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Cardiovascular Research 2007 73(1):164-171; doi:10.1016/j.cardiores.2006.09.015
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Copyright © 2006, European Society of Cardiology

Reoxygenation-induced Ca2+ rise is mediated via Ca2+ influx and Ca2+ release from the endoplasmic reticulum in cardiac endothelial cells

Saskia C. Peters and H. Michael Piper*

Institute of Physiology, Justus-Liebig University Giessen, Aulweg 129, 35392 Giessen, Germany

* Corresponding author. Tel.: +49 641 99 47241; fax: +49 641 99 47239. Email address: michael.piper{at}physiology.med.uni-giessen.de

Received 3 April 2006; revised 5 September 2006; accepted 22 September 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Conditions of ischemia-reperfusion disturb the homoeostasis of cytosolic Ca2+ in cardiac microvascular endothelial cells (CMEC), leading to numerous malfunctions of the endothelium. Reperfusion specifically aggravates the Ca2+ overload developed during sustained ischemia. The aim of this study was to identify the origin of the reperfusion-induced part of the Ca2+ overload. Our hypotheses were that this is either due to a Na+-dependent process, e.g. involving the Na+/H+ exchanger (NHE) and/or the Na+/Ca2+ exchanger (NCX), or a process involving the endoplasmic reticulum (ER) and store-operated channels (SOC).

Methods and results: Cultured CMEC from rats were exposed to conditions of simulated ischemia (hypoxia, pH 6.4) and reperfusion (reoxygenation, pH 7.4). Cytosolic Ca2+ ([Ca2+]i) and cytosolic Na+ ([Na+]i) concentrations and cytosolic pH (pHi) were measured with the use of fluorescent indicators. Removal of Ca2+ from the extracellular media during reoxygenation prevented the [Ca2+]i rise. Neither the activation of the NHE nor of the NCX in reoxygenated CMEC caused a change in this [Ca2+]i rise. Complete or partial removal of Na+ from the external media also had no effect on the [Ca2+]i rise. In contrast, specific inhibition of the inositol trisphosphate (InsP3) receptor by xestospongin C (3 µmol/l), of phospholipase (PLC) by U73122 [GenBank] (1 µmol/l), or of SOC by the inhibitors gadolinium (10 µmol/l) or 2-APB (50 µmol/l) lowered or abolished the reoxygenation-induced [Ca2+]i rise.

Conclusion: In CMEC exposed to reperfusion conditions, the enhanced Ca2+ overload is due to Ca2+ influx. The influx is not mediated by a Na+-dependent mechanism, but rather is due to activation of the InsP3 receptor of the ER and activation of SOC.

KEYWORDS Calcium (cellular); Reoxygenation; Na/Ca-exchanger; Na/H-exchanger; SR (function)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Endothelial cells (EC) form a selective barrier between the blood and the interstitial space. In the heart, ischemia-reperfusion causes impairment of the physiological barrier function of coronary endothelium and, subsequently, causes edema and malfunction of the organ [1]. Post-ischemic malfunction of the endothelium is partly due to the damage that occurs during ischemia, partly due to interactions with activated leukocytes during reperfusion, but also partly due to reperfusion-induced changes at the level of the EC themselves. Previous results from our group showed that reperfusion conditions induce a disturbance of the endothelial barrier function due to an activation of the endothelial contractile machinery [2]. The latter results from the reenergization of EC in a state of cytosolic Ca2+ overload. Ca2+ overload itself is further aggravated during reperfusion. Since Ca2+ overload is harmful to the cells and causes many malfunctions, the present study was undertaken (i) to analyse if the aggravation of Ca2+ overload itself is a phenomenon of ‘reperfusion injury’, i. e. brought about by specific causes under reperfusion conditions, and (ii) to identify the cation transporter mechanisms involved in Ca2+ overload under reperfusion conditions.

