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Am J Physiol Heart Circ Physiol 279: H2310-H2318, 2000;
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Vol. 279, Issue 5, H2310-H2318, November 2000

Hypoxia-induced alterations in Ca2+ mobilization in brain microvascular endothelial cells

Chiwaka Kimura, Masahiro Oike, and Yushi Ito

Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca2+ concentration ([Ca2+]i) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca2+ oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation for 45 min, the basal level of [Ca2+]i was increased from 32.4 to 63.3 nM, and ATP did not induce Ca2+ oscillations. Hypoxia/reoxygenation also inhibited capacitative Ca2+ entry (CCE), which was evoked by thapsigargin (Delta [Ca2+]i-CCE: control, 62.3 ± 3.1 nM; hypoxia/reoxygenation, 17.0 ± 1.8 nM). The impairments of Ca2+ oscillations and CCE, but not basal [Ca2+]i, were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O2-)-sensitive luciferin derivative MCLA, we confirmed that the production of O2- was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/reoxygenation generates O2- at mitochondria and impairs some Ca2+ mobilizing properties in BBEC.

superoxide anion; adenosine 5'-triphosphate; capacitative calcium entry; mitochondria


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN CEREBRAL MICROCIRCULATION, endothelial cells are supposed to control blood flow by influencing the tonus of smooth muscles of precapillary arterioles (12, 19). Another important role of cerebral microvascular endothelial cells is to control the transportation of intravascular materials to the brain through the blood-brain barrier (5). To accomplish each of these endothelial functions, fine control of the intracellular Ca2+ concentration ([Ca2+]i) is essentially important (7, 26, 32), so both cerebral microcirculation and permeation are under the influence of factors that modulate [Ca2+]i in microvascular endothelial cells.

Ischemic cerebrovascular events induce various complications including brain edema (20, 31), which is at least partially caused by the increase in capillary permeability (14, 20). Therefore, considering the important roles of Ca2+ in the regulation of capillary permeability and microcirculation (1, 7), it would be significantly important to investigate the ischemia-induced alterations of Ca2+ mobilization in cerebral microvascular endothelium.

Interruption of blood flow due to ischemia would reduce the supply of nutrients, especially glucose and oxygen, to the tissues, therefore these changes may be responsible for the ischemia-induced alterations of microcirculation. However, we have previously clarified that the reduction of glucose concentration to one-tenth of the normal concentration does not affect the resting [Ca2+]i and the histamine-induced Ca2+ transient at least up to 2 h in bovine brain microvascular endothelial cells (BBEC) (18). We speculated that the abundant presence of mitochondria in cerebral endothelium (15) might provide tolerance for the reduced glucose concentration to this tissue (18). In contrast, it has been reported that the intracellular ATP concentration is reduced by hypoxia in the endothelium because of the suppression of aerobic cellular respiration (2). Sarco(endo) plasmic Ca2+-ATPase (SERCA) plays an essential role in maintaining intracellular Ca2+ sequestration (33), and the transport of Ca2+ by SERCA requires the hydrolysis of ATP (8). Therefore, the overall energy state of the cell influences [Ca2+]i, and the reduction of the intracellular ATP concentration in an hypoxic environment would alter [Ca2+]i in the endothelium. For instance, it has been reported in the BBEC (18) and coronary endothelium (23) that the inhibition of mitochondrial ATP production with CN- increased [Ca2+]i due to the reduction of stored Ca2+.

To further clarify the detailed cellular mechanisms of hypoxia-induced and the subsequent reoxygenation-induced alterations of Ca2+ mobilization in vitro, we have investigated the effects of hypoxia/reoxygenation on ATP- and thapsigargin-induced Ca2+ mobilization in BBEC. Obtained results show the first evidence that the hypoxia/reoxygenation-induced generation of the superoxide anion (O2-) impairs some Ca2+-mobilizing properties in BBEC.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The brains of 1-yr-old calves were obtained from the local slaughterhouse. Microvascular endothelial cells from the brain gray matter were then prepared by a Percoll gradient separation method (4). Collected endothelial cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Rockville, MD) supplemented with 10% fetal calf serum as previously described (18). To avoid the alkalization of the medium during hypoxic challenge, the pH of the culture medium was adjusted to 7.40 with both bicarbonate and 10 mM HEPES-NaOH. Cells of the second subculture were used for the present experiments. Cells were grown on coverslips and a 96-well culture plate for [Ca2+]i and O2- measurements, respectively. Identification of the endothelial cells was confirmed by the specific uptake of acetylated low-density lipoprotein (21).

