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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
Transient plasma membrane disruption induced calcium waves in mouse and human corneal epithelial cells
Zhong Chen1, Xiaowen Lu1, Mitchell A Watsky1
1Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA, United States of America.
Abstract:
The purpose of this study was to examine transient plasma membrane disruptions (TPMDs) and TPMD-induced Ca++ waves (TPMD Ca++ Wvs) in human and mouse corneal epithelium (HCEC and MCEC). A multi-photon microscope was used to create laser-induced TPMDs in single cultured cells and in intact ex vivo and in vivo MCECs and ex vivo human cornea rim HCECs. Eye rubbing-induced TPMDs were studied by gentle rubbing with a cotton tipped applicator over a closed eyelid in ex vivo and in vivo MCECs. Ca++ sources for TPMD-induced Ca++ waves were explored using Ca++ channel inhibitors and Ca++-free media. TPMDs and TPMD Ca++ Wvs were observed in all cornea epithelial models examined, often times showing oscillating Ca++ levels. The sarcoplasmic reticulum Ca++ ATPase inhibitors thapsigargin and CPA reduced TPMD Ca++ Wvs. TRP V1 antagonists reduced TPMD Ca++ Wvs in MCECs but not HCECs. Ca++-free medium, 18α-GA (gap junction inhibitor), apyrase (hydrolyzes ATP), and AMTB (TRPM8 inhibitor) did not affect TPMD Ca++ Wvs. These results provide a direct demonstration of corneal epithelial cell TPMDs and TPMDs in in vivo cells from a live animal. TPMDs were observed following gentle eye rubbing, a routine corneal epithelial cell mechanical stress, indicating TPMDs and TPMD Ca++ Wvs are common features in corneal epithelial cells that likely play a role in corneal homeostasis and possibly pathophysiological conditions. Intracellular Ca++ stores are the primary Ca++ source for corneal epithelial cell TPMD Ca++ Wvs, with TRPV1 Ca++ channels providing Ca++ in MCECs but not HCECs. Corneal epithelial cell TPMD Ca++ Wv propagation is not influenced by gap junctions or ATP.
Insights
Transient plasma membrane disruptions (TPMDs) and associated calcium waves occur in corneal epithelial cells during normal activities like eye rubbing. Intracellular calcium stores are the main source, with TRPV1 channels involved in mouse cells.
Area of Science:
- Ophthalmology
- Cell Biology
- Biophysics
Background:
- Corneal epithelial cells form the eye's protective outer layer.
- Mechanical stresses, such as eye rubbing, can impact corneal epithelial cell integrity.
- Calcium signaling plays a critical role in cellular functions and responses to stimuli.
Purpose of the Study:
- To investigate transient plasma membrane disruptions (TPMDs) and calcium waves (TPMD Ca++ Wvs) in human and mouse corneal epithelial cells (HCECs and MCECs).
- To identify the sources of calcium for TPMD-induced calcium waves.
- To determine the role of TPMDs and associated calcium signaling in corneal homeostasis and potential disease states.
Main Methods:
- Utilized multi-photon microscopy to induce and observe laser-induced TPMDs in cultured and intact corneal epithelial cells (ex vivo and in vivo).
- Studied eye rubbing-induced TPMDs in mouse corneal epithelial cells (MCECs) using gentle rubbing.
- Employed calcium channel inhibitors, calcium-free media, and specific inhibitors (gap junction, ATPase, etc.) to explore calcium sources and signaling pathways.
Main Results:
- TPMDs and TPMD Ca++ Wvs were observed in all examined corneal epithelial models, often with oscillating calcium levels.
- Intracellular calcium stores, particularly those sensitive to sarcoplasmic reticulum Ca++ ATPase inhibitors, were the primary source for TPMD Ca++ Wvs.
- TRPV1 channel antagonists reduced TPMD Ca++ Wvs in MCECs but not HCECs; gap junctions and ATP did not influence wave propagation.
Conclusions:
- Directly demonstrated TPMDs and TPMD Ca++ Wvs in corneal epithelial cells, including in vivo.
- Gentle eye rubbing induces TPMDs, suggesting these events are common and relevant to corneal homeostasis.
- Intracellular calcium stores are key for TPMD Ca++ Wvs, with TRPV1 channels contributing in MCECs, highlighting species-specific differences.
