Mechanisms of ionomycin-induced endothelial cell barrier dysfunction

Area of Science:

• Endothelial cell biology
• Vascular permeability
• Cell signaling

Background:

• Myosin light chain (MLC) phosphorylation by MLCK is key to endothelial cell gap formation.
• Thrombin-induced barrier dysfunction involves MLCK activation.
• The role of Ca2+ ionophores in endothelial barrier function requires further elucidation.

Purpose of the Study:

• To investigate whether the Ca2+ ionophore ionomycin stimulates MLCK-dependent endothelial cell contraction and permeability.
• To determine the mechanisms underlying ionomycin-induced endothelial cell barrier dysfunction.

Main Methods:

• Utilized bovine pulmonary microvascular and macrovascular endothelial cell monolayers.
• Measured albumin clearance and electrical resistance to assess barrier function.
• Quantified levels of phosphorylated MLC and activities of various signaling enzymes (MLCK, protein kinase A, tyrosine kinases, nitric oxide synthase, phospholipase A2).

Main Results:

• Ionomycin increased albumin clearance and decreased electrical resistance, similar to thrombin.
• Contrary to expectations, ionomycin decreased phosphorylated MLC levels, indicating phosphatase activation.
• Ionomycin inhibited protein kinase A and tyrosine kinase activities, including p125 focal adhesion kinase, and reduced phosphotyrosine content.

Conclusions:

• Ionomycin induces endothelial cell barrier dysfunction through MLCK-independent pathways.
• Mechanisms involve reduced endothelial cell tethering forces due to inhibition of protein kinase A and tyrosine kinase signaling.
• p125 focal adhesion kinase is a key target in ionomycin-induced barrier dysfunction.