- Email: [email protected]
Mechanisms of Ca2+store depletion in single endothelial cells in a Ca2+-free environment
Cell Calcium (1999) 25 (5), 345–353 © Harcourt Brace & Co. Ltd 1999 Article No. ceca.1999.0038
Research
Mechanisms of Ca2+ store depletion in single endothelial cells in a Ca2+-free environment J. Paltauf-Doburzynska, M. Frieden, W. F. Graier Department of Medical Biochemistry, Karl-Franzens University of Graz, Graz, Austria
Summary Depletion of agonist-sensitive Ca2+ stores results in activation of capacitative Ca2+ entry (CCE) in endothelial cells. The proportion of Ca2+ stores contributing to the regulation of CCE is unknown. In fura-2/am loaded single endothelial cells freshly isolated from bovine left circumflex coronary arteries, we investigated whether a resting period in a Ca2+-free environment results in emptying of bradykinin-sensitive Ca2+ stores (BsS) and activation of CCE. In a Ca2+-free environment, depletion of BsS occurred in a time-dependent manner (59% after 10 min in Ca2+-free solution). This effect was prevented by inhibition of the Na+–Ca2+ exchange but not by a blockade of ryanodine-sensitive Ca2+ release (RsCR). In contrast to BsS, mitochondrial Ca2+ content remained unchanged in the Ca2+-free environment. Remarkably, activity of CCE (monitored as Mn2+ influx) did not increase after depletion of BsS in the Ca2+-free environment. In contrast to Mn2+ influx, the effect of re-addition of Ca2+ to elevate bulk Ca2+ concentration ([Ca2+]b) decreased with the time the cells rested in Ca2+-free buffer. This decrease was prevented by an inhibition of RsCR. In low Na+ conditions the effect of Ca2+ on [Ca2+]b was reduced while it did not change the time the cells rested in Ca2+-free solution. After a 2 min period in low Na+ conditions, ryanodine-induced Ca2+ extrusion was markedly diminished. Inhibition of RsCR re-established the effect of Ca2+ on [Ca2+]b in low Na+ conditions. Collapsing subplasmalemmal Ca2+ stores with nocodazole, increased the effect of Ca2+ on [Ca2+]b. In nocodazole-treated cells, the effect of Ca2+ on [Ca2+]b was not reduced in Ca2+-free environment. These data indicate that activation of CCE is not associated with the agonist-sensitive Ca2+ pools that deplete rapidly in a Ca2+-free environment. Subplasmalemmal ryanodine-sensitive Ca2+ stores (RsS) are emptied in Ca2+-free /low Na+ solution and re-sequester Ca2+ which enters the cells prior an increase in [Ca2+]b occurs. Thus, in endothelial cells there are differences in the functions of various subplasmalemmal Ca2+ stores (i.e. BsS and RsS), which include either activation of CCE or regulation of subplasmalemmal Ca2+.
INTRODUCTION In endothelial cells the autacoid-induced release of vasoactive compounds, such as prostacycline and nitric oxide (NO), is triggered by an elevation in the cytoplasmic Ca2+ concentration [1,2,3]. The hypothesis of endothelial Ca2+ homeostasis was recently updated by findings of insulated Ca2+ signaling in the cell nucleus [4,5], the mitochondria [6,7] and the subplasmalemmal Received 21 January 1999 Revised 7 May 1999 Accepted 7 May 1999 CCorrespondence to: Professor Dr Wolfgang F. Graier, Department of Medical Biochemistry, Karl-Franzens University of Graz, Harrachgasse 21/III, A-8010 Graz, Austria. Tel.: +43 316 380 7560; fax: +43 316 380 9615; e-mail: [email protected]
area [8]. Nevertheless, owing to technical limitations, most laboratories mainly investigate endothelial Ca2+ signaling using fura-2, which monitors bulk Ca2+ concentration [Ca2+]b. A frequently used protocol is to stimulate the endothelial cells in the nominal absence of extracellular Ca2+, to measure agonist-induced intracellular Ca2+ release, followed by the re-addition of extracellular Ca2+ [9,10] to monitor activity of the so-called capacitative Ca2+ entry (CCE [11]) originally described by Putney [12,13]. Recently, evidence was provided that during re-addition of extracellular Ca2+ to agonist-stimulated endothelial cells, the observed increase in [Ca2+]b does not exclusively reflect CCE, but includes ryanodinesensitive Ca2+ release (RsCR) [14]. RsCR has been previously shown to contribute to agonist-induced elevation in [Ca2+]b [15,16] and localized Ca2+ signaling [8,17] in endothelial cells. In addition, although the activity of 345
346 J Paltauf-Doburzynska, M Frieden, W F Graier
CCE is closely linked to the filling state of internal Ca2+ stores [18], there is evidence that only a certain part of the intracellular Ca2+ stores are involved in regulation of CCE [19]. It has been reported that activation of RsCR by either caffeine [20] or ryanodine [19] facilitates CCE rather than activating it [17]. Thus, it remains unclear, which compartments of the internal Ca2+ stores are responsible for activation of CCE. This study was designed to correlate depletion of endothelial Ca2+ stores with CCE activity. The mechanisms and kinetics of the depletion of agonist-sensitive Ca2+ stores in Ca2+-free environment were examined and the activity of CCE was monitored by measuring Mn2+ influx. In addition, the mechanism of depletion of the ryanodine-sensitive Ca2+ stores during a resting time in a Ca2+-free environment and the role of ryanodine-sensitive Ca2+ stores on the increases in [Ca2+]b in response to the re-addition of extracellular Ca2+ was assessed. MATERIALS AND METHODS Materials Petri dishes were purchased from Corning, Vienna, Austria. Fura-2/AM, fura dextran and ER-Traker BlueWhite DPX was from Molecular Probes, Leiden, The Netherlands. Sera were obtained from PAA laboratories, Linz, Austria. Cell culture media and chemicals were purchased from Life Technologies, Vienna, Austria. All other materials were from Sigma Chemicals, Vienna, Austria. Cells and Cell culture Endothelial cells were freshly isolated from the bovine left circumflex artery as previously described [21]. Briefly, just after the death of the animal, the heart was extracted and the left circumflex artery was isolated and put in chilled storage buffer (SB: DMEM containing 2% horse serum and 0.1% of a vitamin mixture and 0.2% essential amino acids) for transport to the laboratory. Under sterile conditions, vessels were cut longitudinally, fixed with the endothelium on top and incubated for 2 h in SB containing 200 U/ml collagenase (type II) and 1 mg/ml trypsin inhibitor (soybean type I) at 37°C. After cell isolation, cells were kept in SB for fura-2/AM loading. Parts of the collagenase solution were aspirated after 30 min, centrifuged and the cells were cultured in small petri dishes in Opti-MEM containing 2% fetal calf serum. After 10 days, cells were harvested by trypsin (0.02% in phosphate-buffered solution), centrifuged and seeded in a customized glass dish (ø 1 cm) for 1 day in culture medium. Typical cobblestone morphology and the lack of smooth muscle α-actin were tested to control cell culture purity of primary cultures.
Cell Calcium (1999) 25(5), 345–353
The data obtained in endothelial cells freshly isolated from the bovine left circumflex artery were compared with that measured in the endothelial cell line EA.hy 926, which is originally derived from a human umbilical vein [22]. This cell line was a gift from Dr Cora-Jean S. Edgell, Pathology Department, University of North Carolina, Chapel Hill, NC, USA. Cells from the EA.hy 926 cell line were grown in DMEM containing 10% fetal calf serum, 4.5 mg l–1 D-glucose and 1% HAT (5 mM hypoxanthin, 20 µM aminopterin, 0.8 mM thymidine). Measurement of intracellular free Ca2+ concentration For measuring the bulk Ca2+ concentration ([Ca2+]b), conventional fura-2 experiments were performed in single endothelial cells as described previously [8,21]. Briefly, cells were incubated for 45 min at room temperature in the dark with 2 µM fura-2/AM, centrifuged, washed twice and resuspended in SB. After an equilibration period of 10 min, cells were transferred into an experimental chamber which allowed a constant superfusion of 1 ml/min. Cells were perfused for 2 min in Hepes buffer containing in mM: 145 NaCl, 5 KCl, 2.5 Ca2Cl, 1 MgCl2 and 10 Hepes acid, pH adjusted at 7.4 (HBS/Ca), followed with an incubation in nominal Ca2+-free Hepes-buffer (145HBS containing in mM: 145 NaCl, 5 KCl, 1 MgCl2, 10–5 M EGTA and 10 Hepes acid, pH adjusted at 7.4) or in nominal Ca2+-free, low Na+ Hepes buffer (19HBS containing in mM: 19 NaCl, 5 KCl, 1 MgCl2, 126 choline chloride, 10–5 EGTA, 10 Hepes-acid, pH=7.4) as indicated. The [Ca2+]b concentration was monitored by sampling fluorescence intensity at 510 nm emission alternatively at 360 (i.e. Ca2+-insensitive wavelength) and 380 nm excitation (i.e. Ca2+ sensitive wavelength) excitation. Fluorescence intensity for each pair of excitation/emission wavelength was converted to analog by an optical processor [23] and registered by a PC running AxoBASIC® 1.0 (Axon Instruments, Foster City, CA, USA). In view of the reported errors of the [Ca2+]b calibration in our system [8,21] and the general uncertainties of the calibration techniques [15], [Ca2+]b is expressed in ratio units (ratio (F360/F380)). For evaluation of Ca2+ entry activity, Mn2+ quench experiments were performed as previously described [24]. The reduction of fluorescence intensity at F360 was monitored and expressed as % decrease of the initial intensity of F360/min. For measurement of Ca2+ extrusion, cells were resuspended in 145HBS containing 5 µmol l–1 fura dextran potassium salt (MW 70 000) and extrusion of Ca2+ was measured as previously described [17]. Deconvolution microscopy For image analysis of the architectural changes of the endoplasmic reticulum (ER), high-resolution image
© Harcourt Brace & Co. Ltd 1999
Mechanisms of Ca2+ store depletion in single endothelial cells 347
analysis was performed using a deconvolution microscope (Nikon Eclipse TE 300, Nikon, Vienna, Austria) as recently described [8,17]. This set-up provides in combination with a scientific-grade CCD camera (Quantix, Photometrics, Munich, Germany) 3D-images using conventional lamp illumination (150 W XBO; Opti Quip, Albany Turnpike, NY, USA) based on computer algorithms to calculate the out-of-focus light by a 3D point spread function for each microscope objective lens [25,26]. Movements in z-axis were achieved with a z-stage motor controlled by the Ludl-z-stage device (Ludl, Inc., Fairfield Imaging, Tumbridge Wells, UK) controlled by Volume Scan® (VayTek, Inc., Fairfield Imaging, Tumbridge Wells, UK). In combination of a cooled camera head at –40°C and a gain level of 2 this set-up provides excellent sensitivity with high signal-to-noise ratio. Images were collected with a CFI Plan Fluor 40x oil immersion objective (N.A.=1.4; 0.171 µm/pixel, Nikon, Vienna, Austria) and the slice interval was 0.5 µm. Images were sampled using Image Pro 3.0 (Media Cybemetics, Fairfield Imagining, Tumbridge Wells, UK) and out-offocus fluorescence was removed using Micro Tome® (VayTek, Inc., Fairfield Imaging, Tumbridge Wells, UK) using the advanced constrained iterative deconvolution algorithm (5 iterations). Statistics Analysis of variance (ANOVA) was performed and statistical significance of differences was evaluated using the Scheffe’s F test. Level of significance was defined as P<0.05.