Measurement of [Ca2+]i . [Ca2+]i was measured by using an Attofluor digital fluorescence microscopy system (Atto Instruments, Rockville, MD). Cells were loaded with 2 µM of the acetoxymethyl ester form of fura 2 (fura 2-AM, Dojindo, Kumamoto, Japan). The coverslip with fura 2-loaded cells was placed in a 0.5-ml volume chamber and mounted on an inverted microscope (Axiovert 135, Carl Zeiss, Jena, Germany). Fura 2 was excited alternatively at two wavelengths (340 nm and 380 nm), and the fura 2 fluorescence images were recorded on a rewritable optical disc recorder (LQ-4100A, Panasonic, Osaka, Japan) at a rate of ~1 Hz. Fluorescence images of each cell were converted to a fluorescence ratio and to an apparent Ca2+ concentration using a measured dissociation constant (Kd) value of 138.6 nM as previously described (25).

All experiments were performed at room temperature (20-25°C).

Exposure of the cells to hypoxic environment. An hypoxic environment was obtained by using a commercial kit (Oxygen Absorbing System; ISO, Yokohama, Japan), consisting of an oxygen absorber (A-500HS, ISO), an oxygen indicator tablet (K-500M, Tokiwa Industries, Sagamihara, Japan), and a sealing bag made of K-nylon and polyethylene. Cells grown on a coverslip in a 35-mm culture dish or a 96-well culture plate were put into the sealing bag together with an oxygen absorber and an oxygen indicator. The oxygen absorber (A-500HS), the main components of which are iron powder and NaCl, reduces oxygen concentration of 100 ml of normal air to <0.1% within 3 h, and the effect persists for more than 48 h (data provided by ISO, personal communication). According to the manufacturer's description, the color of the oxygen indicator (K-500M) changes from violet to pink when the oxygen concentration becomes <0.1%. We normally started the incubation with ~100 ml of 5% CO2-95% air in the sealing bag, and it took ~2 h for the air in the bag to reach 0.1% hypoxia, judging by the color of the oxygen indicator. Therefore, to expose the cells for 6 h to hypoxia, we incubated the cells for 8 h in the hypoxic bag.

The oxygen absorber used in the present study also absorbs CO2, and 5% CO2 will be reduced to about 2% after 8 h (data provided by ISO, personal communication). Therefore, because the air volume of the flexible sealing bag was decreased in proportion to the absorption of oxygen and CO2, the concentrations of N2 and CO2 inside the bag were supposed to be 98 and 2% after 8 h, respectively. Because the culture medium was buffered with HEPES, reduction of CO2 concentration did not alter pH (control, 7.40 ± 0.03; after the incubation with the oxygen absorber, 7.42 ± 0.02).

It was necessary to slightly open the bag to load the cells with fura 2, and this procedure immediately turned the color of the oxygen indicator into violet, suggesting that the air in the bag was reoxygenated. Therefore, it should be noted that it was technically difficult to examine the effects of "hypoxia" alone and that the treatment was inevitably "6 h of hypoxia and the following reoxygenation (hypoxia/reoxygenation)" in the present experiment. The "reoxygenation period", i.e., time needed until starting the Ca2+ measurement, was 45 min on average.

Measurement of O2- generation. We measured the accumulation of O2- in the extracellular space by using an O2--sensitive luciferin derivative 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA; Tokyo Kasei Kogyo, Tokyo, Japan) (22). Cells were cultured on a 96-well culture plate, and the plate was exposed to the hypoxic environment for 6 h. Immediately or after being exposed to normal air for 20, 45, or 90 min, the culture medium was then replaced with 50 µl of 1 µM MCLA-containing Krebs solution. The plate was then immediately put into a dark box and the emitted photon was counted for 10 min by a luminescence detection system (Argus-50/2D luminometer; Hamamatsu Photonics, Hamamatsu, Japan). Obtained data were analyzed with Argus-50 software (Hamamatsu Photonics). Because it is difficult to convert the MCLA chemiluminescence into the absolute amount of O2-, we expressed the amount of O2- by the corresponding concentration of xanthine oxidase, which reacts with 100 µM xanthine and generates O2- in a concentration-dependent manner (Fig. 1).


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  		Fig. 1.   Concentration-photon counting relationships between xanthine oxidase and O2--induced MCLA chemiluminescence. Indicated concentration of xanthine oxidase was added to xanthine solution (100 µM), and the emitted photon was counted for 10 min.