The mechanisms leading to the abnormal elevation of intracellular [Ca2+] ([Ca2+]i) in reperfused endothelium of the coronary system or other vascular regions are only poorly understood. In contrast, we have considerable knowledge of the causes of the loss of [Ca2+]i control in the predominant cell type of the heart, i.e. the cardiomyocyte. It is well known for this cardiac cell type that Na+-dependent processes of Ca2+ influx are activated during reperfusion. This involves the Na+/H+ exchanger (NHE), which carries Na+ into the cells when intracellular pH (pHi) is normalized during reperfusion, and the reverse mode of the Na+/Ca2+ exchanger (NCX), which carries Ca2+ into the cells when cytosolic [Na+] is high and the plasmalemma is depolarized [3–5]. Since EC also express these plasmalemmal exchanger proteins [6–8], one may hypothesize that Na+-dependent mechanisms may be responsible for Ca2+ overload in reperfused EC.

A second potential mechanism for Ca2+ influx in reperfused EC consists of an activation of store-operated cation channels in the plasmalemma, triggered by the emptying of the endoplasmic reticulum [9]. In EC, store-operated channels (SOC) play an important role in Ca2+ signalling. This mechanism is therefore another likely cause of reperfusion-induced [Ca2+]i rise in EC. In order to investigate the mechanism causing the additional [Ca2+]i increase during reperfusion, we used a culture model of cardiac microvascular endothelial cells (CMEC) exposed to hypoxia and reoxygenation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Cell culture
CMEC were isolated from 3-month-old male Wistar rats (250–300 g). The animals were sedated with 100% CO2 inhalation (<40 s) and humanely killed via cervical dislocation in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The heart was immediately removed, and CMEC were isolated as previously described [10]. Briefly, the heart was dipped 30 s in 100% ice-cold ethanol in order to damage the epicardial cell surface. Then the heart was perfused at 37 °C for 40 min with basic perfusion medium (containing in mmol/l: 110.0 NaCl, 2.6 KCl, 1.2 KHPO4, 1.2 MgSO4, 25.0 NaHCO3, 1.3 CaCl2, 11 glucose and 25.0 HEPES) with collagenase (500 mg/l, Worthington class II, Biochrom, Serva, Germany). Atria and large vessels were removed andthe tissue was chopped with a razor blade to a size of 0.3x0.3-mm pieces. Then, the tissue was incubated for additional 10 min in basic perfusion medium containing collagenase and was centrifuged at 30 xg for 3 min. The supernatant containing EC was incubated in trypsin (250 mg/l) for 5 min at 37 °C and then an equal volume of Medium 199 was added. This solution was then centrifuged for 8 min at 260 xg and the pellet was resuspended in 20 ml Medium 199 and transferred to four 100-mm Petri dishes (PrimariaTM) with 10 ml Medium 199 and 20% fetal/neonatal calf serum (1:1). After 1 h unattached cells were removed by washing the dishes vigorously. Five to six days later the primary culture formed a confluent monolayer. As determined by DiI-ac-LDL (Paesel and Lorei, Duisburg, Germany) uptake the purity of these cultures was > 95% [10]. CMEC in primary culture were trypsinised and seeded on coverslips. Experiments were performed with confluent monolayers, 2–3 days after seeding.

