NADPH Oxidase Activation Increases the Sensitivity of Intracellular Ca2+ Stores to Inositol 1,4,5-Trisphosphate in Human Endothelial Cells

Abstract

Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca2+, but whether NADPH oxidase activation directly affects Ca2+ signaling is unknown. NADPH stimulated the production of superoxide anion and H2O2 in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H2O2 each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP3) required to release intracellularly stored Ca2+ and shifted the InsP3-Ca2+ release dose-response curve to the left. Concentrations of H2O2 as low as 3 μm increased the sensitivity of intracellular Ca2+ stores to InsP3 and decreased the InsP3 EC50 from 423.2 ± 54.9 to 276.9 ± 14.4 nm. The effect of NADPH on InsP3-stimulated Ca2+ release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H2O2 increases the sensitivity of intracellular Ca2+ stores to InsP3 in human endothelial cells. Since Ca2+-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction. Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca2+, but whether NADPH oxidase activation directly affects Ca2+ signaling is unknown. NADPH stimulated the production of superoxide anion and H2O2 in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H2O2 each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP3) required to release intracellularly stored Ca2+ and shifted the InsP3-Ca2+ release dose-response curve to the left. Concentrations of H2O2 as low as 3 μm increased the sensitivity of intracellular Ca2+ stores to InsP3 and decreased the InsP3 EC50 from 423.2 ± 54.9 to 276.9 ± 14.4 nm. The effect of NADPH on InsP3-stimulated Ca2+ release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H2O2 increases the sensitivity of intracellular Ca2+ stores to InsP3 in human endothelial cells. Since Ca2+-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction. human aortic endothelial cell(s) inositol 1,4,5-trisphosphate diphenyleneiodonium not significant superoxide dismutase The endothelial cell membrane contains an NADPH oxidase-like H2O2-generating enzyme (1.Bayraktutan U. Draper N. Lang D. Shah A.M. Cardiovasc Res. 1998; 38: 256-262Crossref PubMed Scopus (163) Google Scholar, 2.Jones S.A. O'Donnell V.B. Wood J.D. Broughton J.P. Hughes E.J. Jones O.T. Am. J. Physiol. 1996; 271: H1626-H1634PubMed Google Scholar) that is stimulated by posthypoxic reoxygenation (3.Zulueta J.J., Yu, F-S. Hertig I.A. Thannickal V.J. Hassoun P.M. Am. J. Respir. Cell Mol. Biol. 1995; 12: 41-49Crossref PubMed Scopus (142) Google Scholar), cyclic stretch (4.Hishikawa K. Luscher T.F. Circulation. 1997; 96: 3610-3616Crossref PubMed Scopus (162) Google Scholar, 5.Howard A.B. Alexander R.W. Nerem R.M. Griendling K.K. Taylor W.R. Am. J. Physiol. 1997; 272: C421-C427Crossref PubMed Google Scholar), and low density lipoprotein (6.Holland J.A. Meyer J.W. Schmitt M.E. Sauro M.D. Johnson D.K. Abdul-Karim R.W. Patel V. Ziegler L.M. Schillinger K.J. Small R.F. Lemanski L.F. Endothelium. 1997; 5: 191-207Crossref PubMed Scopus (27) Google Scholar). The signal transduction pathways stimulated following activation of the NADPH oxidase in the vascular endothelium have not been completely characterized. Many stimuli that activate the oxidase also increase endothelial cytosolic calcium concentration ([Ca2+]i) (7.Sokabe M. Naruse K. Sai S. Yamada T. Kawakami K. Inoue M. Murase K. Miyazu M. Heart Vessels Suppl. 1997; 12: 191-193Google Scholar, 8.Honda H.M. Leitinger N. Frankel M. Goldhaber J.I. Natarajan R. Nadler J.L. Weiss J.N. Berliner J.A. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 680-686Crossref PubMed Scopus (76) Google Scholar, 9.Allen S. Khan S. Al-Mohanna F. Batten P. Yacoub M. J. Clin. Invest. 