Materials. Modified Krebs solution was used as the standard extracellular solution, containing (in mM) 132 NaCl, 5.9 KCl, 1.2 MgCl2, 1.5 CaCl2, 11.5 glucose, and 11.5 HEPES; pH was adjusted to 7.3 with NaOH. Ca2+-free solution was made by replacing CaCl2 with 1 mM EGTA. The bath was perfused continuously with these solutions at a rate of 1.5 ml/min.

ATP (Sigma, St. Louis, MO) and thapsigargin (Sigma) were used to release Ca2+ from the intracellular Ca2+ storage sites. SOD, rotenone, and thenoyltrifluoroacetone (TTFA) were also purchased from Sigma.

Data analysis. Data are given as means ± SE. Statistical significance between control and hypoxia/reoxygenation-treated cells was determined using Student's unpaired t-test for resting [Ca2+]i and thapsigargin-induced Ca2+ leak and and by Mann-Whitney's U-test for the frequency of Ca2+ oscillation and the amplitude of capacitative Ca2+ entry, which showed deviated distribution.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of hypoxia/reoxygenation on the basal level of [Ca2+]i. First, we observed the effects of hypoxia/reoxygenation on the basal level of [Ca2+]i. In control cells, which were kept in normal oxygen concentration throughout the experiment, the basal value of [Ca2+]i was 32.4 ± 1.7 nM (n = 285). In contrast, when cells were incubated in a hypoxic environment (<0.1%) for 6 h and followed by reoxygenation for 45 min, the value was significantly elevated to 63.3 ± 1.6 nM (n = 208, P < 0.01). Samples were randomly selected for each condition.

Effects of hypoxia/reoxygenation on ATP-induced Ca2+ oscillations. ATP is known to be released from the endothelium as a physiological mediator in response to mechanical stress (3, 25). We therefore examined the effects of ATP on control and hypoxia/reoxygenation-treated cells. In control cells, ATP induced Ca2+ transient with a threshold concentration of 0.3 µM (Fig. 2C). At higher concentrations of ATP, Ca2+ oscillations were observed as shown in Fig. 2A. Ca2+ oscillations were not observed without ATP (not shown), and the frequency of Ca2+ oscillations was increased in a concentration-dependent manner at least up to 30 µM (Fig. 2C). Furthermore, ATP-induced Ca2+ transient was abolished by phospholipase C inhibitors (neomycin and U-73122) and P2 antagonist (suramin), suggesting that ATP-induced Ca2+ transient was generated by P2 receptor-mediated D-myo-inositol (1,4,5)-trisphosphate (IP3) production (not shown). In contrast, when cells were exposed to hypoxia for 6 h and to the subsequent reoxygenation for 45 min, the threshold of ATP-induced Ca2+ transient was shifted to a higher concentration (1 µM). Furthermore, a higher concentration of ATP (10 µM) induced a single Ca2+ transient (Fig. 2B) and did not evoke Ca2+ oscillations up to 30 µM (Fig. 2C).


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  		Fig. 2.   Effects of hypoxia/reoxygenation on ATP-induced Ca2+ oscillations. A: in control cells, application of 10 µM ATP induced Ca2+ oscillations in normal Krebs solution. A representative trace from a single cell is shown. [Ca2+]i, intracellular Ca2+ concentration. B: when cells were treated with hypoxia for 6 h and the following reoxygenation for 45 min, Ca2+ oscillations were not induced by 10 µM ATP. A representative trace is shown. C: concentration-response relationships of the frequency of ATP-induced Ca2+ oscillations. In control cells (open circles), the frequency of Ca2+ oscillations were increased in a concentration-dependent manner. In hypoxia/reoxygenation-treated cells (closed circles), almost no Ca2+ oscillations were observed at any concentrations of ATP. Symbols are means ± SE values of the cells indicated in the parentheses.

Because Ca2+ oscillations are generated as a balance of Ca2+ release from the intracellular storage sites, Ca2+ extrusion, and Ca2+ entry from the extracellular space (28), we then examined the effects of hypoxia/reoxygenation on the amount of stored Ca2+ and Ca2+ entry in BBEC.