2.2. Media
CMEC seeded on coverslips were mounted in a perfusion chamber (1 ml volume) and superfused at a flow rate of 0.5 ml/min through gas-tight steel capillaries with a modified Tyrode solution (containing in mmol/l 140.0 NaCl, 2.6 KCl, 1.2 KHPO4, 1.2 MgSO4, 1.3 CaCl2, 2.5 glucose and 25.0 HEPES). pH was adjusted at 30 °C either to 7.4 for normoxic or to 6.4 for hypoxic solutions with NaOH. In media with different pH the extracellular Na+ concentration ([Na+]o) for hypoxic and normoxic medium was equally adjusted to 140 mM. For Na+-free (0 mmol/l) experiments Na+ was substituted equimolarly with the impermeant organic cation N-methyl-D-glucamine (NMDG), and for low [Na+]o (19 mmol/l) about 90% of Na+ was replaced in this manner; pH was adjusted with HCl. Medium was made hypoxic by autoclaving as described previously [11]. pO2 in the hypoxic medium was less than 22 µPa as determined with resazurin [11]. The hypoxic medium was without glucose or serum and was equilibrated with 100% N2 during the experiments. The Na+-free medium contained (mmol/l): 145.0 NMDG, 2.6 KCl, 1.2 KHPO4, 1.2 MgSO4, 1.3 CaCl2, 2.5 glucose and 25.0 HEPES. The low Na+ medium contained (mmol/l): 126.0 NMDG, 19.0 NaCl, 2.6 KCl, 1.2 KHPO4, 1.2 MgSO4, 1.3 CaCl2, 2.5 glucose and 25.0 HEPES. For Ca2+-free experiments Ca2+-free solution with addition of 0.5 mmol/l EGTA was used.

2.3. Measurement of intracellular Ca2+, Na+ and pH and experimental protocol
In order to record the cytosolic Ca2+ ([Ca2+]i) or H+ (pHi) concentration, CMEC were loaded in the modified Tyrode solution with addition of 2% fetal/newborn calf serum (1:1) at 30 °C for 60 min with acetoxymethyl ester of fura-2 (2.5 µmol/l) or 2',7'-bis(2-carboxyethyl)-5,6-carboxy-fluorescein (BCECF) (1 µmol/l). To measure the [Na+]i time course, CMEC were loaded for 45 min with the sodium-binding benzofuran isophthalate (SBFI, 10 µmol/l) and 0.2% Pluronic F127. After loading of the CMEC with fura-2 they were incubated for a further 15–30 min without dyes. Calibration of the BCECF ratio was performed according to Koop and Piper with 10 µg/ml nigericin, a K+/H+ ionophore, and incubation at various pH values [12].

The chamber was mounted on an inverted microscope (IX 70, Olympus, Hamburg, Germany) equipped with a video-imaging-system and a monochromator (Polychrom IV, TILL Photonics GmBH, Graefelfing; Germany). Ratio measurements were performed at 340 and 380 nm for fura-2 and SBFI or 450 and 490 nm for BCECF at a cycle frequency 0.05–0.15 Hz. Emitted light was detected at 490–510 nm for fura-2 and SBFI or 520–560 nm for BCECF. All images were corrected for background. Fura-2 fluorescence was calibrated according to the methods described by Grynkiewicz et al. [13]. For this purpose, cells were exposed to 5 µM ionomycin in modified HEPES buffer containing 3 mM Ca2+ or 5 mM EGTA to obtain the maximum (Rmax) and minimum (Rmin) of the ratio of fluorescence (R), respectively. [Ca2+]i was calculated according to the equation



Formula

with use of the dissociation constant (Kd) of fura-2 determined in intact cells, as previously described [2]. β is the ratio of the 380 nm excitation signal of ionomycin-treated cells at 5 mM EGTA and at 3 mM Ca2+.

Under standard conditions CMEC were exposed to acidic hypoxia at pH 6.4 for 40 min followed by 40 min reoxygenation in normoxic medium with 2.5 mmol/l glucose at pH 7.4 as described previously [2]. All other substances or solutions were supplied during reoxygenation: cariporide (10 µmol/l), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA, 1 µmol/l), N-methyl-isobutyl-amiloride (MIA, 1 µmol/l), U73122 [GenBank] (1 µmol/l), U73433 [GenBank] (1 µmol/l), ouabain (20 µmol/l), xestospongin C (3 µmol/l), gadolinium chloride (GdCl3, 10 µmol/l), 2-aminoethoxydiphenyl borate (2-APB, 50 µmol/l), lanthanum (La3+, 2 mmol/l), and EGTA (0.5 mmol/l); for Na+-free or low Na+ experiments CMEC were superfused with the corresponding modified Tyrode solution. For Na+-free or low Na+ experiments during hypoxia, CMEC were superfused 10–15 min before the onset of hypoxia.