1998; 101: 1064-1075Crossref PubMed Scopus (108) Google Scholar, 10.Zhao B. Ehringer W.D. Dierichs R. Miller F.N. Eur J. Clin. Invest. 1997; 27: 48-54Crossref PubMed Scopus (38) Google Scholar), but whether activation of the NADPH oxidase affects endothelial Ca2+signaling is unknown. We recently showed that H2O2 stimulates [Ca2+]i oscillations in human aortic endothelial cells (HAEC)1 (11.Hu Q. Corda S. Zweier J.L. Capogrossi M.C. Ziegelstein R.C. Circulation. 1998; 97: 268-275Crossref PubMed Scopus (112) Google Scholar). In contrast to other agonists that stimulate [Ca2+]ioscillations in endothelial cells like bradykinin (12.Carter T.D. Bogle R.G. Bjaaland T. Biochem. J. 1991; 278: 697-704Crossref PubMed Scopus (29) Google Scholar), histamine (13.Jacob R. Merritt J.E. Hallam T.J. Rink T.J. Nature. 1988; 335: 40-45Crossref PubMed Scopus (307) Google Scholar), and adenosine trisphosphate (12.Carter T.D. Bogle R.G. Bjaaland T. Biochem. J. 1991; 278: 697-704Crossref PubMed Scopus (29) Google Scholar), H2O2 does not increase levels of inositol 1,4,5-trisphosphate (InsP3) at the concentrations that produce [Ca2+]ioscillations (14.Vercellotti G.M. Severson S.P. Duane P. Moldow C.F. J. Lab. Clin. Med. 1991; 117: 15-24PubMed Google Scholar). Since the H2O2-generating enzyme xanthine oxidase has been shown to decrease luminal Ca2+ content in vascular endothelial cells (15.Wesson D.E. Elliott S.J. Microcirculation. 1995; 2: 195-203Crossref PubMed Scopus (32) Google Scholar) and since redox sensitivity of the InsP3 receptor has been demonstrated in other cell types (16.Renard D.C. Seitz M.B. Thomas A.P. Biochem J. 1992; 284: 507-512Crossref PubMed Scopus (75) Google Scholar, 17.Nomura T. Higashi K. Hoshino M. Saso K. Itou M. Hoek J.B. Alcohol Clin. Exp. Res. 1996; 20 (suppl.): 325A-329ACrossref Google Scholar), we hypothesized that activation of the NADPH oxidase affects endothelial Ca2+signaling by increasing the sensitivity of intracellular Ca2+ stores to InsP3-stimulated Ca2+ release. To test this hypothesis, the effect of NADPH oxidase stimulation on intracellular Ca2+ stores was examined in permeabilized HAEC using the low affinity (micromolar range) Ca2+-sensitive fluorescent indicator Mag-indo 1. To characterize the specific role of NADPH oxidase stimulation, diphenyleneiodonium was used to pharmacologically inhibit the oxidase, and studies were performed using HAEC transiently expressing Rac1N17 (18.Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1449) Google Scholar), a dominant negative allele of the small GTP-binding protein Rac1 that is required for oxidase activity. HAEC were obtained as proliferating quaternary cultures (Clonetics, San Diego, CA) and were grown to confluence to passage 5–9 in endothelial cell growth medium supplemented with 2% fetal bovine serum, 10 μg/liter human-recombinant epidermal growth factor, 1 mg/liter hydrocortisone, 50 μg/ml gentamicin, 50 ng/ml amphotericin-B, and 12 μg/ml bovine brain extract (Clonetics) in a 37 °C humidified atmosphere of 95% air, 5% CO2. For Ca2+ measurements, HAEC were plated at an approximate concentration of 1 × 105/ml on 25-mm diameter circular glass coverslips (VWR Scientific, Media, PA), which were precoated with 2% gelatin solution (Sigma) and washed three times with phosphate-buffered saline (Quality Biological, Inc., Gaithersburg, MD) before cell seeding. Cells were used for experiments after reaching 70% confluence after incubation for 1–2 days at 37 °C in a humidified atmosphere of 95% air, 5% CO2. Superoxide (O⨪2) production by suspensions of permeabilized HAEC (∼8 × 105/ml) was detected by lucigenin luminescence using a Berthold Multi-Biolumat LB-9505C luminometer (Nashua, NH). Lucigenin luminescence was