RESULTS The time-dependent depletion of IP3-sensitive Ca2+ stores in nominal Ca2+-free solution was assessed by measuring bradykinin-induced intracellular Ca2+ release in the cells which rested 1, 3, 7 and 10 min in 145HBS. As shown in Figure 1A intracellular Ca2+ release to 100 nM bradykinin reduced with the time the cells remained in the Ca2+-free environment prior to stimulation. In contrast to the timedependent depletion of bradykinin-sensitive Ca2+ stores (BsS) in 145HBS, prevention of Na+–Ca2+ exchange (NCX) by reducing extracellular Na2+ concentration ([Na2+]e) to 19 mM (i.e. 19HBS) abolished time-dependent depletion of the BsS (Fig. 1B). There was no difference in bradykinin-evoked intracellular Ca2+ release in single endothelial cells incubated for 1, 3, 7 or 10 min in 19HBS. Identical findings were obtained in EA.hy 926 cells using 10 µM histamine to evoke IP3-mediated intracellular Ca2+ release (data not shown). To compare the capacity of bradykinin-mediated intracellular Ca2+ release after various times in nominal © Harcourt Brace & Co. Ltd 1999
Fig. 1 Time-dependent reduction of the effect of bradykinin on [Ca2+]b by incubation for 1, 3, 7 and 10 min in a Ca2+-free environment. Cells were freshly isolated from the bovine left circumflex coronary artery and loaded with fura-2/AM as described under Methods. At times 0 min, HBS/Ca was replaced by nominal Ca2+-free 145HBS (A) or 19HBS (B). After 1, 3, 7 or 10 in the Ca2+free solution cells were stimulated with 100 nM bradykinin (Bk). Points represent the mean±SEM (panel A: n=28; panel B: n=23). *P<0.05 vs the effect of bradykinin after 1 min in Ca2+-free solution.
Ca2+-free solution under normal and low [Na2+]e conditions, single endothelial cells were incubated in 145HBS and 19HBS for 1, 3, 7 and 10 min, followed by the addition of 100 nM bradykinin in 19HBS to prevent NCX activity (Fig. 2A). Analysis of differences between the bradykinin-induced intracellular Ca2+ release indicate a reduction of IP3-releasable pool by 0±0.4, 11.8±0.6, 29.4±0.9 and 58.8±3.0% after 1, 3, 7 and 10 min in 145HBS compared with cells in 19HBS. The decrease in bradykinin-induced intracellular Ca2+ release by repetitive stimulation in nominal Ca2+-free solution was prevented when the cells were exposed to 2.5 mM Ca2+ containing solution between each single stimulation (Fig. 2B). Inhibition of RsCR by 25 µM ryanodine while the cells were incubated in 145HBS did not preserve the effect of Cell Calcium (1999) 25(5), 345–353
348
J Paltauf-Doburzynska, M Frieden, W F Graier
Fig. 3 Time-dependent reduction of the effect of bradykinin on [Ca2+]b by incubation for 1, 3, 7 and 10 min in 145HBS without (open columns) and with ryanodine (filled columns). Endothelial cells were freshly isolated from the bovine left circumflex coronary artery and loaded with fura-2/AM as described under Methods. At times 0 min, HBS/Ca was replaced by nominal Ca2+-free 145HBS containing solvent control (DMSO) or 25 µM ryanodine. After 1, 3, 7 or 10 in the Ca2+-free environment, cells were stimulated with 100 nM bradykinin (Bk). Columns represent the mean±SEM (n=24). *P<0.05 vs. the effect of bradykinin after 1 min in Ca2+-free solution without ryanodine and #P<0.05 vs. the effect of bradykinin after 1 min in 145HBS containing ryanodine.
Fig. 2 Direct comparison of the time-dependent reduction of the effect of bradykinin on [Ca2+]b by incubation for 1, 3, 7 and 10 min in a Ca2+-free environment (A) and refilling of IP3-sensitive Ca2+ stores by extracellular Ca2+ (B). (A) Fura-2/AM loaded endothelial cells were incubated for 1 min in HBS/Ca, which was replaced at time 1 min by nominal Ca2+-free 145HBS (open circles) or 19HBS (open squares). After 1, 3, 7 or 10 in either 145HBS or 19HBS cells were stimulated with 100 nM bradykinin (Bk) in 19HBS. Tracings are representative single cell recordings and the points represent the mean±SEM (n=18). (B) Incubation of endothelial cells with 2.5 mM extracellular Ca2+ after bradykinin stimulation prevented the decrease in intracellular Ca2+ release in response to repetitive agonist stimulation. As bars indicate endothelial cells were stimulated with either 100 nM bradykinin in the nominal absence of extracellular Ca2+ or were incubated in 2.5 mM Ca2+ containing solution (n=8).
bradykinin over the time the cells were incubated in the Ca2+-free environment (Fig. 3). In contrast to the depletion of BsS in 145HBS, the amount of mitochondrial Ca2+ releasable with 10 µM carbonyl cyanide p-(trilfeuor-methoxy) phenylhydrazone (FCCP) remained constant during incubation in 145HBS for up to 10 min (Fig. 4). Identical findings were obtained when cells were incubated over 1, 3, 7 and 10 min in 19HBS prior addition of FCCP (data not show). To investigate whether CCE activity varies in cells after Cell Calcium (1999) 25(5), 345–353
Fig. 4 Lack of the effect of incubation for 1, 3, 7 and 10 min in 145HBS for FCCP-releasable mitochondrial Ca2+. At time 0 min 145HBS replaced HBS/Ca. After 1, 3, 7 or 10 in the Ca2+-free environment, mitochondrial Ca2+ was released by 10 µFCCP. Tracings are original recordings in single endothelial cells and points represent the mean±SEM (n=12).
incubation for several time periods in 145HBS, the rate of Mn2+ entry was assessed in cells after 1, 3, 7 and 10 min in the Ca2+-free 145HBS. As shown in Figure 5A, the resting period in nominal Ca2+-free solution did not alter Mn2+ entry monitored. The observed Mn2+ quench was significantly smaller than that measured in cells after © Harcourt Brace & Co. Ltd 1999
Mechanisms of Ca2+ store depletion in single endothelial cells 349
Fig. 6 Time-dependent reduction of the effect of CaE on [Ca2+]b by incubation for 1, 3, 7 and 10 min in 145HBS (A) or 19HBS (B) without (open columns) and with ryanodine (filled columns). Endothelial cells were loaded with fura-2/AM as described under Methods. At times 0 min, HBS/Ca was replaced by nominal Ca2+free 145HBS (A) or 19 HBS (B) containing solvent control (DMSO) or 25 µM ryanodine. After 1, 3, 7 or 10, 2.5 mM Ca2+ was added. Columns represent the mean±SEM (A: n=8–11; B: n=12). *P<0.05 vs. the effect of CaE after 1 min in 145HBS/19HBS without ryanodine and #P<0.05 vs the effect of CaE in 145HBS without ryanodine.
Fig. 5 CCE activity measured with Mn2+ (A) and the effect of a readdition of Ca2+ (CaE) (B) after incubation for 1, 3, 7 and 10 min in nominal Ca2+-free solution. (A and B) At times 0 min, 145HBS replaced HBS/Ca. At time points 1, 3, 7 or 10 150 µM Mn2+ (panel A) or 2.5 mM Ca2+ (panel B) were added. The continuous lines in panel A indicate the average Mn2+ quench induced by addition of 150 µM Mn2+ while the dotted lines indicate the bradykinin induced Mn2+ quench (time 1 and 7 min). Tracings are representative recordings from single cells and the points represent the mean±SEM (A: n=14; B: n=36). *P<0.05 vs the effect of CaE after 1 min in Ca2+-free solution. (C) At times 0 min, HBS/Ca was replaced by 19HBS followed by the re-addition of 2.5 mM Ca2+ at time points 1, 3, 7 or 10. Tracings are representative recordings from single cells and the points represent the mean±SEM (n=26).