2.4. Materials
Medium 199 was purchased from Gibco; newborn and fetal calf serum from PAA; acetoxymethyl ester of fura-2, BCECF, SBFI were from Molecular Probes; nigericin, GdCl3, thapsigargin were from Sigma; 2-APB was from Tocris; EIPA, MIA, U73122 [GenBank] , U73433 [GenBank] were from Calbiochem; xestospongin C was from Cayman; Cariporide was a gift from Sanofi–Aventis, Frankfurt, Germany. All other chemicals were from Merck or Sigma and of the highest purity available.

2.5. Statistics
Data were shown as mean values±SE. For each experimental procedure 10 to 25 individual regions of the endothelial monolayer were used from each of at least 4 independent cell preparations. The comparison of means between the groups was performed by one-way analysis of variance (ANOVA) followed by a Bonferroni post-hoc test. Changes in parameters within the same group were assessed by multiple ANOVA analysis and a statistical significance was accepted when p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Importance of Na+ and pHi for Ca2+ overload
Under conditions of simulated ischemia, [Ca2+]i of EC rose in a biphasic manner. An initial rapid increase of the fura-2 ratio was followed by a slow, progressive rise (Fig. 1). The normoxic ratio corresponds to [Ca2+]i 56±2 nM, and the end-anoxic ratio corresponds to [Ca2+]i 106±3 nM. Readdition of oxygen and glucose, i.e. simulation of reperfusion, caused an additional increase of the fura-2 signal (Fig. 1). The ratio after 40 min of reoxygenation corresponds to [Ca2+]i 148±5 nM. This increase in [Ca2+]i was significantly beyond the level observed under continued ischemia. Thus, reoxygenation aggravates the cytosolic Ca2+ overload. In order to identify the source of the additional [Ca2+]i rise during reoxygenation, we performed experiments in Ca2+-free solutions. Under these conditions, the additional increase during reoxygenation was abolished, indicating that the reoxygenation-induced increase is due to Ca2+ influx from the extracellular space (Fig. 1).


Figure 1
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  		Fig. 1 Change of fura-2 ratio during hypoxia (black bar, 40 min) and subsequent reoxygenation. After hypoxia in [Ca2+] containing medium, cells were either left under continued hypoxia in [Ca2+] containing medium (-{diamond}-), reoxygenated with normal [Ca2+] medium (-•-) or reoxygenated in Ca2+-free medium (-{circ}-). Data represent means±S.E.M. of 6 separate experiments with independent cell preparations. *p<0.05 vs. Ca2+-free conditions, #p<0.05 vs. continuous hypoxia.

 
The question was raised if [Na+]i- and pHi-dependent changes are responsible for the reoxygenation-induced Ca2+-overload. As shown in Fig. 2A, pHi decreased during 40 min hypoxia from 7.41±0.06 to 6.41±0.05 and recovered to the control level during the first 20 min of reoxygenation. As indicated by the change in SBFI fluorescence (Fig. 2B), [Na+]i was significantly elevated after 40 min hypoxia. It also returned to the control level within 20 min of reoxygenation.


Figure 2
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  		Fig. 2 Effect of hypoxia (black bar, 40 min) and subsequent reoxygenation (40 min) on pH (A, C) and SBFI fluorescence ratio, indicating the Na+ content in CMEC (B, D). In panels C and D, cariporide (10 µmol/l) was added during reoxygenation while pH and SBFI ratio was monitored. Data represent means±S.E.M. of 4 separate experiments with independent cell preparations. *p<0.05 vs. normoxic conditions, #p<0.05 vs. end-hypoxia.

