Hypoxic Modulation of Ca2+ Signaling in Human Venous Endothelial Cells

The effects of hypoxia (pO2 ∼25 mm Hg) on Ca2+ signaling stimulated by extracellular ATP in human saphenous vein endothelial cells were investigated using fluorimetric recordings from Fura-2 loaded cells. In the absence of extracellular Ca2+, ATP-evoked rises of cytosolic Ca2+ concentration ([Ca2+]i) because of mobilization from the endoplasmic reticulum (ER). These responses were reduced by prior exposure to hypoxia but potentiated during hypoxia. Hypoxia itself liberated Ca2+ from the ER, but unlike the effects of ATP this effect was not inhibited by blockade of the inositol trisphosphate receptor. By contrast, ryanodine blocked the effects of hypoxia but not those of ATP. Antioxidants abolished the effects of hypoxia but potentiated the effects of ATP. Inhibition of NADPH oxidase also augmented ATP-evoked responses but was without effect on hypoxia-evoked rises of [Ca2+]i. However, either uncoupling mitochondrial electron transport or inhibiting complex I markedly suppressed the actions of hypoxia yet exerted only small inhibitory effects on ATP-evoked rises of [Ca2+]i. Both hypoxia and ATP were able to activate capacitative Ca2+ entry. Our results indicate that hypoxia regulates intracellular Ca2+ signaling via two distinct pathways. First, it modulates agonist-evoked liberation of Ca2+ from the ER primarily through regulation of reactive oxygen species generation from NADPH oxidase. Second, it liberates Ca2+ from the ER via ryanodine receptors, an effect requiring mitochondrial reactive oxygen species generation. These findings suggest that local O2 tension is a major determinant of Ca2+ signaling in the vascular endothelium, a finding that is likely to be of both physiological and pathophysiological importance. The effects of hypoxia (pO2 ∼25 mm Hg) on Ca2+ signaling stimulated by extracellular ATP in human saphenous vein endothelial cells were investigated using fluorimetric recordings from Fura-2 loaded cells. In the absence of extracellular Ca2+, ATP-evoked rises of cytosolic Ca2+ concentration ([Ca2+]i) because of mobilization from the endoplasmic reticulum (ER). These responses were reduced by prior exposure to hypoxia but potentiated during hypoxia. Hypoxia itself liberated Ca2+ from the ER, but unlike the effects of ATP this effect was not inhibited by blockade of the inositol trisphosphate receptor. By contrast, ryanodine blocked the effects of hypoxia but not those of ATP. Antioxidants abolished the effects of hypoxia but potentiated the effects of ATP. Inhibition of NADPH oxidase also augmented ATP-evoked responses but was without effect on hypoxia-evoked rises of [Ca2+]i. However, either uncoupling mitochondrial electron transport or inhibiting complex I markedly suppressed the actions of hypoxia yet exerted only small inhibitory effects on ATP-evoked rises of [Ca2+]i. Both hypoxia and ATP were able to activate capacitative Ca2+ entry. Our results indicate that hypoxia regulates intracellular Ca2+ signaling via two distinct pathways. First, it modulates agonist-evoked liberation of Ca2+ from the ER primarily through regulation of reactive oxygen species generation from NADPH oxidase. Second, it liberates Ca2+ from the ER via ryanodine receptors, an effect requiring mitochondrial reactive oxygen species generation. These findings suggest that local O2 tension is a major determinant of Ca2+ signaling in the vascular endothelium, a finding that is likely to be of both physiological and pathophysiological importance. The vascular endothelium plays a central role in the control of vascular function, exerting important influences on vital functions as diverse as coagulation, inflammation, vessel permeability, angiogenesis, and vascular tone (reviewed by Refs. 1.Tran Q.K. Ohashi K. Watanabe H. Cardiovasc. Res. 2000; 48: 13-22Crossref PubMed Scopus (155) Google Scholar, 2.Adams D.J. Hill M.A. J. Cardiovasc. Electrophysiol. 