© Harcourt Brace & Co. Ltd 1999
stimulation with 100 nM bradykinin (7.3±2.2% of the initial fluorescence at 360 nm excitation; n=4; P<0.05 vs non-stimulated cells; data not shown). In contrast to the Mn2+ entry, in single endothelial cells resting for 1, 3, 7 and 10 min in 145HBS, increases in [Ca2+]b to the addition of 2.5 mM extracellular Ca2+ (CaE) became reduced with the time the cells rested in the Ca2+-free environment (Fig. 5B). In cells prestimulated with 100 nM bradykinin, [Ca2+]b increased by 0.48 ratio F360/F380 units (n=23; P<0.05 vs non-stimulated cells; data not shown). To investigate the contribution of NCX to the observed reduction of CaE-induced elevation of [Ca2+]b, single endothelial cells were incubated in 19HBS for various time periods, followed by the addition of 2.5 mM Ca2+ (Fig. 5C). In cells resting for 1, 3, 7 and 10 min in 19HBS, no time-dependent reduction of the increases of [Ca2+]b in response to CaE was found (Fig. 5C). However, the effect of CaE was reduced in 19HBS and did not differ from the effect of CaE in cells preincubated in 145HBS for 10 min. Cell Calcium (1999) 25(5), 345–353
350 J Paltauf-Doburzynska, M Frieden, W F Graier
Fig. 7 Effect of a 2 min exposure to 19HBS on the ryanodineinduced Ca2+ extrusion from endothelial cells in 145HBS measured by fura dextran potassium salt [14]. Suspended endothelial cells were pretreated for 2 min in 145HBS (Control; open circles, n=5) or 19HBS (Pretreated with 19 [Na+]e; open squares, n=5), centrifuged and resuspended in 145HBS containing 5 µM fura dextran potassium salt. After 2 min, cells were stimulated with 200 nM ryanodine and extrusion of Ca2+ was monitored by measuring the Ca2+-sensitive wavelength of fura dextran. *P<0.05 vs. control.
Fig. 8 Relative distribution of the ER in endothelial cells without and with nocodazole treatment. Endothelial cells were isolated from the bovine left circumflex coronary artery, cultured in primary culture for 10 days and transferred in a sterile glass-bottom chamber for further 24 h and were incubated in DMEM containing solvent control (0.1% DMSO) or 10 µM nocodazole at 37°C. After 16 h cells were washed three times and incubated for 40 min in DMEM containing 2 µM ER-Traker Blue-White DPX (Molecular Probes, Leiden, The Netherlands) in the dark. The ER-network of the endothelial cell was monitored in middle depth. Haze was removed using the advanced constrained iterative algorithm (5 iterations; MicroTome, Vaytek, Tumbridge Wells, UK). Results were expressed as the ratio of the average fluorescence intensities of two, 2 µm thick, circles beneath the cell membrane (i.e. subplasmalemmal fluorescence; lsub.) and around the cell nucleus (perinuclear fluorescence; (lperi.) region. *P<0.05 vs. solvent control.
Inhibition of RsCR by 25 µM ryanodine while incubation in 145HBS abolished time-dependent reduction of the effect of CaE (Fig. 6A). Inhibition of RsCR by ryanodine during the incubation in 19HBS re-established the effect of CaE while no time-dependent reduction of the Cell Calcium (1999) 25(5), 345–353
Fig. 9 Time-dependent reduction of the effect of CaE on [Ca2+]b by incubation for 1, 3, 7 and 10 min in 145HBS in control cells (open columns) and in cells with nocodazole-collapsed ER (filled columns). Cultured endothelial cells were incubated for 16 h in DMEM containing 0.1% DMSO (solvent control) or 10 µM nocodazole in a sterile chamber with glass bottom. After washing the cells endothelial cells were loaded with fura-2/AM. At times 0 min, HBS/Ca was replaced by 145HBS and at times 1, 3, 7 or 10 min, 2.5 mM Ca2+ was added. Columns represent the mean±SEM (n=12). *P<0.05 vs the effect of CaE after 1 min in 145HBS and #P<0.05 vs the effect of CaE in control cells.
effect of CaE was observed (Fig. 6B). In agreement with these findings, a preincubation of endothelial cells in 19HBS blunted ryanodine-induced (200 nM) Ca2+ extrusion in 145HBS (Fig. 7). To investigate whether the architectural organization of the superficial ER contributes to the observed reduction of the effect of CaE in Ca2+-free buffer, superficial ER was collapsed by a 16 h incubation with 10 µM nocodazole as recently described [8]. However, the lack of specificity of the ER-marker used (BODIPY FL-X ryanodine) made a re-evaluation of the effect of nocodazole necessary. Therefore, to monitor selectively ER domains, the high selective ER marker, ER-Traker Blue-White DPX [27] was used. As shown in Figure 8, treatment with nocodazole collapsed the ER as indicated by the decrease in the ratio of subplasmalemmal (lsub.) and perinuclear (lperi.) fluorescence. The treatment with nocodazole increased elevation of [Ca2+]b in response to CaE by 55%, while no time-dependent reduction of the effect of CaE was found in 145HBS (Fig. 9). DISCUSSION In Ca2+-free solution, the bradykinin-sensitive Ca2+ stores (BsS) became depleted with the time the cells rested in the Ca2+-free buffer (i.e. 145HBS). The emptying of BsS was prevented when NCX activity was inhibited in low © Harcourt Brace & Co. Ltd 1999
Mechanisms of Ca2+ store depletion in single endothelial cells 351
Fig. 10 Schematic view on the proposed depletion of bradykinin(BsS) and ryanodine-sensitive Ca2+ stores (RsS) in endothelial cells in nominal Ca2+-free solution and the distribution of Ca2+ entering the cells. Upper panel: Under normal conditions (i.e. 145 mM [Na+]e; active NCX), the Ca2+ pools are depleted by leak and/or basal activity of either the IP3 or the ryanodine receptor and the Ca2+ is extruded by the NCX. By the addition of extracellular Ca2+, Ca2+ leaks into the cell and is sequestered into both Ca2+ stores prior elevating [Ca2+]b. Thus, the effect of Ca2+ entering the cell depends on the rate of Ca2+ sequestration into subplasmalemmal Ca2+ stores. Lower panel: At low extracellular [Na+] (19 mM; NCX activity is inhibited) in nominal Ca2+-free solution, Ca2+ released from the BsS and the RsS cannot be extruded and, thus, in turn, initiates activation of Ca2+-induced Ca2+ release, resulting in a pronounced depletion of the RsS. If extracellular Ca2+ is added, Ca2+ leaks into the cell and gets sequested from subplasmalemmal Ca2+ pools prior elevating [Ca2+]b. Since the RsS is more depleted it ‘buffers’ the Ca2+ influx, resulting in a reduced elevation of [Ca2+]b to the addition of extracellular Ca2+.