 
To investigate a potential role of the NHE, which extrudes protons in exchange for Na+, experiments were performed in nominally HCO3-free, HEPES-buffered solution. Under these conditions, application of 10 µmol/l cariporide, an inhibitor of the NHE, blocked the recovery of pHi during reoxygenation (Fig. 2C), indicating the presence of an active NHE in these cells. There was no effect on the [Na+]i recovery (Fig. 2D) nor on the [Ca2+]i overload during reoxygenation (Fig. 3). Other NHE inhibitors like EIPA (1 µmol/l) and MIA (1 µmol/l) had the same effect (data not shown). This shows that NHE, albeit active in reoxygenated endothelial cells, is not involved in the causal mechanism of reoxygenation-induced [Ca2+]i rise. If ouabain (20 µmol/l) was present during reoxygenation, the recovery of [Na+]i was prevented, indicating that the [Na+]i recovery is driven by an active Na+/K+ ATPase (Fig. 3A). Even in the presence of ouabain, [Ca2+]i overload was not significantly higher than compared to control conditions (Fig. 3B).


Figure 3
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  		Fig. 3 Effect of hypoxia (Hypox, after 40 min) and reoxygenation (Reox, after 40 min) on SBFI ratio (A) and fura-2 ratio (B), indicating the Na+ and Ca2+ content, respectively. The NHE inhibitor cariporide (10 µmol/l) or the Na+/K+ ATPase inhibitor ouabain (20 µmol/l) was applied during reoxygenation. Data show means±S.E.M. of 5–7 separate experiments with independent cell preparations. *p<0.05 vs. end-hypoxia.

 
To identify whether Na+-dependent processes are at all involved in reoxygenation-induced [Ca2+]i overload at large, we performed experiments where [Na+]o was removed. NaCl was substituted by NMDG. One set of the experiments was performed in nominally Na+-free media, the other in media with reduced [Na+]o (19 mmol/l), to reduce the activity of the NCX close to its reversal potential. Initially, we tested under normoxic control conditions if the NCX can be activated by removal of Na+o in CMEC (Fig. 4A). Na+o removal, which activates the reverse mode of the NCX, led to a transient peak of [Ca2+]i followed by sustained [Ca2+]i elevation at a lower level. Readdition of Na+o, which activates the NCX forward mode, brought [Ca2+]i back to baseline. These results show that CMEC possess an active NCX. The decline in [Ca2+]i after the transient peak was blocked and the [Ca2+]i remained elevated (Fig. 4B) when La3+ (2 mmol/l), which inhibits the plasmalemmal Ca2+ ATPase (PMCA), was added 5 min before Na+o removal. This shows that the transient Ca2+ rise caused by abrupt removal of Na+o activates the PMCA.


Figure 4
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  		Fig. 4 (A) Change of fura-2 ratio during Na+ removal (0 Na) and readdition of Na+ (+ Na) under normoxic conditions in order to activate the NCX. Data show means±S.E.M. of 6 separate experiments with independent cell preparations. (B) Change of fura-2 ratio during Na+ removal (0 Na) under normoxic conditions and simultaneous inhibition of the PMCA by La3+ (+ La, 2 mmol/l) applied 5 min before Na+ removal. Data show means±S.E.M. of 6 separate experiments with independent cell preparations. (C) Effect of Na+ removal (+ Na, -•-) on fura-2 ratio at the beginning of reoxygenation and simultaneous administration of La3+ (+ La, -{circ}-) during reoxygenation compared to control conditions (-{square}-). Data show means±S.E.M. of 6 separate experiments with independent cell preparations.

 
When the abrupt removal of Na+o was performed upon reoxygenation, a transient rise of [Ca2+]i also occurred, pointing to activation of the NCX (Fig. 4C). The subsequent rapid return of [Ca2+]i to the level of Ca2+ overload seen under normal reperfusion conditions also seems to be due to a rapid and transient activation of the PMCA, since inhibition of the PMCA (2 mmol/l La3+) during reperfusion blocked the decline in [Ca2+]i after the initial rise and led to a markedly enhanced [Ca2+]i overload. These results indicate that the PMCA can be activated during reoxygenation, when Ca2+ influx is rapidly stimulated as by reverse mode activation of the NCX. Interestingly, inhibition of the PMCA alone during reperfusion (2 mmol/l La3+) had no effect on the slowly progressive Ca2+ overload compared to normal reperfusion conditions (fura ratio at 20 min: 1.38±0.02, with La3+ vs. 1.38±0.03 without; ns.). This indicates that the PMCA is not very active during the slowly progressive Ca2+ overload.