2004; 15: 598-610Crossref PubMed Scopus (46) Google Scholar, 3.Yao X. Huang Y. Trends Pharmacol. Sci. 2003; 24: 263-266Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 4.Nilius B. Droogmans G. Physiol. Rev. 2001; 81: 1415-1459Crossref PubMed Scopus (756) Google Scholar). Many of these functions, such as production of vasoactive agents (5.Martin T.W. Michaelis K.C. Biochim. Biophys. Acta. 1990; 1054: 159-168Crossref PubMed Scopus (9) Google Scholar, 6.Lin S. Fagan K.A. Li K.X. Shaul P.W. Cooper D.M. Rodman D.M. J. Biol. Chem. 2000; 275: 17979-17985Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), rely on regulated changes of intracellular Ca2+ concentration ([Ca2+]i). As in other non-excitable cells, Ca2+ homeostasis in endothelial cells involves uptake and release of Ca2+ into intracellular organelles (particularly the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; IP3, inositol trisphosphate; 2-APB, 2-aminoethoxydiphenyl borate; RyR, ryanodine receptor; cADPR, cyclic ADP-ribose; ROS, reactive oxygen species; trolox, 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid; TEMPO, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl; CCE, capacitative Ca2+ entry; TRP, transient receptor potential; TRPC, canonical TRP. 1The abbreviations used are: ER, endoplasmic reticulum; IP3, inositol trisphosphate; 2-APB, 2-aminoethoxydiphenyl borate; RyR, ryanodine receptor; cADPR, cyclic ADP-ribose; ROS, reactive oxygen species; trolox, 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid; TEMPO, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl; CCE, capacitative Ca2+ entry; TRP, transient receptor potential; TRPC, canonical TRP.) as well as controlled influx from the extracellular environment (1.Tran Q.K. 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Cell Sci. 2001; 114: 2223-2229Crossref PubMed Google Scholar, 11.Putney J.W. Mol. Intervent. 2001; 1: 84-94PubMed Google Scholar, 12.Putney Jr., J.W. McKay R.R. BioEssays. 1999; 21: 38-46Crossref PubMed Scopus (357) Google Scholar, 13.Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1046) Google Scholar), which has been linked specifically to the activation of nitricoxide synthase (6.Lin S. Fagan K.A. Li K.X. Shaul P.W. Cooper D.M. Rodman D.M. J. Biol. Chem. 2000; 275: 17979-17985Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Although the vascular endothelium can be considered a syncytium, it clearly experiences different environments in different regions of the vasculature. The most striking difference is between arterial and venous environments; clearly, venous endothelial cells experience an environment which, as compared with those of the arterial vessels, is of much lower pressure and is also relatively hypoxic and hypercapnic. Of these parameters, we have focused on the effects of hypoxia on Ca2+ signaling in venous endothelial cells. This is a poorly studied area that deserves investigation for several reasons. First, most in vitro studies of Ca2+ signaling in endothelial (and other) cells have been conducted using perfusate equilibrated with room air (∼150 mm Hg), which is hyperoxic even for the arterial endothelium. Second, where any effects of hypoxia have been studied, they have usually been studied in combination with other altered parameters such as glucose removal (to mimic ischemia/reperfusion conditions) and/or have been studied over very prolonged time courses (14.Arnould T. Michiels C. Alexandre I. Remacle J. J. Cell. Physiol. 1992; 152: 215-221Crossref PubMed Scopus (126) Google Scholar, 15.Kimura C. Oike M. Ito Y. Am. J. Physiol. 2000; 279: H2310-H2318Crossref PubMed Google Scholar, 16.Hu Q. Ziegelstein R.C. Circulation. 2000; 102: 2541-2547Crossref PubMed Scopus (49) Google Scholar). Third, whereas the effects of acute hypoxia on ion channel function have been studied in depth in a variety of cell types (17.Kemp P.J. Searle G.J. Hartness M.E. Lewis A. Miller P. Williams S. Wootton P. Adriaensen D. Peers C. Anat. Rec. 2003; 270: 41-50Crossref Scopus (38) Google Scholar, 18.Peers C. Trends Pharmacol. Sci. 1997; 18: 405-408Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 19.Lopez-Barneo J. Pardal R. Ortega-Saenz P. Annu. Rev. Physiol. 