P2Y receptor-mediated Ca(2+) signaling increases human vascular endothelial cell permeability.
We investigated the effects of P2-receptor agonists on cell size, intracellular calcium levels ([Ca(2+)](i)), and permeation of FITC-labeled dextran (FD-4) as well as the relationship between these effects in human umbilical vein endothelial cells (HUVEC). FD-4 concentration, cell size, and [Ca(2+)](i) were analyzed by HPLC with fluorescence, phase contrast microscopic imaging, and fluorescent confocal microscopic imaging, respectively. The P2Y(1)-receptor agonists 2-methylthio ATP (2meS-ATP) and ADP decreased cell size and increased [Ca(2+)](i) in HUVEC. The P2Y(2)-receptor agonist UTP increased [Ca(2+)](i), but did not influence cell size. The P2X-receptor agonist alpha,beta-methylene ATP did not induce either response. The decrease in size and increase in [Ca(2+)](i) by 2meS-ATP were blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, P2Y(1)-antagonist), thapsigargin (Ca(2+)-pump inhibitor), and U73122 (phospholipase C inhibitor). Furthermore, 2meS-ATP (P2Y(1)-receptor agonist) enhanced permeation of FD-4 through the endothelial cell monolayer. The 2meS-ATP-induced enhancement of the permeation was also prevented by PPADS, thapsigargin, and U73122. These results indicate that activation of P2Y receptors induces a decrease in cell size, an increase in [Ca(2+)](i), and may participate in facilitating macromolecular permeability in HUVEC.
Tanaka
Naoko
N
Department of Pharmacology, School of Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan.
Kawasaki
Kumiko
K
Nejime
Namie
N
Kubota
Yoko
Y
Nakamura
Kazuki
K
Kunitomo
Masaru
M
Takahashi
Koichi
K
Hashimoto
Michio
M
Shinozuka
Kazumasa
K
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D048429:Cell Size; D002614:Chelating Agents / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology*; D004963:Estrenes / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D058910:Purinergic P2 Receptor Agonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D002614:Chelating Agents; D004963:Estrenes; D005456:Fluorescent Dyes; D058910:Purinergic P2 Receptor Agonists; D011760:Pyrrolidinones; D018048:Receptors, Purinergic P2; D013873:Thionucleotides; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium; C008501:2-methylthio-ATP