© Harcourt Brace & Co. Ltd 1999
Na+ solution (i.e. 19HBS) or by incubation with Ca2+containing medium between the repetitive stimulation. In contrast, inhibition of ryanodine receptors failed to reduce the time-dependent depletion of the BsS in 145HBS. These data suggest that NCX extrudes the Ca2+ that leaks out of the BsS, while RsCR is not involved in the depletion of BsS under these conditions (Fig. 10). The comparison of the effect of bradykinin in cells pretreated in 145HBS or 19HBS (Fig. 2A) allows estimating the amount of depletion of the BsS in Ca2+-free solution. Interestingly, while there was a time-dependent depletion of the BsS in 145HBS that reached 58% after 10 min, no activation of CCE monitored using Mn2+ flux technique could be found. These data suggest that the depletion of certain BsS in Ca2+-free environment does not initiate CCE activation. This is in contrast to the report of Hofer et al. who described that the activity of CCE is closely linked to the filling state of internal Ca2+ stores [18]. Since inhibition of NCX completely abolished the emptying of BsS under the Ca2+-free environment, one might speculate that subplasmalemmal BsS which are coupled with NCX, as described in smooth muscle cells [28], became depleted, while the Ca2+ content of the BsS responsible for activation of CCE remains constant. These findings suggest that the BsS responsible for CCE activation constitutes less than 40% of the whole BsS. This is consistent with the findings of Sasajima et al. [19] who reported that only a small compartment of the agonist-sensitive ER is involved in the activation of CCE. Similar to the time-dependent reduction of bradykinininduced elevation in [Ca2+]b in Ca2+-free environment, the effect of a re-addition of 2.5 mM Ca2+ (CaE) became reduced in a time-dependent manner. The mechanisms of CaE on [Ca2+]b might involve leak Ca2+ entry, CCE and RsCR [14]. Since the Mn2+ entry remained constant over time, it seems unlikely that a reduced Ca2+ entry is responsible for the reduction of the elevation in [Ca2+]b in response to CaE. On the other hand, inhibition of RsCR did not attenuate the effect of CaE, suggesting that RsCR is not involved in the effect of CaE under these experimental conditions. However, our findings that inhibition of RsCR prevented the time-dependent reduction of the effect of CaE, might indicate that RsCR contributes to the observed attenuation of the effect of CaE (Fig. 10). Considering the constant influx of Ca2+ as indicated in our Mn2+ flux experiments, one might suggest that in the Ca2+-free environment subplasmalemmal ryanodine-sensitive Ca2+ stores (RsS) become depleted via RsCR. This might result in a rapid sequestration of the Ca2+ entering the cells into depleted subplasmalemmal Ca2+ pools. Such ‘Ca2+ steal’ by the subplasmalemmal Ca2+ stores would attenuate elevation of [Ca2+]b in response to CaE. This hypothesis is supported by our findings that an inhibition of NCX by 19HBS prevented the time-dependent Cell Calcium (1999) 25(5), 345–353
352 J Paltauf-Doburzynska, M Frieden, W F Graier
reduction of the effect of CaE, while the effect of CaE was markedly reduced. Since inhibition of RsCR re-established the effect of CaE even in 19HBS while no timedependent reduction in the effect of CaE was observed, we suggest that in 19HBS depletion of subplasmalemmal Ca2+ stores via RsCR is pronounced (Fig. 10). This hypothesis is supported by our findings that ryanodineinduced Ca2+ extrusion in 145HBS was strongly reduced in cells that were pre-incubated in 19HBS for just 2 min. Our findings that in the Ca2+-free environment subplasmalemmal Ca2+ stores sensitive to bradykinin (BsS) or ryanodine (RsS) are depleted by different pathways (NCX and RsCR respectively) suggest that, at least in the subplasmalemmal area, BsS and RsS are two distinct Ca2+ stores (Fig. 10). This is in agreement with our previous work in porcine coronary endothelial cells, where we suggested that BsS and Ca2+ stores sensitive to caffeine, a well-known activator of ryanodine receptors are two different compartments of the ER [20]. Recently, the involvement of subplasmalemmal mitochondria in the localized Ca2+ signaling [7,8] and the existence of a functioning Ca2+-induced Ca2+ release mechanisms on the mitochondrial membrane [29] have been described. Thus, it is possible that the increases in [Ca2+]b in response to CaE is due, at leat in part, to Ca2+induced Ca2+ release from the mitochondria. Although we cannot rule out this possibility, our findings that the effect of FCCP [7] remained constant during the time in Ca2+-free solution may indicate that a depletion of the mitochondrial Ca2+ pool is not responsible for the reduction of the effect of CaE in Ca2+-free environment. Our hypothesis that the time-dependent reduction in the elevation in [Ca2+]b to CaE is due to RsCR-mediated depletion of subplasmalemmal Ca2+ stores, which, in turn, re-sequester Ca2+ during CaE, and thus an attenuated increase in [Ca2+]b is observed, is further supported by our experiments with nocodazole. We have previously reported that nocodazole treatment disrupts the subplasmalemmal Ca2+ control unit (SCCU [8]) by remodeling the ER network as shown using BODIPY FL-X ryanodine [8]. Unfortunately, this staining is not selectively for the ER. Therefore, in the present work ER network was exclusively stained using ER-Traker Blue-White DPX [27] and the nocodazole-induced changes in the architectural organization of the cell was observed. Consistent with our hypothesis, the effect of CaE was increased in nocodazole-treated cells, while no time-dependent reduction in the Ca2+-free environment could be observed. These data suggest that removal of subplasmalemmal Ca2+ stores results in the lack of a sequestration of the Ca2+ entering the cell by subplasmalemmal Ca2+ stores. In conclusion, our findings provide new information on the functional organization of endothelial Ca2+ stores and their contribution to CCE activation and subcellular Cell Calcium (1999) 25(5), 345–353
Ca2+ distribution. Thus, in addition to our previous findings that the SCCU controls localized Ca2+ signaling [8,17] and spatial activation of constitutive nitric oxide synthase [17], new evidence are provided herein that the SCCU contributes to the distribution of Ca2+ entering the cell from the extracellular space. ACKNOWLEDGEMENTS We thank Mrs Beatrix Petschar for her excellent technical assistance. This work was supported by the Austrian Science Funds (P-12341-Med and SFB 714), the Austrian Nationalbank (P7542) and the Franz Lanyar Foundation. REFERENCES 1. Busse R, Mülsch A, Flemming I, Hecker M. Mechanisms of nitric oxide release from the vascular endothelium. Circulation 1993; 87 (suppl V): V18–V25. 2. Graier WF, Sturek M, Kukovetz WR. Ca2+ regulation and endothelial vascular function. Endothelium 1994;1: 223–236. 3. van Breemen C, Skarsgard P, Laher I, McMannus B, Wang X. (1997) Endothelium-smooth muscle interactions in blood vessels. Clin Exp Pharmacol Physiol 1997; 24: 989–992. 4. Burnier M, Centeno G, Burki E, Brunner HR. Confocal microscopy to analyze cytosolic and nuclear calcium in cultured vascular cells. Am J Physiol 1994; 266: C1118–C1127. 5. Ikeda M, Ariyoshi H, Kambayashi J, Shinoki N, Sakon M, Kawasaki T, Monden M. Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells. J Cell Biochem 1996; 63: 23–36. 6. Donnadieu E, bourguignon LYW. Ca2+ signaling in endothelial cells stimulated by bradykinin: Ca2+ measurement in the mitochondria and the cytosol by confocal microscopy. Cell Calcium 1996; 20: 53–61. 7. Lawrie AM, Rizzuto R, Pozzan T, Simpson AW. A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells. J Biol Chem 1996; 271: 10753–10759. 8. Graier WF, Paltauf-Doburzynska J, Hill B, Fleischhacker E, Hoebel BG, Kostner GM, Sturek M. Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane. J Physiol Lond 1998; 506: 109–125. 9. Schilling WP, Ritchie AK, Navarro LT, Eskin SG. Bradykininstimulated calcium influx in cultured bovine aortic endothelial cells. Am J Physiol 1998; 255: H219–H227. 10. Graier WF, Schmidt K, Kukovetz WR. Bradykinin-induced Ca2+-influx into cultured endothelial cells is not regulated by inositol 1,4,5-trisphosphate or inositol 1,3,4,5tetrakisphosphate. Second Messengers and Phosphoproteins 1991; 187–197. 11. Schilling WP, Cabello OA, Rajan L. Depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store in vascular endothelial cells activates the agonist-sensitive Ca2+-influx pathway. Biochem J 1992; 284: 521–530. 12. Putney JW. Capacitative calcium entry revisited. Cell Calcium 1990; 11: 611–624. 13. Putney JW. A model for receptor-regulated calcium-entry. Cell Calcium 1986; 7: 1–12. 14. Mozhayeva MG. [Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-indused Ca2+ release can involve both IP3-, and ryanodine-sensitive Ca2+ release. Pflügers Arch 1996; 433: 180–187.
© Harcourt Brace & Co. Ltd 1999
Mechanisms of Ca2+ store depletion in single endothelial cells 353
15. Wang X, Lau F, Li L, Yoshikawa A, VanBreemen C. Acetylcholine-sensitive intracellular Ca2+ store in fresh endothelial cells and evidence for ryanodine receptors. Circ. Res 1995; 77: 37–42. 16. Mozhayeva MG, Mozhayeva GN. Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+. Pflügers Arch. -Eur J Physiol 1996; 432: 614–622. 17. Paltauf-Doburzynska J, Posch K, Paltauf G, Graier WF. Stealth ryanodine-sensitive Ca2+ release contributes to activity of capacitative Ca2+ entry and nitric oxide synthase in bovine endothelial cells. J Physiol Lond 1998; 513: 369–379. 18. Hofer AM, Fasolato C, Pozzan T. Capacitative Ca2+ entry is closely linked to the filling state of internal Ca2+ stores: a study using simultaneous measurements of ICRAC and intraluminal [Ca2+]i. J Cell Biol 1998; 140: 325–334. 19. Sasajima H, Wang X, van Breemen C. Fractional Ca2+ release from the endoplasmic reticulum activates Ca2+ entry in freshly isolated rabbit aortic endothelial cells. Biochem Biophys Res Commun 1997; 241: 471–475. 20. Graier WF, Simecek S, Bowles DK, Sturek M. Heterogeneity of caffeine and bradykinin-sensitive stores in vascular endothelial cells. Biochem J 1994; 300: 637–641. 21. Graier WF, Simecek S, Sturek M. Cytochrome P450 monooxygenase-regulated signaling of endothelial Ca2+ entry. J Physiol London 1995; 482: 259–274. 22. Edgell CJS, McDonald CC, Graham JB. Permanent cell line expressing factor VIII related antigen established by hybridization. Proc Natl Acad Sci USA 1983; 80: 3734–3737.
© Harcourt Brace & Co. Ltd 1999
23. Sturek M, Smith P, Stehno-Bittle L. In vitro models of vascular endothelial cell calcium regulation. In: Sperelakis N., Kuriyama H. (Eds). Ion Channels of Vascular Smooth Muscle Cells and Endothelial Cells New York: Elsevier, 1991; 349–364. 24. Paltauf-Dobruzynska J, Graier WF. Temperature dependence of agonist-stimulated Ca2+ signaling in cultured endothelial cells. Cell Calcium 1997; 21: 43–51. 25. Carrington WA, Lynch RM, Moore EDW, Isenberg G, Fogarty KE, Fay FS. Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. Science 1995; 268: 1483–1487. 26. Waters D, Brown C. New high-resolution 3-D microscope – Avoids damage to live samples. Biophotonics International 1996; 9/10, 40–44. 27. Diwu Z, Lu Y, Zhang C, Klaubert DH, Haugland RP. Fluorescent Molecular Probes II. The Synthesis; Spectral Properties and Use of Fluorescent Solvatochromic Dayoxyl Dyes. Photochem Photobiol 1997; 66: 424–431. 28. Moore EDW, Etter EF, Philipson KD, Carrington WA, Fogarty KE, Lifshitz LM, Fay FS. Coupling of the Na+/Ca2+ exchanger, Na+/K+ pump and sarcoplasmic reticulum in smooth muscle. Nature 1993; 365: 657–660. 29. Wood PG, Gillespie JI. Evidence for mitochondrial Ca2+-induced Ca2+ release in permeabilised end endothelial cells. Biochem Biophys Res Commun 1998; 246: 543–548.