2001; 63: 259-287Crossref PubMed Scopus (485) Google Scholar), their actions on other ion flux mechanisms, such as those responsible for Ca2+ homeostasis in non-excitable cells, are poorly understood. Finally, although local venous O2 levels clearly influence physiological functions (such as release of nitric oxide (20.Pearson P.J. Evora P.R. Discigil B. Schaff H.V. Ann. Thorac. Surg. 1998; 65: 1220-1225Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar)), hypoxia may be important in the development of specific vascular disease states, such as varicoses. It has been proposed that hypoxic “activation” of endothelial cells (which can occur via blood stasis, particularly in leg veins) requires mobilization of Ca2+ and initiates a cascade of events leading to varicose formation (21.Michiels C. Arnould T. Thibaut-Vercruyssen R. Bouaziz N. Janssens D. Remacle J. Int. Angiol. 1997; 16: 134-141PubMed Google Scholar, 22.Michiels C. Arnould T. Remacle J. Angiology. 1993; 44: 639-646Crossref PubMed Scopus (58) Google Scholar). Thus, we have investigated the effects of acute hypoxia on Ca2+ homeostasis in primary cultures of human saphenous vein endothelial cells and compared responses to those of the well characterized Ca2+ mobilizing agent, extracellular ATP (23.Pirotton S. Communi D. Motte S. Janssens R. Boeynaems J.M. J. Auton. Pharmacol. 1996; 16: 353-356Crossref PubMed Scopus (34) Google Scholar). Isolation and Culture of Saphenous Vein Endothelial Cells—The isolation of primary cultures of saphenous vein endothelial cells was adapted from methods described previously (24.Budd J.S. Allen K.E. Bell P.R. Br. J. Surg. 1991; 78: 878-882Crossref PubMed Scopus (23) Google Scholar). Saphenous vein samples were collected from patients undergoing coronary bypass grafting following local ethical permission and informed, written patient consent. Tissue from a total of 36 patients was used, 10 female (28%) and 26 male (72%). The age range was 44–79, the median age was 67, and the mean age was 65.7 ± 1.5 years. All patients were undergoing elective coronary artery bypass surgery, and any patients with potentially confounding conditions (e.g. diabetes) were excluded. Individual samples ranging from 10–30 mm in length were opened longitudinally and pinned, lumen uppermost, onto silicone elastomer-coated 60-mm Petri dishes using A1 Minuten pins. The tissue sample was then incubated in 1 mg/ml Type II collagenase (Worthington) dissolved in Medium 199 (37 °C, 15 min). The collagenase solution was collected along with 2 × 10 ml of wash solution (minimal essential medium supplemented with 5% fetal calf serum and 1% antibiotic/antimycotic), which was used to detach any residual endothelial cells from the tissue. The suspension was centrifuged for 6 min at 600 × g, the supernatant was removed, and the pellet was resuspended in 25 ml of wash solution and recentrifuged. The supernatant was once again removed, and the final pellet was resuspended in 4 ml of complete endothelial culture medium (M199) supplemented with 20% fetal calf serum, 1% penicillin-streptomycin, 1% glucose, 1 m HEPES (Invitrogen), heparin (5 units/ml, Leo Laboratories), endothelial growth factor (15 μg/ml), and pyruvate (1 μm, Sigma-Aldrich, Poole, Dorset, UK). This mixture was then plated into a 25 cm2 flask and maintained in a humidified incubator at 37 °C (95% air, 5% CO2). 2 days following plating, cells received a full medium change to remove non-adherent cells. Culture medium was then half changed every 2–3 days, resulting in a confluent flask within 2–3 weeks. This was designated passage 0; cells were subcultured using trypsin and used for experiments up to passage 3. Measurement of [Ca2+]i—Cells were plated onto glass coverslips in 24-well culture plates and grown to ∼80% confluence. Coverslips onto which cells had grown were incubated in 2 ml of culture medium containing 4μm Fura-2AM (Molecular Probes, Cambridge, UK) for 40 min. at 37 °C in the dark and then left to de-esterify for 15 min in control solution. Fragments of coverslips were then transferred into an 80-μl recording chamber mounted on the stage of an inverted microscope where cells were continuously perfused under gravity at a rate of ∼5 ml/min. Control perfusate was composed of: 135 mm NaCl, 5 mm KCl, 1.2 mm MgSO4, 2.5 mm CaCl2, 5 mm HEPES, and 10 mm glucose (pH 7.4, osmolarity adjusted to 300 mosm with sucrose, 21–24 °C). Solutions were made hypoxic where indicated by with for at min prior to of cells, which in was the using a J. Physiol. 