Extracellular β-nicotinamide adenine dinucleotide (β-NAD) promotes the endothelial cell barrier integrity via PKA- and EPAC1/Rac1-dependent actin cytoskeleton rearrangement

Umapathy N Siddaramappa, Evgeny Alexandrovich Zemskov, Joyce N Gonzales, Boris A Gorshkov, Supriya Sridhar, Trinad Chakraborty, Rudolf Lucas, Alexander Dmitriyevich Verin

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)215-223
Number of pages9
JournalJournal of Cellular Physiology
Volume223
Issue number1
DOIs
StatePublished - Apr 1 2010

Fingerprint

Endothelial cells
Actin Cytoskeleton
NAD
Actins
Endothelial Cells
Purinergic Receptors
Acoustic impedance
Monomeric GTP-Binding Proteins
Cyclic AMP-Dependent Protein Kinases
Electric Impedance
Purinergic P2Y Receptor Agonists
Permeability
Purinergic P2Y1 Receptors
Bacterial Toxins
Myosin Light Chains
Phosphorylation
Inositol
Fluorescence Microscopy
Thrombin
Pulmonary Artery

ASJC Scopus subject areas

  • Medicine(all)
  • Physiology
  • Clinical Biochemistry
  • Cell Biology

Cite this

@article{56996003cb764b04b6466779d8d9a349,
title = "Extracellular β-nicotinamide adenine dinucleotide (β-NAD) promotes the endothelial cell barrier integrity via PKA- and EPAC1/Rac1-dependent actin cytoskeleton rearrangement",
abstract = "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.",
author = "Siddaramappa, {Umapathy N} and Zemskov, {Evgeny Alexandrovich} and Gonzales, {Joyce N} and Gorshkov, {Boris A} and Supriya Sridhar and Trinad Chakraborty and Rudolf Lucas and Verin, {Alexander Dmitriyevich}",
year = "2010",
month = "4",
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doi = "10.1002/jcp.22029",
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volume = "223",
pages = "215--223",
journal = "Journal of Cellular Physiology",
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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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