Cell Calcium (1999) 25(5), 345–353
Research
Mechanisms of Ca2+ store depletion in single endothelial cells in a Ca2+-free environment J. Paltauf-Doburzynska, M. Frieden, W. F. Graier Department of Medical Biochemistry, Karl-Franzens University of Graz, Graz, Austria
Summary Depletion of agonist-sensitive Ca2+ stores results in activation of capacitative Ca2+ entry (CCE) in endothelial cells. The proportion of Ca2+ stores contributing to the regulation of CCE is unknown. In fura-2/am loaded single endothelial cells freshly isolated from bovine left circumflex coronary arteries, we investigated whether a resting period in a Ca2+-free environment results in emptying of bradykinin-sensitive Ca2+ stores (BsS) and activation of CCE. In a Ca2+-free environment, depletion of BsS occurred in a time-dependent manner (59% after 10 min in Ca2+-free solution). This effect was prevented by inhibition of the Na+–Ca2+ exchange but not by a blockade of ryanodine-sensitive Ca2+ release (RsCR). In contrast to BsS, mitochondrial Ca2+ content remained unchanged in the Ca2+-free environment. Remarkably, activity of CCE (monitored as Mn2+ influx) did not increase after depletion of BsS in the Ca2+-free environment. In contrast to Mn2+ influx, the effect of re-addition of Ca2+ to elevate bulk Ca2+ concentration ([Ca2+]b) decreased with the time the cells rested in Ca2+-free buffer. This decrease was prevented by an inhibition of RsCR. In low Na+ conditions the effect of Ca2+ on [Ca2+]b was reduced while it did not change the time the cells rested in Ca2+-free solution. After a 2 min period in low Na+ conditions, ryanodine-induced Ca2+ extrusion was markedly diminished. Inhibition of RsCR re-established the effect of Ca2+ on [Ca2+]b in low Na+ conditions. Collapsing subplasmalemmal Ca2+ stores with nocodazole, increased the effect of Ca2+ on [Ca2+]b. In nocodazole-treated cells, the effect of Ca2+ on [Ca2+]b was not reduced in Ca2+-free environment. These data indicate that activation of CCE is not associated with the agonist-sensitive Ca2+ pools that deplete rapidly in a Ca2+-free environment. Subplasmalemmal ryanodine-sensitive Ca2+ stores (RsS) are emptied in Ca2+-free /low Na+ solution and re-sequester Ca2+ which enters the cells prior an increase in [Ca2+]b occurs. Thus, in endothelial cells there are differences in the functions of various subplasmalemmal Ca2+ stores (i.e. BsS and RsS), which include either activation of CCE or regulation of subplasmalemmal Ca2+.
INTRODUCTION In endothelial cells the autacoid-induced release of vasoactive compounds, such as prostacycline and nitric oxide (NO), is triggered by an elevation in the cytoplasmic Ca2+ concentration [1,2,3]. The hypothesis of endothelial Ca2+ homeostasis was recently updated by findings of insulated Ca2+ signaling in the cell nucleus [4,5], the mitochondria [6,7] and the subplasmalemmal Received 21 January 1999 Revised 7 May 1999 Accepted 7 May 1999 CCorrespondence to: Professor Dr Wolfgang F. Graier, Department of Medical Biochemistry, Karl-Franzens University of Graz, Harrachgasse 21/III, A-8010 Graz, Austria. Tel.: +43 316 380 7560; fax: +43 316 380 9615; e-mail: [email protected]
area [8]. Nevertheless, owing to technical limitations, most laboratories mainly investigate endothelial Ca2+ signaling using fura-2, which monitors bulk Ca2+ concentration [Ca2+]b. A frequently used protocol is to stimulate the endothelial cells in the nominal absence of extracellular Ca2+, to measure agonist-induced intracellular Ca2+ release, followed by the re-addition of extracellular Ca2+ [9,10] to monitor activity of the so-called capacitative Ca2+ entry (CCE [11]) originally described by Putney [12,13]. Recently, evidence was provided that during re-addition of extracellular Ca2+ to agonist-stimulated endothelial cells, the observed increase in [Ca2+]b does not exclusively reflect CCE, but includes ryanodinesensitive Ca2+ release (RsCR) [14]. RsCR has been previously shown to contribute to agonist-induced elevation in [Ca2+]b [15,16] and localized Ca2+ signaling [8,17] in endothelial cells. In addition, although the activity of 345
346 J Paltauf-Doburzynska, M Frieden, W F Graier
CCE is closely linked to the filling state of internal Ca2+ stores [18], there is evidence that only a certain part of the intracellular Ca2+ stores are involved in regulation of CCE [19]. It has been reported that activation of RsCR by either caffeine [20] or ryanodine [19] facilitates CCE rather than activating it [17]. Thus, it remains unclear, which compartments of the internal Ca2+ stores are responsible for activation of CCE. This study was designed to correlate depletion of endothelial Ca2+ stores with CCE activity. The mechanisms and kinetics of the depletion of agonist-sensitive Ca2+ stores in Ca2+-free environment were examined and the activity of CCE was monitored by measuring Mn2+ influx. In addition, the mechanism of depletion of the ryanodine-sensitive Ca2+ stores during a resting time in a Ca2+-free environment and the role of ryanodine-sensitive Ca2+ stores on the increases in [Ca2+]b in response to the re-addition of extracellular Ca2+ was assessed. MATERIALS AND METHODS Materials Petri dishes were purchased from Corning, Vienna, Austria. Fura-2/AM, fura dextran and ER-Traker BlueWhite DPX was from Molecular Probes, Leiden, The Netherlands. Sera were obtained from PAA laboratories, Linz, Austria. Cell culture media and chemicals were purchased from Life Technologies, Vienna, Austria. All other materials were from Sigma Chemicals, Vienna, Austria. Cells and Cell culture Endothelial cells were freshly isolated from the bovine left circumflex artery as previously described [21]. Briefly, just after the death of the animal, the heart was extracted and the left circumflex artery was isolated and put in chilled storage buffer (SB: DMEM containing 2% horse serum and 0.1% of a vitamin mixture and 0.2% essential amino acids) for transport to the laboratory. Under sterile conditions, vessels were cut longitudinally, fixed with the endothelium on top and incubated for 2 h in SB containing 200 U/ml collagenase (type II) and 1 mg/ml trypsin inhibitor (soybean type I) at 37°C. After cell isolation, cells were kept in SB for fura-2/AM loading. Parts of the collagenase solution were aspirated after 30 min, centrifuged and the cells were cultured in small petri dishes in Opti-MEM containing 2% fetal calf serum. After 10 days, cells were harvested by trypsin (0.02% in phosphate-buffered solution), centrifuged and seeded in a customized glass dish (ø 1 cm) for 1 day in culture medium. Typical cobblestone morphology and the lack of smooth muscle α-actin were tested to control cell culture purity of primary cultures.
Cell Calcium (1999) 25(5), 345–353
The data obtained in endothelial cells freshly isolated from the bovine left circumflex artery were compared with that measured in the endothelial cell line EA.hy 926, which is originally derived from a human umbilical vein [22]. This cell line was a gift from Dr Cora-Jean S. Edgell, Pathology Department, University of North Carolina, Chapel Hill, NC, USA. Cells from the EA.hy 926 cell line were grown in DMEM containing 10% fetal calf serum, 4.5 mg l–1 D-glucose and 1% HAT (5 mM hypoxanthin, 20 µM aminopterin, 0.8 mM thymidine). Measurement of intracellular free Ca2+ concentration For measuring the bulk Ca2+ concentration ([Ca2+]b), conventional fura-2 experiments were performed in single endothelial cells as described previously [8,21]. Briefly, cells were incubated for 45 min at room temperature in the dark with 2 µM fura-2/AM, centrifuged, washed twice and resuspended in SB. After an equilibration period of 10 min, cells were transferred into an experimental chamber which allowed a constant superfusion of 1 ml/min. Cells were perfused for 2 min in Hepes buffer containing in mM: 145 NaCl, 5 KCl, 2.5 Ca2Cl, 1 MgCl2 and 10 Hepes acid, pH adjusted at 7.4 (HBS/Ca), followed with an incubation in nominal Ca2+-free Hepes-buffer (145HBS containing in mM: 145 NaCl, 5 KCl, 1 MgCl2, 10–5 M EGTA and 10 Hepes acid, pH adjusted at 7.4) or in nominal Ca2+-free, low Na+ Hepes buffer (19HBS containing in mM: 19 NaCl, 5 KCl, 1 MgCl2, 126 choline chloride, 10–5 EGTA, 10 Hepes-acid, pH=7.4) as indicated. The [Ca2+]b concentration was monitored by sampling fluorescence intensity at 510 nm emission alternatively at 360 (i.e. Ca2+-insensitive wavelength) and 380 nm excitation (i.e. Ca2+ sensitive wavelength) excitation. Fluorescence intensity for each pair of excitation/emission wavelength was converted to analog by an optical processor [23] and registered by a PC running AxoBASIC® 1.0 (Axon Instruments, Foster City, CA, USA). In view of the reported errors of the [Ca2+]b calibration in our system [8,21] and the general uncertainties of the calibration techniques [15], [Ca2+]b is expressed in ratio units (ratio (F360/F380)). For evaluation of Ca2+ entry activity, Mn2+ quench experiments were performed as previously described [24]. The reduction of fluorescence intensity at F360 was monitored and expressed as % decrease of the initial intensity of F360/min. For measurement of Ca2+ extrusion, cells were resuspended in 145HBS containing 5 µmol l–1 fura dextran potassium salt (MW 70 000) and extrusion of Ca2+ was measured as previously described [17]. Deconvolution microscopy For image analysis of the architectural changes of the endoplasmic reticulum (ER), high-resolution image
© Harcourt Brace & Co. Ltd 1999
Mechanisms of Ca2+ store depletion in single endothelial cells 347
analysis was performed using a deconvolution microscope (Nikon Eclipse TE 300, Nikon, Vienna, Austria) as recently described [8,17]. This set-up provides in combination with a scientific-grade CCD camera (Quantix, Photometrics, Munich, Germany) 3D-images using conventional lamp illumination (150 W XBO; Opti Quip, Albany Turnpike, NY, USA) based on computer algorithms to calculate the out-of-focus light by a 3D point spread function for each microscope objective lens [25,26]. Movements in z-axis were achieved with a z-stage motor controlled by the Ludl-z-stage device (Ludl, Inc., Fairfield Imaging, Tumbridge Wells, UK) controlled by Volume Scan® (VayTek, Inc., Fairfield Imaging, Tumbridge Wells, UK). In combination of a cooled camera head at –40°C and a gain level of 2 this set-up provides excellent sensitivity with high signal-to-noise ratio. Images were collected with a CFI Plan Fluor 40x oil immersion objective (N.A.=1.4; 0.171 µm/pixel, Nikon, Vienna, Austria) and the slice interval was 0.5 µm. Images were sampled using Image Pro 3.0 (Media Cybemetics, Fairfield Imagining, Tumbridge Wells, UK) and out-offocus fluorescence was removed using Micro Tome® (VayTek, Inc., Fairfield Imaging, Tumbridge Wells, UK) using the advanced constrained iterative deconvolution algorithm (5 iterations). Statistics Analysis of variance (ANOVA) was performed and statistical significance of differences was evaluated using the Scheffe’s F test. Level of significance was defined as P<0.05.