Removing or reducing the Na+o in the incubation media before the start of hypoxia and during reoxygenation caused no difference in the reoxygenation-induced [Ca2+]i rise (Fig.5). Removal or reduction of [Na+]o solely during reoxygenation caused no difference in the Ca2+i overload compared to the results with containing normal Na+o. To sum up, these experiments show that Na+-dependent processes are not responsible for the reoxygenation-induced [Ca2+]i rise.


Figure 5
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  		Fig. 5 Effect of different [Na+] concentrations applied at the beginning of hypoxia (Hypox) or at the beginning of reoxygenation (Hypox/Reox) on the fura-2 ratio during 40 min of reoxygenation in order to activate the reverse mode of the NCX (Na 0, 0 mmol/l Na+) or to inactivate the NCX (Na 19, 19 mmol/l Na+). *p<0.05 vs. hypoxia. Data show means±S.E.M. of 5 separate experiments with independent cell preparations.

 
3.2. Importance of the endoplasmic reticulum for Ca2+ overload
In the second part of the experimental program, the hypothesis was tested that there is a release of Ca2+ from the ER and activation of SOC plays a role for reoxygenation-induced [Ca2+]i overload. First, a functional role of the InsP3 receptor Ca2+ release mechanism was investigated. Experiments were performed in the presence of xestospongin C (XeC, 3 µmol/l), a specific inhibitor of the InsP3 receptor [28]. XeC blocked the reoxygenation-induced [Ca2+]i rise (Fig. 6). This effect could be mimicked by application of the phospholipase C inhibitor, U73122 [GenBank] (1 µmol/l). In contrast, the inactive analogue U73433 [GenBank] had no effect compared to the control. Second, a functional role in Ca2+ influx by SOC was investigated with the use of the SOC inhibitor gadolinium and 2-APB, another putative SOC inhibitor. To determine the concentration dependency for gadolinium, EC were first treated with thapsigargin (1 µmol/l) in Ca2+-free solution, which empties the ER and activates SOC. Readdition of Ca2+ then provokes a SOC-driven Ca2+ influx (Fig. 7). This Ca2+ influx could be fully inhibited by Gd3+ at 10 µmol/l. 2-APB is also widely used as a SOC inhibitor, but depending on concentration and cell type it can also act as InsP3 inhibitor and might inhibit SERCA [14]. Therefore, we determined the SOC specificity of 2-APB in an experiment with thapsigargin, the application of which triggers initially a passive emptying of the ER via the IP3 receptor followed by a sustained Ca2+ influx into the cell via SOC. 2-APB was applied at various concentrations for 5 min in the presence of Ca2+ prior to application of thapsigargin (1 µmol/l). At 50 µmol/l 2-APB the initial thapsigargin-induced ER Ca2+ release was unaffected, but the second phase of Ca2+ elevation due to SOC-mediated Ca2+ influx was blocked. At 100 µmol/l 2-APB the ER release was also reduced. Application of 2-APB caused no change in the basal level, indicating that it has not a thapsigargin-like effect on the ER. As shown in Fig. 8, 10 µmol/l Gd3+ and 50 µmol/l 2-APB abolished the additional [Ca2+]i increase during reoxygenation, thus imitating the absence of [Ca2+]o. Combined removal of extracellular Ca2+ to abolish Ca2+ influx, and emptying of the ER passively by inhibiting SERCA via thapsigargin, brought [Ca2+]i back to almost baseline conditions after 40 min (Fura-2 ratio: 1.08±0.03, p<0.05 vs. control, n=98 cells).