1997; Scopus Google and from mm perfusate in experiments of those in 1 mm and was using an UK) with and of were used to to cells. All was controlled by and were to cells via the and experiments were conducted at 21–24 were to the of G. M. J. Biol. Chem. Full Text PDF PubMed Scopus Google in were by the of to levels that results are as ± with and were made using All mean were from the of cells In these were collected from cells of at (and usually different Hypoxia Ca2+ and ATP-evoked Ca2+ rises of in non-excitable cells via Ca2+ mobilization from intracellular stores and Ca2+ these we the of ATP to rises of cells were perfused with a solution with 1 mm to Ca2+ an transient of by the of 10 a well of in endothelial cells, via to inositol trisphosphate (23.Pirotton S. Communi D. Motte S. Janssens R. Boeynaems J.M. J. Auton. Pharmacol. 1996; 16: 353-356Crossref PubMed Scopus (34) Google Scholar). of cells to hypoxia (pO2 a small but transient of in of cells cells were to acute hypoxia by ATP in a this was able to a transient of (e.g. However, responses were those in cells not previously to hypoxia By contrast, exposure of cells to hypoxia exposure to ATP to a of of ATP was during hypoxia responses were in and those in the that during this concentration of ATP was not These findings are in the of in that hypoxia release of Ca2+ from the intracellular as the we cells for min with the ER (1 In cells this ATP hypoxia rises of during with solution (e.g. also investigated the effects of 2-aminoethoxydiphenyl considered an of T. T. S. T. K. J. Biochem. 1997; PubMed Scopus Google but as a of Ca2+ signaling via (e.g. Bootman M.D. Berridge M.J. Roderick H.L. 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In endothelial cells, of Ca2+ to the perfusate prior of intracellular had effect on (e.g. but we cells to to intracellular stores as as and then Ca2+ to the this a of which be inhibited by and in a of stores were by of 10 of Ca2+ to the perfusate also a and influx of Ca2+ and the was stores were by hypoxia the of capacitative Ca2+ entry by these different store depletion the CCE, and this was also the most to which suppressed the influx by at 1 mm and hypoxia-evoked responses were in of and (1 mm of ATP-evoked and of hypoxia-evoked CCE, was clearly by ATP-evoked by and hypoxia-evoked by The that local O2 levels have effects on Ca2+ signaling in vascular endothelial cells and Ca2+ signaling by extracellular ATP. have two distinct by which hypoxia regulates Ca2+ release from the is the mitochondrial in which hypoxia an production of at the to release of Ca2+ from the ER via mitochondrial is the oxidase in which of levels during hypoxia inhibitory influences of on Ca2+ release from the ER oxidase also that is between these two mitochondrial production agonist-evoked Ca2+ The in from the finding that hypoxia Ca2+ from an intracellular that was also to ATP. an effect of hypoxia is in with an endothelial cells J. 1995; Google Scholar). 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Thus, the Ca2+ influx was much in that by ATP or hypoxia and was inhibited by 1 mm In contrast, the of and hypoxia-evoked were to other these agents markedly different of Ca2+ from stores as was the to blockade by to the was of the of store depletion Ca2+ entry is to be by transient receptor of which the canonical are to be B. Droogmans G. R. 2003; PubMed Scopus Google Scholar). Endothelial cells of the of the B. Droogmans G. R. 2003; PubMed Scopus Google Scholar). Our suggest that ATP and hypoxia may activate the but these may be distinct from a those by specific of this in the in this but the relatively to blockade by the of M. Bird G.S. McKay R.R. Jr., J.W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In results indicate that hypoxia is a determinant of intracellular Ca2+ signaling in human venous endothelial cells, agonist-evoked liberation of Ca2+ from the ER through regulation of generation from NADPH oxidase. hypoxia itself liberates Ca2+ from the ER via mitochondrial generation and activation of because hypoxia is also of CCE, results indicate that local O2 levels be considered such in these and other cell effects are likely to be of not only in the of the but also in pathophysiological or hypoxia. D. J. for human saphenous vein