RESULTS The time-dependent depletion of IP3-sensitive Ca2+ stores in nominal Ca2+-free solution was assessed by measuring bradykinin-induced intracellular Ca2+ release in the cells which rested 1, 3, 7 and 10 min in 145HBS. As shown in Figure 1A intracellular Ca2+ release to 100 nM bradykinin reduced with the time the cells remained in the Ca2+-free environment prior to stimulation. In contrast to the timedependent depletion of bradykinin-sensitive Ca2+ stores (BsS) in 145HBS, prevention of Na+–Ca2+ exchange (NCX) by reducing extracellular Na2+ concentration ([Na2+]e) to 19 mM (i.e. 19HBS) abolished time-dependent depletion of the BsS (Fig. 1B). There was no difference in bradykinin-evoked intracellular Ca2+ release in single endothelial cells incubated for 1, 3, 7 or 10 min in 19HBS. Identical findings were obtained in EA.hy 926 cells using 10 µM histamine to evoke IP3-mediated intracellular Ca2+ release (data not shown). To compare the capacity of bradykinin-mediated intracellular Ca2+ release after various times in nominal © Harcourt Brace & Co. Ltd 1999
Fig. 1 Time-dependent reduction of the effect of bradykinin on [Ca2+]b by incubation for 1, 3, 7 and 10 min in a Ca2+-free environment. Cells were freshly isolated from the bovine left circumflex coronary artery and loaded with fura-2/AM as described under Methods. At times 0 min, HBS/Ca was replaced by nominal Ca2+-free 145HBS (A) or 19HBS (B). After 1, 3, 7 or 10 in the Ca2+free solution cells were stimulated with 100 nM bradykinin (Bk). Points represent the mean±SEM (panel A: n=28; panel B: n=23). *P<0.05 vs the effect of bradykinin after 1 min in Ca2+-free solution.
Ca2+-free solution under normal and low [Na2+]e conditions, single endothelial cells were incubated in 145HBS and 19HBS for 1, 3, 7 and 10 min, followed by the addition of 100 nM bradykinin in 19HBS to prevent NCX activity (Fig. 2A). Analysis of differences between the bradykinin-induced intracellular Ca2+ release indicate a reduction of IP3-releasable pool by 0±0.4, 11.8±0.6, 29.4±0.9 and 58.8±3.0% after 1, 3, 7 and 10 min in 145HBS compared with cells in 19HBS. The decrease in bradykinin-induced intracellular Ca2+ release by repetitive stimulation in nominal Ca2+-free solution was prevented when the cells were exposed to 2.5 mM Ca2+ containing solution between each single stimulation (Fig. 2B). Inhibition of RsCR by 25 µM ryanodine while the cells were incubated in 145HBS did not preserve the effect of Cell Calcium (1999) 25(5), 345–353
348
J Paltauf-Doburzynska, M Frieden, W F Graier
Fig. 3 Time-dependent reduction of the effect of bradykinin on [Ca2+]b by incubation for 1, 3, 7 and 10 min in 145HBS without (open columns) and with ryanodine (filled columns). Endothelial cells were freshly isolated from the bovine left circumflex coronary artery and loaded with fura-2/AM as described under Methods. At times 0 min, HBS/Ca was replaced by nominal Ca2+-free 145HBS containing solvent control (DMSO) or 25 µM ryanodine. After 1, 3, 7 or 10 in the Ca2+-free environment, cells were stimulated with 100 nM bradykinin (Bk). Columns represent the mean±SEM (n=24). *P<0.05 vs. the effect of bradykinin after 1 min in Ca2+-free solution without ryanodine and #P<0.05 vs. the effect of bradykinin after 1 min in 145HBS containing ryanodine.
Fig. 2 Direct comparison of the time-dependent reduction of the effect of bradykinin on [Ca2+]b by incubation for 1, 3, 7 and 10 min in a Ca2+-free environment (A) and refilling of IP3-sensitive Ca2+ stores by extracellular Ca2+ (B). (A) Fura-2/AM loaded endothelial cells were incubated for 1 min in HBS/Ca, which was replaced at time 1 min by nominal Ca2+-free 145HBS (open circles) or 19HBS (open squares). After 1, 3, 7 or 10 in either 145HBS or 19HBS cells were stimulated with 100 nM bradykinin (Bk) in 19HBS. Tracings are representative single cell recordings and the points represent the mean±SEM (n=18). (B) Incubation of endothelial cells with 2.5 mM extracellular Ca2+ after bradykinin stimulation prevented the decrease in intracellular Ca2+ release in response to repetitive agonist stimulation. As bars indicate endothelial cells were stimulated with either 100 nM bradykinin in the nominal absence of extracellular Ca2+ or were incubated in 2.5 mM Ca2+ containing solution (n=8).
bradykinin over the time the cells were incubated in the Ca2+-free environment (Fig. 3). In contrast to the depletion of BsS in 145HBS, the amount of mitochondrial Ca2+ releasable with 10 µM carbonyl cyanide p-(trilfeuor-methoxy) phenylhydrazone (FCCP) remained constant during incubation in 145HBS for up to 10 min (Fig. 4). Identical findings were obtained when cells were incubated over 1, 3, 7 and 10 min in 19HBS prior addition of FCCP (data not show). To investigate whether CCE activity varies in cells after Cell Calcium (1999) 25(5), 345–353
Fig. 4 Lack of the effect of incubation for 1, 3, 7 and 10 min in 145HBS for FCCP-releasable mitochondrial Ca2+. At time 0 min 145HBS replaced HBS/Ca. After 1, 3, 7 or 10 in the Ca2+-free environment, mitochondrial Ca2+ was released by 10 µFCCP. Tracings are original recordings in single endothelial cells and points represent the mean±SEM (n=12).
incubation for several time periods in 145HBS, the rate of Mn2+ entry was assessed in cells after 1, 3, 7 and 10 min in the Ca2+-free 145HBS. As shown in Figure 5A, the resting period in nominal Ca2+-free solution did not alter Mn2+ entry monitored. The observed Mn2+ quench was significantly smaller than that measured in cells after © Harcourt Brace & Co. Ltd 1999
Mechanisms of Ca2+ store depletion in single endothelial cells 349
Fig. 6 Time-dependent reduction of the effect of CaE on [Ca2+]b by incubation for 1, 3, 7 and 10 min in 145HBS (A) or 19HBS (B) without (open columns) and with ryanodine (filled columns). Endothelial cells were loaded with fura-2/AM as described under Methods. At times 0 min, HBS/Ca was replaced by nominal Ca2+free 145HBS (A) or 19 HBS (B) containing solvent control (DMSO) or 25 µM ryanodine. After 1, 3, 7 or 10, 2.5 mM Ca2+ was added. Columns represent the mean±SEM (A: n=8–11; B: n=12). *P<0.05 vs. the effect of CaE after 1 min in 145HBS/19HBS without ryanodine and #P<0.05 vs the effect of CaE in 145HBS without ryanodine.
Fig. 5 CCE activity measured with Mn2+ (A) and the effect of a readdition of Ca2+ (CaE) (B) after incubation for 1, 3, 7 and 10 min in nominal Ca2+-free solution. (A and B) At times 0 min, 145HBS replaced HBS/Ca. At time points 1, 3, 7 or 10 150 µM Mn2+ (panel A) or 2.5 mM Ca2+ (panel B) were added. The continuous lines in panel A indicate the average Mn2+ quench induced by addition of 150 µM Mn2+ while the dotted lines indicate the bradykinin induced Mn2+ quench (time 1 and 7 min). Tracings are representative recordings from single cells and the points represent the mean±SEM (A: n=14; B: n=36). *P<0.05 vs the effect of CaE after 1 min in Ca2+-free solution. (C) At times 0 min, HBS/Ca was replaced by 19HBS followed by the re-addition of 2.5 mM Ca2+ at time points 1, 3, 7 or 10. Tracings are representative recordings from single cells and the points represent the mean±SEM (n=26).