Figure 6
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  		Fig. 6 Effect of InsP3 receptor inhibitor xestospongin C (XeC, 3 µmol/l) and the PLC inhibitor U73122 (1 µmol/l) and its inactive analogue U73433 (1 µmol/l) on the fura-2 ratio after 40 min of reoxygenation (Reox). Dotted line represents end-hypoxic value. *p<0.05 vs. hypoxia, #p<0.05 vs. U73433. Data show means±S.E.M. of 5 separate experiments with independent cell preparations.

 

Figure 7
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  		Fig. 7 (A) Effect of various concentrations of gadolinium (Gd) on the thapsigargin-induced Ca2+ entry. EC were subjected to Ca2+-free conditions and 1 µmol/l thapsigargin was applied for 10 min to empty the ER and to activate SOC. [Ca2+]i was monitored via fura-2 ratio. Five min before readdition of 1.3 mmol/l Ca2+, GdCl was applied at different concentrations (Gd 0=0 µmol/l, Gd 0.5=0.5 µmol/l, Gd 1.0=1 µmol/l, Gd 10.0=10 µmol/l). Traces show means±S.E.M. of at least 4 different experiments. (B) Effect of various concentrations of 2-APB on ER Ca2+ release and SOC-mediated Ca2+ influx in a thapsigargin challenge assay. 2-APB (0 µmol/l, 10 µmol/l, 50 µmol/l, 100 µmol/l) was applied 5 min before thapsigargin (THG, 1 µM). Traces show means±S.E.M. of at least 4 different experiments.

 

Figure 8
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  		Fig. 8 Effect of (GdCl3, 10 µmol/l), 2-APB (50 µmol/l) and Ca2+-free solution on the fura-2 ratio after 40 min of reoxygenation (Reox). Dotted line represents end-hypoxic value. *p<0.05 vs. Reox. Data show means±S.E.M. with 5 separate experiments of independent cell preparations.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In the present study, we analysed the causes for the enhanced [Ca2+]i overload occurring in CMEC upon reoxygenation. We analysed both Na+-dependent plasmalemmal transport processes and the processes depending on the ER and store-operated channels. The main finding is that Na+-dependent processes do not play a significant role, but ER and SOC do, by a mechanism triggered by reoxygenation of the cells.

The rise of [Ca2+]i is due to an influx of Ca2+ across the plasmalemma. Several studies on whole ischemic/reperfused hearts have shown that inhibitors of the NCX or NHE improve the organ function or prevent endothelial dysfunction after an ischemic insult [15–17]. Cardioprotective effects have also been shown in several studies on whole-perfused hearts by inhibition of the reverse NCX [18–20]. On isolated cardiomyocytes exposed to hypoxia and reoxygenation, it has been shown that the transplasmalemmal [H+]-gradient activates the NHE as a proton extruder during reoxygenation. The ensuing [Na+]i accumulation activates in turn the reverse mode of the plasmalemma NCX, leading to a reoxygenation-induced [Ca2+]i elevation [21].

It has remained unclear if inhibition of NHE or NCX is beneficial directly at the level of the reoxygenated endothelial cells. In this study, we first investigated if CMEC possess functionally the elements for this tandem mechanism that may cause reoxygenation-induced Ca2+ overload, i.e. an active NHE and NCX. We found that activation of the NHE during reoxygenation does not provoke Ca2+ overload, obviously because the Na+/K+ ATPase can cope with the excessive Na+ influx. We also found that forcing the reverse mode activation of the NCX in reoxygenated CMEC does not cause a sustained additional rise of [Ca2+], since PMCA can cope with a sudden influx of Ca2+. But in spite of the compensatory potential of PMCA for a rapid Ca2+ influx, the [Ca2+]i rose gradually under conditions of reoxygenation in CMEC. Although Ca2+ removal suppressed the additional Ca2+ rise, the Ca2+ remained elevated during the recording time of 40 min under reperfusion conditions. One possible explanation is that the PMCA is not significantly activated because the Ca2+ rise occurs slowly and an initial trigger of quickly elevated cytosolic Ca2+ is needed to fully activate the PMCA, as shown by other studies [29].

