Effect of Vanadate on ATP-Induced Increase in Intracellular Calcium Ion Levels in Human Umbilical Vein Endothelial Cells

Abstract

The vascular endothelium participates in various physiological and pathophysiological responses, including blood flow, angiogenesis, blood coagulation, and permeability to fluids and solutes. Many mediators initiate these diverse functions of endothelial cells. ATP is one endogenous modulator that is involved in nitric oxide production, proliferation, migration, apoptosis, and regulation of permeability in these cells. 1,2) We previously found that ATP increases intracellular calcium ion level ([Ca 2ϩ ] i ) of cultured endothelial cells from the rat caudal artery and regulates intercellular permeability.3) Endothelial cells also release ATP and its metabolites during shear stress, a 1 -adrenoceptor activation, and hypoxia. 1,4) Vanadium is a widely distributed element that assumes several experimental oxidation states including that of vanadate (VO 4 Ϫ ). This phosphate analogue is an insulinomimetic, and has been reported to exert beneficial metabolic and cardiovascular effects in several models of diabetes.5-7) On the other hand, Misurski et al. 8) showed that sodium orthovanadate elicited vasorelaxation of the rat mesenteric vascular bed that was mediated by endothelial-derived nitric oxide and endothelial-derived hyperpolarizing factor (EDHF) but not by vasodilator eicosanoids. Vanadium has recently been added to mineral water as a health supplement.Based on the above evidence, we examined the effect of vanadate on ATP-induced increase in [Ca 2ϩ ] i of human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODSEndothelial Cell Culture HUVEC purchased from BioWhittaker (Walkersville, MD, U.S.A.) were cultured in EBM-2 medium supplemented with 5% fetal bovine serum, hydrocortisone, endothelial growth factor, epidermal growth factor, human fibroblast growth factor, insulin-like growth factor, and ascorbic acid. Cells were seeded in 57-cm 2 tissue culture dishes (Cell tight C-1; Sumitomo Bakelite Co., Ltd., Osaka, Japan) and incubated at 37°C in a 95% air/5% CO 2 atmosphere. Cells were removed from the dishes using 0.025% trypsin/0. N,NЈ, to PSS to remove CaCl 2 . Primary cultured endothelial cells on type I collagen-coated glass coverslips were incubated with 5 mmol/l of Calcium Green 1/AM at 37°C for 30 min. Cells were rinsed three times and incubated in PSS at 37°C for an additional 15 min in a 95% air/5% CO 2 atmosphere to complete hydrolysis of any intact ester linkages in the intracellular Calcium Green 1/AM. Loaded coverslips were placed onto the stage of an inverted microscope coupled to a Nipkow disk confocal scanner (CSU10; Yokogawa Electric Corp., Tokyo, Japan) and ARGUS-50 imaging system (Hamamatsu Photonics). An argon-krypton laser (Omnichrome, Chino, CA, U.S.A.) emitted an excitation wavelength of 488 nm. Emitted light was collected with a 510-nm long-pass dichroic reflector and 515-nm long-pass emission filter through a planfluor objective (ϫ100 or ϫ20; NAϭ1.4 and 0.5, respectively). Fluorescence images were acquired by ICCD camera (C2400-87; Hamamatsu Photonics) at a resolu...

Best time window for the use of calcium‐modulating agents to improve functional recovery in injured peripheral nerves—An experiment in rats

“…To determine the Ca 2+ intensity in the nerve segments, we used an established technique employing Calcium Green‐1 Acetoxymethyl (AM) ester (Molecular Probes/Invitrogen, Carlsbad, CA) (Stosiek et al, ; Lu and Paige, ; Tonkikh and Carlen, ; Nejime et al, ). Calcium Green‐1 contains a heavily modified fluorescein moiety attached to a BAPTA (1, 2‐bis (o‐aminophenoxy) ethane‐N,N,N′,N′‐tetraacetic acid) chelator.…”