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

J Cell Physiol. 2010 Apr;223(1):215-23. doi: 10.1002/jcp.22029.


Extracellular beta-NAD is known to elevate intracellular levels of calcium ions, inositol 1,4,5-trisphate and cAMP. Recently, beta-NAD was identified as an agonist for P2Y1 and P2Y11 purinergic receptors. Since beta-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 beta-NAD-activated purinergic receptors upon beta-NAD stimulation. Our data demonstrate that extracellular beta-NAD increases the transendothelial electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration-dependent manner indicating endothelial barrier enhancement. Importantly, beta-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 beta-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 beta-NAD stimulation. Interestingly, inhibitory analysis (pharmacological antagonists and receptor sequence specific siRNAs) indicates the participation of both P2Y1 and P2Y11 receptors in beta-NAD-induced TER increase. beta-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 beta-NAD-induced EC barrier enhancement. With these results, we conclude that beta-NAD regulates the pulmonary EC barrier integrity via small GTPase Rac1- and MLCP- dependent signaling pathways.

Keywords: EPAC1; Gq protein; Gs protein; HPAEC; P2Y antagonists; P2Y1 and P2Y11 receptors; Rac1; cAMP.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism*
  • Antigens, CD / metabolism
  • Bacterial Proteins / pharmacology
  • Cadherins / metabolism
  • Capillary Permeability* / drug effects
  • Cells, Cultured
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Cytoskeleton / drug effects
  • Cytoskeleton / metabolism*
  • Electric Impedance
  • Endothelial Cells / drug effects
  • Endothelial Cells / enzymology*
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Humans
  • Intercellular Junctions / metabolism
  • Lipopolysaccharides / pharmacology
  • Myosin Light Chains / metabolism
  • Myosin-Light-Chain Phosphatase / metabolism
  • NAD / metabolism*
  • Phosphorylation
  • Pulmonary Artery / cytology
  • Pulmonary Artery / drug effects
  • Pulmonary Artery / enzymology*
  • RNA Interference
  • RNA, Messenger / metabolism
  • Receptors, Purinergic P2 / metabolism
  • Receptors, Purinergic P2Y1
  • Signal Transduction
  • Streptolysins / pharmacology
  • Thrombin / metabolism
  • Time Factors
  • rac1 GTP-Binding Protein / genetics
  • rac1 GTP-Binding Protein / metabolism*


  • Actins
  • Antigens, CD
  • Bacterial Proteins
  • Cadherins
  • Guanine Nucleotide Exchange Factors
  • Lipopolysaccharides
  • Myosin Light Chains
  • P2RY1 protein, human
  • RAC1 protein, human
  • RAPGEF3 protein, human
  • RNA, Messenger
  • Receptors, Purinergic P2
  • Receptors, Purinergic P2Y1
  • Streptolysins
  • cadherin 5
  • plY protein, Streptococcus pneumoniae
  • NAD
  • Cyclic AMP-Dependent Protein Kinases
  • Myosin-Light-Chain Phosphatase
  • Thrombin
  • rac1 GTP-Binding Protein