TY - JOUR
T1 - Extracellular β-nicotinamide adenine dinucleotide (β-NAD) promotes the endothelial cell barrier integrity via PKA- and EPAC1/Rac1-dependent actin cytoskeleton rearrangement
AU - Siddaramappa, Umapathy N
AU - Zemskov, Evgeny Alexandrovich
AU - Gonzales, Joyce N
AU - Gorshkov, Boris A
AU - Sridhar, Supriya
AU - Chakraborty, Trinad
AU - Lucas, Rudolf
AU - Verin, Alexander Dmitriyevich
PY - 2010/4/1
Y1 - 2010/4/1
N2 - Extracellular β-NAD is known to elevate intracellular levels of calcium ions, inositol 1,4,5-trisphate and cAMP. Recently, β-NAD was identified as an agonist for P2Y1 and P2Y11 purinergic receptors. Since β-NAD can be released extracellularly from endothelial cells (EC), we have proposed its involvement in the regulation of EC permeability. Here we show, for the first time, that endothelial integrity can be enhanced in EC endogenously expressing β-NAD-activated purinergic receptors upon β-NAD stimulation. Our data demonstrate that extracellular β-NAD increases the transendothelial electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration-dependent manner indicating endothelial barrier enhancement. Importantly, β-NAD significantly attenuated thrombin-induced EC permeability as well as the barrier-compromising effects of Gram-negative and Gram-positive bacterial toxins representing the barrier-protective function of β-NAD. Immunofluorescence microscopy reveals more pronounced staining of cell-cell junctional protein VE-cadherin at the cellular periphery signifying increased tightness of the cell-cell contacts after β-NAD stimulation. Interestingly, inhibitory analysis (pharmacological antagonists and receptor sequence specific siRNAs) indicates the participation of both P2Y1 and P2Y11 receptors in β-NAD-induced TER increase. β-NAD-treatment attenuates the lipopolysaccharide (LPS)-induced phosphorylation of myosin light chain (MLC) indicating its involvement in barrier protection. Our studies also show the involvement of cAMP-dependent protein kinase A and EPAC1 pathways as well as small GTPase Rac1 in β-NAD-induced EC barrier enhancement. With these results, we conclude that β-NAD regulates the pulmonary EC barrier integrity via small GTPase Rac1- and MLCP- dependent signaling pathways.
AB - Extracellular β-NAD is known to elevate intracellular levels of calcium ions, inositol 1,4,5-trisphate and cAMP. Recently, β-NAD was identified as an agonist for P2Y1 and P2Y11 purinergic receptors. Since β-NAD can be released extracellularly from endothelial cells (EC), we have proposed its involvement in the regulation of EC permeability. Here we show, for the first time, that endothelial integrity can be enhanced in EC endogenously expressing β-NAD-activated purinergic receptors upon β-NAD stimulation. Our data demonstrate that extracellular β-NAD increases the transendothelial electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration-dependent manner indicating endothelial barrier enhancement. Importantly, β-NAD significantly attenuated thrombin-induced EC permeability as well as the barrier-compromising effects of Gram-negative and Gram-positive bacterial toxins representing the barrier-protective function of β-NAD. Immunofluorescence microscopy reveals more pronounced staining of cell-cell junctional protein VE-cadherin at the cellular periphery signifying increased tightness of the cell-cell contacts after β-NAD stimulation. Interestingly, inhibitory analysis (pharmacological antagonists and receptor sequence specific siRNAs) indicates the participation of both P2Y1 and P2Y11 receptors in β-NAD-induced TER increase. β-NAD-treatment attenuates the lipopolysaccharide (LPS)-induced phosphorylation of myosin light chain (MLC) indicating its involvement in barrier protection. Our studies also show the involvement of cAMP-dependent protein kinase A and EPAC1 pathways as well as small GTPase Rac1 in β-NAD-induced EC barrier enhancement. With these results, we conclude that β-NAD regulates the pulmonary EC barrier integrity via small GTPase Rac1- and MLCP- dependent signaling pathways.
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U2 - 10.1002/jcp.22029
DO - 10.1002/jcp.22029
M3 - Article
C2 - 20054824
AN - SCOPUS:75749102978
SN - 0021-9541
VL - 223
SP - 215
EP - 223
JO - Journal of Cellular Physiology
JF - Journal of Cellular Physiology
IS - 1
ER -