recorded at room temperature continuously over 30–45 min from test tubes of HAEC with 25 μm lucigenin in intracellular-like medium (ICM) containing 125 mm KCl, 19 mm NaCl, 10 mm HEPES, 1 mm EGTA (Sigma), and 0.33 mm CaCl2 (free Ca2+ concentration was 50 nm, pH adjusted to 7.20 at room temperature with KOH). Chemiluminescence counts were integrated over 3–4 min from the time of the addition of 100 μm NADPH to plateau. In some experiments, HAEC were preincubated for 15 min with either superoxide dismutase (SOD; 200 units/ml) or with the NADPH oxidase inhibitor diphenyleneiodonium (DPI; Color Your Enzyme, Ontario Canada; 10 μm), which were each also present during the chemiluminescence recording. H2O2 production by HAEC was measured fluorometrically using the AmplexTM Red Hydrogen Peroxide Assay Kit (Molecular Probes, Inc., Eugene, OR). AmplexTMRed is a fluorogenic substrate with very low background fluorescence, which reacts with H2O2 with a 1:1 stoichiometry to produce highly fluorescent resorufin (19.Mohanty J.G. Jaffe J.S. Schulman E.S. Raible D.G. J. Immunol. Methods. 1997; 202: 133-141Crossref PubMed Scopus (429) Google Scholar). Measurements of H2O2 production after the addition of NADPH were performed using suspensions (∼105/ml) of permeabilized HAEC in Corning 96-well microplates. Fluorescence intensity was measured in a Cytofluor 2300 System (Millipore Corp., Bedford, MA) at an excitation wavelength of 530 ± 25 nm and an emission wavelength of 590 ± 35 nm at room temperature. After subtracting background fluorescence, cumulative H2O2 concentrations (μm/105 cells) were calculated using a resorufin-H2O2 standard calibration curve generated from cell-free experiments using H2O2and AmplexTM Red. In some experiments, HAEC were preincubated for 30 min prior to NADPH addition with either DPI (10 μm), catalase (1000 units/ml), or SOD (200 units/ml). These inhibitors were also present during fluorescence measurements. HAEC monolayers on glass coverslips were incubated in a HEPES (Sigma)-buffered saline containing 10 μm of the ester derivative (acetoxymethyl ester form) of Mag-indo 1 (Molecular Probes) at room temperature for 45 min. The HEPES-buffered saline contained 137 mm NaCl, 4.9 mm KCl, 1.5 mm CaCl2, 1.2 mm MgSO4, 1.2 mmNaH2PO4, 15 mm d-glucose, 20 mm HEPES (pH adjusted to 7.40 at room temperature with NaOH). All salt solutions were made with pure water (free Ca2+ < 0.05 ppb; PICOpure Water Systems, Research Triangle Park, NC). After Mag-indo 1 loading, coverslips were gently washed with indicator-free HEPES-buffered saline and maintained for an additional 10 min to allow for de-esterification of the indicator. Mag-indo 1-loaded HAEC were for min to HAEC were permeabilized by to containing 30 μg/ml (Sigma) for min at room temperature. was by the release of Mag-indo 1 cytosolic After HAEC were washed with and to 1 mm and concentration for 10 min to allow for of intracellular Ca2+ HAEC were for at 10 min with medium containing 125 mm KCl, 19 mm NaCl, 10 mm HEPES, mm concentration by fluorescence of The pH of the medium was adjusted to 7.20 with Mag-indo 1 fluorescence was recorded at room temperature from HAEC on coverslips in a on the of a Mag-indo 1 fluorescence was at ± 50 nm using a Inc., and wavelength of fluorescence at ± 10 and ± 10 nm, to the and of the fluorescence from permeabilized HAEC was and measured using a The a of to the of the After from HAEC was of Mag-indo 1-loaded HAEC and was from Mag-indo 1 fluorescence Since low affinity Ca2+-sensitive like Mag-indo 1 B. U. S. PubMed Scopus Google Scholar) Ca2+ concentration in the and in intracellular may be used to the Ca2+ content of intracellular Ca2+ stores but are to in cytosolic which in the HAEC monolayers were with