Effects of hypoxia/reoxygenation on thapsigargin-induced Ca2+ mobilizations. In control cells, 1 µM thapsigargin, a specific inhibitor of SERCA (30), induced a transient [Ca2+]i increase in a Ca2+-free solution due to a Ca2+ leak from the intracellular Ca2+ storage sites (Fig. 3A). Intracellular Ca2+ storage sites consist of leaky membrane and therefore Ca2+ is continuously leaking out of the Ca2+ stores (6). Because the activity of SERCA maintains the sequestration of intracellular Ca2+, thapsigargin induces Ca2+ leak from the storage sites (30). The following application of normal Krebs solution induced a further increase in [Ca2+]i (Fig. 3A) due to capacitative Ca2+ entry (CCE) (24). The hypoxia/reoxygenation-treated BBEC also showed a thapsigargin-induced initial Ca2+ transient in the Ca2+-free solution (Fig. 3B). However, the net maximal increase in the initial Ca2+ transient (Delta [Ca2+]i-peak), which reflects the total amount of stored Ca2+, was lower in hypoxia/reoxygenation-treated cells than in control cells (Fig. 3C). Furthermore, Ca2+ reapplication-induced [Ca2+]i increase (Delta [Ca2+]i-CCE) was also much lower in hypoxia/reoxygenation-treated cells than that in control cells (Fig. 3D).


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  		Fig. 3.   Thapsigargin-induced Ca2+ leak and capacitative Ca2+ entry (CCE). A: control cells showed gradual Ca2+ leak from the intracellular storage sites by thapsigargin (1 µM) in Ca2+-free solution. The following perfusion of Ca2+-containing solution induced further increase in [Ca2+]i due to CCE. B: in hypoxia/reoxygenation-treated cells, initial Ca2+ transient was observed by the application of thapsigargin. However, reapplication of extracellular Ca2+ induced little increase in [Ca2+]i. Note that the amplitude of the initial Ca2+ transient is much smaller than control cell. C: net increment of the initial Ca2+ transient (Delta [Ca2+]i-peak) was reduced in hypoxia/reoxygenation-treated cells. Numbers in parentheses indicate the number of cells examined. **P < 0.01, compared with control. Values were measured as indicated in A. D: Ca2+ reapplication-induced [Ca2+]i increase (Delta [Ca2+]i-CCE) was also reduced in hypoxia/reoxygenation-treated cells. Numbers in parentheses indicate the number of cells examined. **P < 0.01, compared with control. Values were measured as indicated in A.

Effects of SOD on the hypoxia/reoxygenation-induced impairment of Ca2+ mobilization. We have previously revealed in bovine aortic endothelial cells (BAEC) that O2- abolishes ATP-induced Ca2+ oscillations partially due to the impairment of CCE (17). It has also been reported that hypoxia/reoxygenation produces O2- in pulmonary artery endothelial cells (29). Therefore the results indicated above suggest the possible involvement of O2- in hypoxia/reoxygenation-induced impairments of Ca2+ mobilization. Therefore, we then examined the effects of SOD, a scavenger of O2-, on Ca2+ mobilizations in hypoxia/reoxygenation-treated BBEC.

When 150 U/ml of SOD was present in the culture medium during the hypoxic and the subsequent fura 2-loading periods, ATP (1 µM) induced Ca2+ oscillations as in the case of control cells (Figs. 4A and 5B). Furthermore, impairments of thapsigargin-induced Ca2+ responses in hypoxia/reoxygenation-treated cells were partially restored by SOD (Fig. 4B). Namely, though the initial Ca2+ transient was not restored by SOD (Fig. 5C), CCE was reversed to the control level (Fig. 5D). However, the basal [Ca2+]i was still significantly higher in hypoxia/reoxygenation/SOD-treated cells than in that in control cells (Fig. 5A).


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  		Fig. 4.   Effects of superoxide dismutase (SOD) on hypoxia/reoxygenation-induced impairments of Ca2+ mobilizations. A: when 150 U/ml of SOD was present during the incubation period, ATP (1 µM) induced Ca2+ oscillations. B: Ca2+ reapplication-induced [Ca2+]i increase was also restored in hypoxia/reoxygenation/SOD-treated cells.



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  		Fig. 5.   Statistical analysis of the resting [Ca2+]i (A), frequency of Ca2+ oscillations (B), thapsigargin-induced initial [Ca2+]i peak (C), and CCE (D). For the analysis of the resting [Ca2+]i (A), values were either taken from Ca2+-free solution (open bars) or Krebs solution (shaded bars). Values were taken from the cells treated with or without SOD or rotenone/TTFA. Rotenone/TTFA was present in the culture medium either after hypoxic period (reoxygenation) or throughout the preincubation periods (throughout). **P < 0.01, *P < 0.05, compared with control cells. Dagger Dagger P < 0.01, between two conditions. Numbers in the parentheses indicate the number of the cells analyzed.