The results with NHE and NCX are further corroborated by the general finding that removal of external Na+ does not alter the net rise in [Ca2+]i in reoxygenated CMEC. To exclude possible side effects by the use of 0 mmol/l Na+, we also performed experiments at low [Na+]o concentrations to bring the NCX near its reversal potential [22,23]. Under either condition of Na+ removal, the [Ca2+]i increase during reoxygenation was unchanged compared to control. In conclusion, therefore, Na+-dependent causes for the Ca2+ overload in reperfusion may be excluded.

Like other non-excitable cells, EC possess a Ca2+ influx pathway that can be activated by emptying the ER, i.e. channels located in the plasma membrane called ‘store-operated channels’ [24,25]. These channels can be blocked by micromolar concentrations of the trivalent cation Gd3+ [24] or by 2-APB [27]. We found that the [Ca2+] rise during reoxygenation could be blocked by either SOC inhibitor. These results suggested that emptying of the ER under conditions of reoxygenation is the trigger event for the observed Ca2+ influx. To elucidate the role of the ER in mediating the Ca2+ entry in CMEC during reoxygenation, we blocked the InsP3 receptor, the main Ca2+ release mechanism of the ER in EC, by the specific inhibitor xestospongin C (XeC) [28]. XeC prevented the reoxygenation-induced [Ca2+] rise. This result was supported by the fact that U73122 [GenBank] , the cell-permeable inhibitor of phospholipase C that catalyses the generation of InsP3, reduced the [Ca2+]i rise in a manner similar to that of the InsP3 receptor inhibitor. Unspecific side effects of U73122 [GenBank] were excluded by comparing the results with those obtained with the use of its inactive analogue, U73433 [GenBank] .

We found that even in the presence of elevated Ca2+, Ca2+ still enters the EC under pathophysiological conditions of reperfusion via the ER-SOC pathway. Although it has been known for the best characterized mechanism among SOC, Icrac, that high cytosolic Ca2+ can exert a negative feedback on the SOC itself, there is increasing experimental evidence that other SOC mechanisms apart from Icrac have different sensitivities to Ca2+ [30,31]. Our data show that SOC are open to some extent even at elevated Ca2+ and this opens a new window to further study SOC under reperfusion conditions.

This study was initiated to answer two questions. One was whether the enhanced Ca2+ overload under reoxygenation conditions is due to causes brought about by these conditions themselves. The answer is yes, since it does not occur under continued hypoxic conditions and can be prevented in reoxygenation by various interactions interfering with the ER-SOC axis. It is left to speculation what could be the initial triggering cause. A likely suggestion would be reactive oxygen species activating PLC. The other question was about the nature of the ion transporters involved.

This has been answered by identifying the ER-SOC mechanism. These results have consequences for the current understanding of cardiac reperfusion injury. It is now generally accepted that the early minutes of reperfusion represent a window of opportunity for treatment of patients with acute coronary occlusion. To date, efforts for new therapeutic strategies are all directed toward the myocardial cells or to leukocyte–vascular interactions. It is now clear that the mechanism of the endothelium's own reperfusion injury also needs to be studied. Both in endothelial and myocardial cells, Ca2+ overload is a decisive element of the pathophysiology of reperfusion. But, as the present study shows, the specific causes and therefore the therapeutic options for these two cell types are different. In cardiomyocytes the "tandem mechanism" of NHE and NCX contributes to reperfusion-induced Ca2+ overload, whereas in EC it is the ER-SOC mechanism. Therefore, strategies to reduce cellular Ca2+ overload in the different constituent cells of the heart should obviously not be based on a single therapeutic principle.


   Acknowledgements
 
We would like to thank Guenther Schade and Otto Becker for manufacturing excellent technical equipment. The technical help of A. Reis and D. Schreiber is gratefully acknowledged.


   Notes
 
* Hiroshi Watanabe of Hamamatsu University School of Medicine (Hamamatsu, Japan) served as Guest Editor for this article.

Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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