).
- Evaluated hemichannel activity through lucifer yellow uptake and ATP release assays.
Main Results:
- Thrombin and PAR-1 agonist TRAP-6 reduced Ca(2+) wave propagation and FRAP.
- These effects were mediated by PAR-1 receptor activation and involved MLC phosphorylation via MLCK, PKC, and Rho kinase pathways.
- Thrombin primarily inhibited ATP-mediated PIC and reduced GJIC to a lesser extent.
Conclusions:
- Thrombin inhibits intercellular Ca(2+) wave propagation in BCECs through PAR-1 receptor activation.
- The mechanism involves MLC phosphorylation and affects both PIC and GJIC pathways.
- Findings highlight thrombin's role in modulating corneal endothelial cell communication.
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