Mag-indo the fluorescence of the indicator was the After incubation in and of the intracellular fluorescence was to of the cell membrane and release of intracellular The fluorescence to in the in like the The fluorescence at and calcium were from 10 μm Mag-indo 1 solutions in containing CaCl2 and 1 mm EGTA or containing 10 and were used as the and min min ± ± for Mag-indo 1 for The approximate intracellular was calculated as B. U. S. PubMed Scopus Google Scholar, M. J. Biol. PubMed Scopus Google Scholar), to the following is the of Mag-indo and are the fluorescence at nm of the and The and intensity of the Mag-indo 1 fluorescence signal was in the of a fluorescence intensity decreased by ± ± the by permeabilized HAEC were to and medium to allow for of intracellular Ca2+ the increase of the Mag-indo 1 was on the Ca2+ concentration in the of and but was not by either and or not These are with the of Mag-indo sensitivity to is to Ca2+ mm for and 35 μm for Ca2+, at pH D.K. Biochem. Res. 1991; PubMed Scopus Google Scholar). The Mag-indo 1 was to of and the of the increase in the Mag-indo 1 during of intracellular Ca2+ stores was inhibited by the inhibitor not the negative Rac1 containing a at was used as (18.Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1449) Google Scholar). of the Rac1N17 was by protein with an to the Inc., are as ± were made using test for the and the of was used were was significant at < HAEC suspensions NADPH generated chemiluminescence in the of 25 μm the addition of 100 μm NADPH to HAEC suspensions a increase in production ± × cells, which at 15 min. of was observed in the of SOD ± × < or DPI ± × < The effect of NADPH on by HAEC was examined by resorufin fluorescence intensity from HAEC suspensions after NADPH addition NADPH stimulated production of H2O2 in a and with a threshold of H2O2 production a after min. The of H2O2 production was and cells after the addition of and H2O2 production was observed in the of H2O2 production was significantly inhibited by with either catalase or with the NADPH oxidase inhibitor DPI at each concentration of NADPH examined In SOD not significantly H2O2 production at NADPH concentration In permeabilized HAEC in which Ca2+ stores are by with and 100 μm not intracellular Ca2+ stores during a ± ± for time In the 3 the addition of InsP3 stimulated a decrease in the content of intracellular Ca2+ concentration of H2O2 mm for 10 in the of InsP3 also not intracellular Ca2+ stores in permeabilized HAEC ± time shown in 3 HAEC were to a concentration of InsP3 the Mag-indo 1 decreased from ± 0.05 to ± 0.05 < and was maintained at this for 10 min of The decrease in the content of intracellular Ca2+ stores stimulated by InsP3 was inhibited by ± with ± for time for 100 not intracellular Ca2+ stores in permeabilized HAEC in the of during an to a InsP3 concentration of nm 100 μm decreased the content of intracellular Ca2+ stores ± in the of H2O2 InsP3 ± in the of InsP3 < effect was also by with ± in the of and time After of in Mag-indo permeabilized a decrease in the content of intracellular Ca2+ stores with a threshold concentration of nm and a concentration of 1 μm a concentration of 10 nm the in the Mag-indo 1 was not significant ± concentrations of InsP3 the content of intracellular Ca2+ stores ± for 100 nm ± for nm, and ± 0.05 for 1 for concentration of 3 not produce an additional decrease in the content of intracellular Ca2+ stores ± 1 The addition of stimulated an additional decrease in the content of intracellular Ca2+ stores following a concentration of the of additional intracellular Ca2+ stores in HAEC H2O2 shifted the InsP3-stimulated intracellular Ca2+ release dose-response curve to the decreased the threshold concentration to