© Harcourt Brace & Co. Ltd 1999
stimulation with 100 nM bradykinin (7.3±2.2% of the initial fluorescence at 360 nm excitation; n=4; P<0.05 vs non-stimulated cells; data not shown). In contrast to the Mn2+ entry, in single endothelial cells resting for 1, 3, 7 and 10 min in 145HBS, increases in [Ca2+]b to the addition of 2.5 mM extracellular Ca2+ (CaE) became reduced with the time the cells rested in the Ca2+-free environment (Fig. 5B). In cells prestimulated with 100 nM bradykinin, [Ca2+]b increased by 0.48 ratio F360/F380 units (n=23; P<0.05 vs non-stimulated cells; data not shown). To investigate the contribution of NCX to the observed reduction of CaE-induced elevation of [Ca2+]b, single endothelial cells were incubated in 19HBS for various time periods, followed by the addition of 2.5 mM Ca2+ (Fig. 5C). In cells resting for 1, 3, 7 and 10 min in 19HBS, no time-dependent reduction of the increases of [Ca2+]b in response to CaE was found (Fig. 5C). However, the effect of CaE was reduced in 19HBS and did not differ from the effect of CaE in cells preincubated in 145HBS for 10 min. Cell Calcium (1999) 25(5), 345–353
350 J Paltauf-Doburzynska, M Frieden, W F Graier
Fig. 7 Effect of a 2 min exposure to 19HBS on the ryanodineinduced Ca2+ extrusion from endothelial cells in 145HBS measured by fura dextran potassium salt [14]. Suspended endothelial cells were pretreated for 2 min in 145HBS (Control; open circles, n=5) or 19HBS (Pretreated with 19 [Na+]e; open squares, n=5), centrifuged and resuspended in 145HBS containing 5 µM fura dextran potassium salt. After 2 min, cells were stimulated with 200 nM ryanodine and extrusion of Ca2+ was monitored by measuring the Ca2+-sensitive wavelength of fura dextran. *P<0.05 vs. control.
Fig. 8 Relative distribution of the ER in endothelial cells without and with nocodazole treatment. Endothelial cells were isolated from the bovine left circumflex coronary artery, cultured in primary culture for 10 days and transferred in a sterile glass-bottom chamber for further 24 h and were incubated in DMEM containing solvent control (0.1% DMSO) or 10 µM nocodazole at 37°C. After 16 h cells were washed three times and incubated for 40 min in DMEM containing 2 µM ER-Traker Blue-White DPX (Molecular Probes, Leiden, The Netherlands) in the dark. The ER-network of the endothelial cell was monitored in middle depth. Haze was removed using the advanced constrained iterative algorithm (5 iterations; MicroTome, Vaytek, Tumbridge Wells, UK). Results were expressed as the ratio of the average fluorescence intensities of two, 2 µm thick, circles beneath the cell membrane (i.e. subplasmalemmal fluorescence; lsub.) and around the cell nucleus (perinuclear fluorescence; (lperi.) region. *P<0.05 vs. solvent control.
Inhibition of RsCR by 25 µM ryanodine while incubation in 145HBS abolished time-dependent reduction of the effect of CaE (Fig. 6A). Inhibition of RsCR by ryanodine during the incubation in 19HBS re-established the effect of CaE while no time-dependent reduction of the Cell Calcium (1999) 25(5), 345–353
Fig. 9 Time-dependent reduction of the effect of CaE on [Ca2+]b by incubation for 1, 3, 7 and 10 min in 145HBS in control cells (open columns) and in cells with nocodazole-collapsed ER (filled columns). Cultured endothelial cells were incubated for 16 h in DMEM containing 0.1% DMSO (solvent control) or 10 µM nocodazole in a sterile chamber with glass bottom. After washing the cells endothelial cells were loaded with fura-2/AM. At times 0 min, HBS/Ca was replaced by 145HBS and at times 1, 3, 7 or 10 min, 2.5 mM Ca2+ was added. Columns represent the mean±SEM (n=12). *P<0.05 vs the effect of CaE after 1 min in 145HBS and #P<0.05 vs the effect of CaE in control cells.
effect of CaE was observed (Fig. 6B). In agreement with these findings, a preincubation of endothelial cells in 19HBS blunted ryanodine-induced (200 nM) Ca2+ extrusion in 145HBS (Fig. 7). To investigate whether the architectural organization of the superficial ER contributes to the observed reduction of the effect of CaE in Ca2+-free buffer, superficial ER was collapsed by a 16 h incubation with 10 µM nocodazole as recently described [8]. However, the lack of specificity of the ER-marker used (BODIPY FL-X ryanodine) made a re-evaluation of the effect of nocodazole necessary. Therefore, to monitor selectively ER domains, the high selective ER marker, ER-Traker Blue-White DPX [27] was used. As shown in Figure 8, treatment with nocodazole collapsed the ER as indicated by the decrease in the ratio of subplasmalemmal (lsub.) and perinuclear (lperi.) fluorescence. The treatment with nocodazole increased elevation of [Ca2+]b in response to CaE by 55%, while no time-dependent reduction of the effect of CaE was found in 145HBS (Fig. 9). DISCUSSION In Ca2+-free solution, the bradykinin-sensitive Ca2+ stores (BsS) became depleted with the time the cells rested in the Ca2+-free buffer (i.e. 145HBS). The emptying of BsS was prevented when NCX activity was inhibited in low © Harcourt Brace & Co. Ltd 1999
Mechanisms of Ca2+ store depletion in single endothelial cells 351
Fig. 10 Schematic view on the proposed depletion of bradykinin(BsS) and ryanodine-sensitive Ca2+ stores (RsS) in endothelial cells in nominal Ca2+-free solution and the distribution of Ca2+ entering the cells. Upper panel: Under normal conditions (i.e. 145 mM [Na+]e; active NCX), the Ca2+ pools are depleted by leak and/or basal activity of either the IP3 or the ryanodine receptor and the Ca2+ is extruded by the NCX. By the addition of extracellular Ca2+, Ca2+ leaks into the cell and is sequestered into both Ca2+ stores prior elevating [Ca2+]b. Thus, the effect of Ca2+ entering the cell depends on the rate of Ca2+ sequestration into subplasmalemmal Ca2+ stores. Lower panel: At low extracellular [Na+] (19 mM; NCX activity is inhibited) in nominal Ca2+-free solution, Ca2+ released from the BsS and the RsS cannot be extruded and, thus, in turn, initiates activation of Ca2+-induced Ca2+ release, resulting in a pronounced depletion of the RsS. If extracellular Ca2+ is added, Ca2+ leaks into the cell and gets sequested from subplasmalemmal Ca2+ pools prior elevating [Ca2+]b. Since the RsS is more depleted it ‘buffers’ the Ca2+ influx, resulting in a reduced elevation of [Ca2+]b to the addition of extracellular Ca2+.
