Store-operated calcium entry and increased endothelial cell permeability

Area of Science:

• Endothelial cell biology
• Calcium signaling
• Molecular mechanisms of cell barrier regulation

Background:

• The endothelial cell barrier is vital for vascular homeostasis.
• Store-operated calcium entry (SOCE) plays a key role in regulating endothelial barrier function.
• The precise mechanisms linking calcium release to SOCE activation remain incompletely understood.

Purpose of the Study:

• To investigate the role of myosin light chain kinase (MLCK) in linking calcium release to SOCE activation.
• To determine how MLCK activity influences endothelial cell permeability and calcium influx.
• To elucidate the relationship between MLCK, SOCE, and thapsigargin-induced barrier dysfunction.

Main Methods:

• Acute inhibition of MLCK using pharmacological agents.
• Measurement of calcium release from inositol trisphosphate-sensitive stores.
• Electrophysiological recordings (voltage-clamped single rat pulmonary artery endothelial cells) to assess calcium currents.
• Assessment of F-actin dynamics.
• Measurement of endothelial cell permeability under varying extracellular calcium conditions.
• Application of thapsigargin to induce store depletion and assess its effects.

Main Results:

• MLCK inhibition caused calcium release from intracellular stores and blocked thapsigargin-induced SOCE.
• Thapsigargin activated an inward calcium current in endothelial cells, which was abolished by MLCK inhibition.
• F-actin disruption activated a calcium current, while F-actin stabilization inhibited the thapsigargin-induced current.
• Thapsigargin increased endothelial cell permeability, dependent on extracellular calcium.
• MLCK inhibition prevented thapsigargin-stimulated calcium entry but did not prevent the increase in permeability, which was exacerbated in low extracellular calcium.

Conclusions:

• MLCK is a critical mediator linking store depletion to the activation of SOCE channels in endothelial cells.
• While MLCK regulates calcium entry, its inhibition is insufficient to prevent thapsigargin-induced increases in endothelial cell permeability.
• These findings highlight a complex interplay between MLCK, calcium signaling, and endothelial barrier integrity, with implications for vascular physiology and disease.