nm and decreased the EC50 in to InsP3 In the of 100 a significant decrease in the Mag-indo 1 was observed during to 10 ± < 10 nm InsP3 permeabilized HAEC were to 100 and nm InsP3 in the of 100 μm the decrease in the content of intracellular Ca2+ stores was that observed in the of H2O2 ± ± for 100 nm ± ± for < 0.05 at each In the of 100 the decrease in the Mag-indo 1 stimulated by 1 μm InsP3 ± and by 3 μm InsP3 ± was not significantly from that stimulated by concentrations of InsP3 in the of H2O2 for each the in the Mag-indo 1 stimulation by was not in the ± the of H2O2 ± for shown in H2O2 a decrease in the EC50 of a concentration of 100 μm the EC50 of InsP3-stimulated was decreased from 423.2 ± 54.9 nm to ± nm < significant effect of H2O2 on InsP3-stimulated Ca2+ release was observed at a concentration of H2O2 as low as 3 this H2O2 decreased the EC50 of InsP3-stimulated by to 276.9 ± 14.4 < additional effect on the EC50 of InsP3 was observed at H2O2 concentrations 100 To the effect of NADPH oxidase stimulation on intracellular Ca2+ Mag-indo 1-loaded HAEC were stimulated by the addition of 100 μm concentration of NADPH has been used to stimulate the oxidase in endothelial cells S.A. O'Donnell V.B. Wood J.D. Broughton J.P. Hughes E.J. Jones O.T. Am. J. Physiol. 1996; 271: H1626-H1634PubMed Google J.J., Yu, F-S. Hertig I.A. Thannickal V.J. Hassoun P.M. Am. J. Respir. Cell Mol. Biol. 1995; 12: 41-49Crossref PubMed Scopus (142) Google Scholar, P.M. Am. J. Physiol. Google Scholar). The addition of 100 μm NADPH to permeabilized HAEC and decreased the Mag-indo 1 by ± over 10 to an increase in the fluorescence intensity of Mag-indo 1 at the to be an effect on the indicator an effect on intracellular Ca2+ since in a cell-free the of Mag-indo 1 (10 μm in 100 μm was decreased ± NADPH shifted the InsP3 dose-response to the the threshold concentration of and decreased the EC50 in to a concentration of 10 nm which does not decrease the content of intracellular Ca2+ stores in HAEC in the of the Mag-indo 1 decreased by ± in the of 100 μm < 10 The of 100 and nm InsP3 on intracellular were by 100 μm NADPH ± for 100 < 100 nm InsP3 and ± 0.05 for nm < 0.05 nm InsP3 for each The decrease in the content of intracellular Ca2+ stores stimulated by 1 and 3 μm InsP3 was not by the of shown in NADPH a decrease in the EC50 of which significant at a concentration of 100 this NADPH decreased the EC50 in to InsP3 by from 423.2 ± 54.9 to ± nm. additional effect on the EC50 of InsP3 was observed at NADPH concentrations 100 The effect of 100 μm NADPH on InsP3-stimulated intracellular Ca2+ release was blocked by with either catalase ± nm, or by 10 μm DPI ± nm, but was by 200 SOD ± < 0.05 100 μm NADPH SOD the sensitivity to InsP3-stimulated intracellular Ca2+ release EC50 ± for ± nm for ± nm for for To characterize the role of NADPH oxidase activation in the production of H2O2 and increasing the sensitivity to InsP3-stimulated intracellular Ca2+ studies were performed in HAEC transiently expressing a dominant negative allele of Rac1 (18.Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1449) Google Scholar). cells were stimulated with H2O2 production was significantly in with at three NADPH concentrations 100 μm H2O2 production in ± cells) was to that observed in cells ± cells, In H2O2 production in HAEC after to 100 μm NADPH was significantly decreased ± < To the importance of functional Rac1 protein in the increased sensitivity of intracellular Ca2+ stores to InsP3 stimulated by HAEC and cells were permeabilized and with 100 μm NADPH for 30 min before stimulated by shown in the decrease in the EC50 to InsP3 by 100 in cells ± ± nm, < was not observed in HAEC ± ± nm, In the effect of 100 on intracellular Ca2+ release was not in and that NADPH oxidase activation stimulates the production of H2O2 and increases the sensitivity of intracellular Ca2+ stores to InsP3 in human endothelial cells. vascular oxidase is a of reactive oxygen species in the K.K. J.D. Alexander R.W. Res. 