Differential mechanisms of adenosine‐ and ATPγS‐induced microvascular endothelial barrier strengthening

• Published in: Journal of cellular physiology Vol. 234; no. 5; pp. 5863 - 5879
• Main Authors: Bátori, Róbert, Kumar, Sanjiv, Bordán, Zsuzsanna, Cherian‐Shaw, Mary, Kovács‐Kása, Anita, MacDonald, Justin A., Fulton, David J. R., Erdődi, Ferenc, Verin, Alexander D.
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.05.2019
• Subjects: A Kinase Anchor Proteins - genetics; A Kinase Anchor Proteins - metabolism; Activation; Adenosine; Adenosine monophosphate; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - pharmacology; ATP; ATPγS; Capillary Permeability - drug effects; Contraction; Cyclic AMP; Cyclic AMP - metabolism; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; Depletion; Electric Impedance; endothelial barrier protection; Endothelial cells; Guanine; Guanine nucleotide exchange factor; Guanine Nucleotide Exchange Factors - genetics; Guanine Nucleotide Exchange Factors - metabolism; HEK293 Cells; Homeostasis; Humans; Kinases; Lungs; Membrane Proteins - genetics; Membrane Proteins - metabolism; Microvasculature; Microvessels - drug effects; Microvessels - metabolism; Myosin; myosin light chain; Myosin Light Chains - metabolism; Myosin-Light-Chain Phosphatase - genetics; Myosin-Light-Chain Phosphatase - metabolism; Myosin-light-chain-phosphatase; Permeability; Phosphatase; Phosphorylation; PKA; Preservation; Protein kinase A; Proteins; Purinergic P1 Receptor Agonists - pharmacology; Purines; Receptors, Purinergic P1 - drug effects; Receptors, Purinergic P1 - genetics; Receptors, Purinergic P1 - metabolism; Restoration; Signal Transduction; adenosine; myosin light chain; ATPγS; endothelial barrier protection; PKA
• DOI: 10.1002/jcp.26419
• ISSN: 0021-9541

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5′‐[γ‐thio]‐triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS‐induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP‐independent pathway of PKA activation. Furthermore, ATPγS‐induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA‐anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase‐targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS‐induced increases in transendothelial electrical resistance. Ado‐induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP‐dependent manner. In summary, ATPγS‐induced enhancement of the EC barrier is EPAC1‐independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP‐independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation. In the present study we define and compare the molecular mechanisms linking adenosine‐ and ATPγ‐induced purinergic receptor activation and barrier strengthening in HLMVECs. We found that ATPγS‐induced EC barrier enhancement is EPAC1‐independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP‐independent mechanism, to activate MLCP and dephosphorylate MLC20 resulting in reduced EC contraction.