© Harcourt Brace & Co. Ltd 1999
Na+ solution (i.e. 19HBS) or by incubation with Ca2+containing medium between the repetitive stimulation. In contrast, inhibition of ryanodine receptors failed to reduce the time-dependent depletion of the BsS in 145HBS. These data suggest that NCX extrudes the Ca2+ that leaks out of the BsS, while RsCR is not involved in the depletion of BsS under these conditions (Fig. 10). The comparison of the effect of bradykinin in cells pretreated in 145HBS or 19HBS (Fig. 2A) allows estimating the amount of depletion of the BsS in Ca2+-free solution. Interestingly, while there was a time-dependent depletion of the BsS in 145HBS that reached 58% after 10 min, no activation of CCE monitored using Mn2+ flux technique could be found. These data suggest that the depletion of certain BsS in Ca2+-free environment does not initiate CCE activation. This is in contrast to the report of Hofer et al. who described that the activity of CCE is closely linked to the filling state of internal Ca2+ stores [18]. Since inhibition of NCX completely abolished the emptying of BsS under the Ca2+-free environment, one might speculate that subplasmalemmal BsS which are coupled with NCX, as described in smooth muscle cells [28], became depleted, while the Ca2+ content of the BsS responsible for activation of CCE remains constant. These findings suggest that the BsS responsible for CCE activation constitutes less than 40% of the whole BsS. This is consistent with the findings of Sasajima et al. [19] who reported that only a small compartment of the agonist-sensitive ER is involved in the activation of CCE. Similar to the time-dependent reduction of bradykinininduced elevation in [Ca2+]b in Ca2+-free environment, the effect of a re-addition of 2.5 mM Ca2+ (CaE) became reduced in a time-dependent manner. The mechanisms of CaE on [Ca2+]b might involve leak Ca2+ entry, CCE and RsCR [14]. Since the Mn2+ entry remained constant over time, it seems unlikely that a reduced Ca2+ entry is responsible for the reduction of the elevation in [Ca2+]b in response to CaE. On the other hand, inhibition of RsCR did not attenuate the effect of CaE, suggesting that RsCR is not involved in the effect of CaE under these experimental conditions. However, our findings that inhibition of RsCR prevented the time-dependent reduction of the effect of CaE, might indicate that RsCR contributes to the observed attenuation of the effect of CaE (Fig. 10). Considering the constant influx of Ca2+ as indicated in our Mn2+ flux experiments, one might suggest that in the Ca2+-free environment subplasmalemmal ryanodine-sensitive Ca2+ stores (RsS) become depleted via RsCR. This might result in a rapid sequestration of the Ca2+ entering the cells into depleted subplasmalemmal Ca2+ pools. Such ‘Ca2+ steal’ by the subplasmalemmal Ca2+ stores would attenuate elevation of [Ca2+]b in response to CaE. This hypothesis is supported by our findings that an inhibition of NCX by 19HBS prevented the time-dependent Cell Calcium (1999) 25(5), 345–353
352 J Paltauf-Doburzynska, M Frieden, W F Graier
reduction of the effect of CaE, while the effect of CaE was markedly reduced. Since inhibition of RsCR re-established the effect of CaE even in 19HBS while no timedependent reduction in the effect of CaE was observed, we suggest that in 19HBS depletion of subplasmalemmal Ca2+ stores via RsCR is pronounced (Fig. 10). This hypothesis is supported by our findings that ryanodineinduced Ca2+ extrusion in 145HBS was strongly reduced in cells that were pre-incubated in 19HBS for just 2 min. Our findings that in the Ca2+-free environment subplasmalemmal Ca2+ stores sensitive to bradykinin (BsS) or ryanodine (RsS) are depleted by different pathways (NCX and RsCR respectively) suggest that, at least in the subplasmalemmal area, BsS and RsS are two distinct Ca2+ stores (Fig. 10). This is in agreement with our previous work in porcine coronary endothelial cells, where we suggested that BsS and Ca2+ stores sensitive to caffeine, a well-known activator of ryanodine receptors are two different compartments of the ER [20]. Recently, the involvement of subplasmalemmal mitochondria in the localized Ca2+ signaling [7,8] and the existence of a functioning Ca2+-induced Ca2+ release mechanisms on the mitochondrial membrane [29] have been described. Thus, it is possible that the increases in [Ca2+]b in response to CaE is due, at leat in part, to Ca2+induced Ca2+ release from the mitochondria. Although we cannot rule out this possibility, our findings that the effect of FCCP [7] remained constant during the time in Ca2+-free solution may indicate that a depletion of the mitochondrial Ca2+ pool is not responsible for the reduction of the effect of CaE in Ca2+-free environment. Our hypothesis that the time-dependent reduction in the elevation in [Ca2+]b to CaE is due to RsCR-mediated depletion of subplasmalemmal Ca2+ stores, which, in turn, re-sequester Ca2+ during CaE, and thus an attenuated increase in [Ca2+]b is observed, is further supported by our experiments with nocodazole. We have previously reported that nocodazole treatment disrupts the subplasmalemmal Ca2+ control unit (SCCU [8]) by remodeling the ER network as shown using BODIPY FL-X ryanodine [8]. Unfortunately, this staining is not selectively for the ER. Therefore, in the present work ER network was exclusively stained using ER-Traker Blue-White DPX [27] and the nocodazole-induced changes in the architectural organization of the cell was observed. Consistent with our hypothesis, the effect of CaE was increased in nocodazole-treated cells, while no time-dependent reduction in the Ca2+-free environment could be observed. These data suggest that removal of subplasmalemmal Ca2+ stores results in the lack of a sequestration of the Ca2+ entering the cell by subplasmalemmal Ca2+ stores. In conclusion, our findings provide new information on the functional organization of endothelial Ca2+ stores and their contribution to CCE activation and subcellular Cell Calcium (1999) 25(5), 345–353
Ca2+ distribution. Thus, in addition to our previous findings that the SCCU controls localized Ca2+ signaling [8,17] and spatial activation of constitutive nitric oxide synthase [17], new evidence are provided herein that the SCCU contributes to the distribution of Ca2+ entering the cell from the extracellular space. ACKNOWLEDGEMENTS We thank Mrs Beatrix Petschar for her excellent technical assistance. This work was supported by the Austrian Science Funds (P-12341-Med and SFB 714), the Austrian Nationalbank (P7542) and the Franz Lanyar Foundation. REFERENCES 1. Busse R, Mülsch A, Flemming I, Hecker M. Mechanisms of nitric oxide release from the vascular endothelium. Circulation 1993; 87 (suppl V): V18–V25. 2. Graier WF, Sturek M, Kukovetz WR. Ca2+ regulation and endothelial vascular function. Endothelium 1994;1: 223–236. 3. van Breemen C, Skarsgard P, Laher I, McMannus B, Wang X. (1997) Endothelium-smooth muscle interactions in blood vessels. Clin Exp Pharmacol Physiol 1997; 24: 989–992. 4. Burnier M, Centeno G, Burki E, Brunner HR. Confocal microscopy to analyze cytosolic and nuclear calcium in cultured vascular cells. Am J Physiol 1994; 266: C1118–C1127. 5. Ikeda M, Ariyoshi H, Kambayashi J, Shinoki N, Sakon M, Kawasaki T, Monden M. Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells. J Cell Biochem 1996; 63: 23–36. 6. Donnadieu E, bourguignon LYW. Ca2+ signaling in endothelial cells stimulated by bradykinin: Ca2+ measurement in the mitochondria and the cytosol by confocal microscopy. Cell Calcium 1996; 20: 53–61. 7. Lawrie AM, Rizzuto R, Pozzan T, Simpson AW. A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells. J Biol Chem 1996; 271: 10753–10759. 8. Graier WF, Paltauf-Doburzynska J, Hill B, Fleischhacker E, Hoebel BG, Kostner GM, Sturek M. Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane. J Physiol Lond 1998; 506: 109–125. 9. Schilling WP, Ritchie AK, Navarro LT, Eskin SG. Bradykininstimulated calcium influx in cultured bovine aortic endothelial cells. Am J Physiol 1998; 255: H219–H227. 10. Graier WF, Schmidt K, Kukovetz WR. Bradykinin-induced Ca2+-influx into cultured endothelial cells is not regulated by inositol 1,4,5-trisphosphate or inositol 1,3,4,5tetrakisphosphate. Second Messengers and Phosphoproteins 1991; 187–197. 11. Schilling WP, Cabello OA, Rajan L. Depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store in vascular endothelial cells activates the agonist-sensitive Ca2+-influx pathway. Biochem J 1992; 284: 521–530. 12. Putney JW. Capacitative calcium entry revisited. Cell Calcium 1990; 11: 611–624. 13. Putney JW. A model for receptor-regulated calcium-entry. Cell Calcium 1986; 7: 1–12. 14. Mozhayeva MG. [Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-indused Ca2+ release can involve both IP3-, and ryanodine-sensitive Ca2+ release. Pflügers Arch 1996; 433: 180–187.
© Harcourt Brace & Co. Ltd 1999
Mechanisms of Ca2+ store depletion in single endothelial cells 353
15. Wang X, Lau F, Li L, Yoshikawa A, VanBreemen C. Acetylcholine-sensitive intracellular Ca2+ store in fresh endothelial cells and evidence for ryanodine receptors. Circ. Res 1995; 77: 37–42. 16. Mozhayeva MG, Mozhayeva GN. Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+. Pflügers Arch. -Eur J Physiol 1996; 432: 614–622. 17. Paltauf-Doburzynska J, Posch K, Paltauf G, Graier WF. Stealth ryanodine-sensitive Ca2+ release contributes to activity of capacitative Ca2+ entry and nitric oxide synthase in bovine endothelial cells. J Physiol Lond 1998; 513: 369–379. 18. Hofer AM, Fasolato C, Pozzan T. Capacitative Ca2+ entry is closely linked to the filling state of internal Ca2+ stores: a study using simultaneous measurements of ICRAC and intraluminal [Ca2+]i. J Cell Biol 1998; 140: 325–334. 19. Sasajima H, Wang X, van Breemen C. Fractional Ca2+ release from the endoplasmic reticulum activates Ca2+ entry in freshly isolated rabbit aortic endothelial cells. Biochem Biophys Res Commun 1997; 241: 471–475. 20. Graier WF, Simecek S, Bowles DK, Sturek M. Heterogeneity of caffeine and bradykinin-sensitive stores in vascular endothelial cells. Biochem J 1994; 300: 637–641. 21. Graier WF, Simecek S, Sturek M. Cytochrome P450 monooxygenase-regulated signaling of endothelial Ca2+ entry. J Physiol London 1995; 482: 259–274. 22. Edgell CJS, McDonald CC, Graham JB. Permanent cell line expressing factor VIII related antigen established by hybridization. Proc Natl Acad Sci USA 1983; 80: 3734–3737.
© Harcourt Brace & Co. Ltd 1999
23. Sturek M, Smith P, Stehno-Bittle L. In vitro models of vascular endothelial cell calcium regulation. In: Sperelakis N., Kuriyama H. (Eds). Ion Channels of Vascular Smooth Muscle Cells and Endothelial Cells New York: Elsevier, 1991; 349–364. 24. Paltauf-Dobruzynska J, Graier WF. Temperature dependence of agonist-stimulated Ca2+ signaling in cultured endothelial cells. Cell Calcium 1997; 21: 43–51. 25. Carrington WA, Lynch RM, Moore EDW, Isenberg G, Fogarty KE, Fay FS. Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. Science 1995; 268: 1483–1487. 26. Waters D, Brown C. New high-resolution 3-D microscope – Avoids damage to live samples. Biophotonics International 1996; 9/10, 40–44. 27. Diwu Z, Lu Y, Zhang C, Klaubert DH, Haugland RP. Fluorescent Molecular Probes II. The Synthesis; Spectral Properties and Use of Fluorescent Solvatochromic Dayoxyl Dyes. Photochem Photobiol 1997; 66: 424–431. 28. Moore EDW, Etter EF, Philipson KD, Carrington WA, Fogarty KE, Lifshitz LM, Fay FS. Coupling of the Na+/Ca2+ exchanger, Na+/K+ pump and sarcoplasmic reticulum in smooth muscle. Nature 1993; 365: 657–660. 29. Wood PG, Gillespie JI. Evidence for mitochondrial Ca2+-induced Ca2+ release in permeabilised end endothelial cells. Biochem Biophys Res Commun 1998; 246: 543–548.
Cell Calcium (1999) 25(5), 345–353