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Hughes E.J. Jones O.T. Am. J. Physiol. 1996; 271: H1626-H1634PubMed Google Scholar, J.J., Yu, F-S. Hertig I.A. Thannickal V.J. Hassoun P.M. Am. J. Respir. Cell Mol. Biol. 1995; 12: 41-49Crossref PubMed Scopus (142) Google Scholar, P.M. Am. J. Physiol. Google Scholar) H2O2 in a and NADPH stimulated H2O2 production NADPH oxidase activation is by the that H2O2 production was inhibited by DPI and was significantly in cells lacking a functional GTP-binding protein Rac1, which is required for oxidase activity. NADPH oxidase stimulation generated in the present and this is the of oxidase-derived NADPH H2O2 intracellular Ca2+ each stimulated additional release of intracellular Ca2+ stores during an to a concentration of In the of either H2O2 or the threshold concentration of InsP3-stimulated intracellular Ca2+ release was decreased to nm, the InsP3 dose-response curve was shifted to the and the EC50 to InsP3 was significantly a inhibitor of Ca2+ release J. Biol. 1988; PubMed Google Scholar), blocked the effect of H2O2 in the of a concentration of that the addition of H2O2 or the of H2O2 by NADPH affects intracellular Ca2+ stores by increasing the sensitivity of the InsP3 receptor in The that the effect of NADPH on the sensitivity of intracellular Ca2+ stores to InsP3 was blocked by catalase but was not by SOD a specific effect of H2O2 on the InsP3 sensitivity of the has been in other cell types (16.Renard D.C. Seitz M.B. Thomas A.P. Biochem J. 1992; 284: 507-512Crossref PubMed Scopus (75) Google Scholar, 17.Nomura T. Higashi K. Hoshino M. Saso K. Itou M. Hoek J.B. Alcohol Clin. Exp. Res. 1996; 20 (suppl.): 325A-329ACrossref Google Scholar). The present that small in the intracellular concentration of H2O2 are to have significant on intracellular Ca2+ the on the EC50 of InsP3 of small in the concentration of either H2O2 or a and that the intracellular concentration of H2O2 is in Since the concentrations of H2O2 that are generated by vascular cells as a of NADPH oxidase activation are not is to the concentrations of H2O2 observed following NADPH addition to permeabilized cell suspensions in the present with that which in concentrations of H2O2 in the low increased the sensitivity of intracellular to InsP3 in that NADPH oxidase-derived H2O2 the threshold concentration for intracellular Ca2+ release in endothelial cells to nm Since intracellular levels of InsP3 are in the 50 nm Taylor J. Physiol. 1992; PubMed Scopus (112) Google Scholar, M. S. J. Biol. PubMed Google Scholar), stimuli that activate the oxidase may endothelial levels of may be of in this that we recently showed that at concentrations that not increase levels of InsP3 (14.Vercellotti G.M. Severson S.P. Duane P. Moldow C.F. J. Lab. Clin. Med. 1991; 117: 15-24PubMed Google Scholar), stimulate in human endothelial cells (11.Hu Q. Corda S. Zweier J.L. Capogrossi M.C. Ziegelstein R.C. Circulation. 1998; 97: 268-275Crossref PubMed Scopus (112) Google Scholar). Since intracellular Ca2+ oscillations at levels of InsP3 M. Nature. PubMed Scopus Google Scholar), the increased sensitivity of intracellular Ca2+ stores to InsP3 from NADPH oxidase activation may the of intracellular Ca2+ oscillations or the of an effect may in growth vascular cells. In this cell growth intracellular Ca2+ oscillations B. J. Biol. 1991; PubMed Google Scholar), and stimulates the of the T. V.B. B. R. 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