Differential role of PKA/MLCP axis in ATP‐and adenosine‐induced microvascular endothelial barrier enhancement
Authors: Batori Robert Karoly , Kumar Sanjiv , Kasa Anita , Verin Alexander D. 
Published: November 2023

Endothelial cells (EC) are in close contact to each other and act as a semi‐selective barrier between the vessel lumen and the underlying tissues. Any breach of the EC barrier leads to severe lung diseases such as acute lung injury (ALI). Our group has previously shown that extracellular purines, ATP and adenosine are able to preserve vascular barrier in murine model of LPS‐induced ALI and enhance EC barrier in human pulmonary artery EC (HPAEC). We also have shown that ATP‐ and adenosine‐induced HPAEC barrier enhancement involves activation of myosin light chain (MLC) phosphatase (MLCP). MLCP activation may involve PKA‐dependent phosphorylation of the MLCP targeting subunit 1 (MYPT1) at S695, which may activate MLCP directly or indirectly by preventing its Rho kinase‐mediated phosphorylation of the subsequent T696 residue. Both ATP and adenosine increase PKA activity in HPAEC and we have demonstrated that PKA is involved in ATP‐induced barrier strengthening in these cells, however, direct link between MLCP, PKA and purine‐induced EC barrier enhancement was not established.To further define the molecular mechanisms of ATP‐ and adenosine‐induced EC barrier strengthening we employed human lung microvascular EC (HLMVEC) model. Both adenosine and stable analog of ATP, ATPγS, induce HLMVEC barrier enhancement which is accompanied by PKA activation and decrease in MLC and MYPT‐T696 (inhibitory site) phosphorylation suggesting MLCP activation. To directly link PKA activation and EC barrier enhancement induced by extracellular purines we depleted PKA catalytic subunit (PKAc), and then activate HLMVEC with either adenosine or ATPγS and measure transendothelial electrical resistance (TER) of EC monolayers. Surprisingly, depletion of PKAc significantly attenuates ATPγS‐, but not adenosine‐induced increase in TER indicating that PKA activation is involved in ATP‐, but not in adenosine‐induced EC barrier enhancement in microvascular EC. Further, depletion of PKAc leads to increase in MLC and MYPT‐T696 phosphorylation in ATPγS‐, but not in adenosine‐challenged EC supporting the role of PKA in MLCP activation induced by ATP, but not adenosine. To further elucidate the role of PKA signaling in ATP‐induced HLMVEC barrier enhancement we depleted several PKA‐anchoring proteins (AKAPs) and TER was measured after ATPγS or adenosine stimulation. Depletion of AKAP2 and 9, but not AKAP12 significantly attenuates TER increase induced by ATPγS indicating the involvement of these AKAPs in ATP‐induced EC barrier strengthening. Interestingly, none of the above mentioned AKAPs is involved in adenosine effect. Further, our results demonstrate that AKAP 2, but not AKAPs 3, 12, 79 and 350 co‐immunoprecipitates with MYPT1 in HEK293 cells suggesting specific association of AKAP2 with MLCP. This interaction was confirmed in HLMVEC by proximity ligation assay. Therefore, our data suggest that direct AKAP2/MLCP interaction may be involved in EC barrier enhancement induced by ATP. Collectively, our results demonstrate that ATP‐ and adenosine‐induced microvascular EC barrier enhancement required different signaling with PKA promoting ATP‐, but not adenosine‐induced EC barrier strengthening.