In contrast, the addition of SOD for the same period did not affect any of these Ca2+-mobilizing properties in control cells (Fig. 5). Therefore, these effects of SOD on hypoxia/reoxygenation-treated cells were not due to its direct effects on Ca2+-mobilizing pathways.

Involvement of mitochondrial electron transport system in hypoxia/reoxygenation-induced generation of O2-. It has been reported that mitochondrial electron transport, which is a possible candidate for the site of O2- production, is accelerated by reoxygenation after hypoxic challenge (10, 11). Therefore, we then examined the effects of the inhibitors of mitochondrial electron transport, rotenone (10 µM), and TTFA (30 µM), on hypoxia/reoxygenation-induced alterations in Ca2+ mobilization.

Some reports have suggested that O2- is generated in the mitochondria during the reoxygenation period (11), therefore, we added these two agents to the culture medium either only during the reoxygenation period (i.e., fura 2-loading step) or throughout the hypoxic and reoxygenation periods. As shown in Fig. 6A, left, when rotenone and TTFA were present only during the reoxygenation period, low concentrations of ATP (1 µM) induced Ca2+ oscillations. Its frequency was significantly larger than hypoxia/reoxygenation alone but not restored completely (Fig. 5B). In contrast, when rotenone and TTFA were present throughout the pretreatment period with hypoxia/reoxygenation, the frequency of 1 µM ATP-induced Ca2+ oscillations was completely restored to the control level as shown in Figs. 5B and 6B.


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  		Fig. 6.   Effects of the inhibitors of mitochondrial electron transport rotenone and thenoyltrifluoroacetone (TTFA) on hypoxia/reoxygenation-induced alterations in Ca2+ mobilization. A: when rotenone (10 µM) and TTFA (30 µM) were added to the culture medium only during the reoxygenation period, i.e., starting after 6 h of exposure to hypoxia, both ATP (1 µM)-induced Ca2+ oscillations (left) and CCE (right) were restored. B: when these agents were present throughout the preincubation periods, i.e., hypoxia and the following reoxygenation periods, ATP (1 µM) induced Ca2+ oscillations of higher frequency than in A (left). Furthermore, CCE after thapsigargin (1 µM)-induced store depletion was also restored completely.

Furthermore, addition of rotenone and TTFA during the reoxygenation period partially restored CCE (Figs. 5D and 6A). This was also completely restored when rotenone and TTFA were present throughout the incubation period (Figs. 5D and 6B).

However, as in the case of SOD-treated cells, basal [Ca2+]i and thapsigargin-induced Delta [Ca2+]i-peak were not restored by the pretreatment with rotenone and TTFA (Fig. 5, A and C). We also observed that rotenone and TTFA, added for 45 min, did not affect Ca2+-mobilizing properties in control cells (Fig. 5).

Hypoxia/reoxygenation-induced production of O2-. To confirm that O2- is generated by hypoxia/reoxygenation in the BBEC as a result of mitochondrial electron transport, we measured O2- by using MCLA, an O2--sensitive chemiluminescence. In control cells, accumulation of O2- in the extracellular space in 10 min was equivalent to that produced by 0.028 ± 0.002 mU/ml of xanthine oxidase (n = 10). In contrast, when cells were exposed to hypoxia for 6 h and then reoxygenation for 45 min, a significantly larger amount of O2- was released into the extracellular space (equivalent to 0.090 ± 0.002 mU/ml of xanthine oxidase, n = 10, P < 0.01 compared with control, Fig. 7A). As expected, generated O2- was completely scavenged by SOD (below the detection limit, n = 10, Fig. 7A), thereby indicating that MCLA chemiluminescence was closely related to O2-. Because MCLA detects the released O2- in real time, we examined the effects of rotenone and TTFA on MCLA chemiluminescence by adding them during the reoxygenation period. These agents prevented the excess production of O2- by hypoxia/reoxygenation (equivalent to 0.026 ± 0.003 mU/ml of xanthine oxidase, n = 10, P > 0.05 compared with control, Fig. 7A). Therefore, the hypoxia/reoxygenation environment generates O2- indeed, and the increased generation of O2- by hypoxia/reoxygenation can be attributed to the mitochondrial electron transportation probably during the reoxygenation period.


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  		Fig. 7.   Measurement of the generation of O2- in hypoxia-treated cells by using MCLA. O2- released from bovine brain endothelial cells (BBEC) was counted for 10 min and converted into corresponding xanthine oxidase concentration using the standard curve shown in Fig. 1. A: cells were exposed to normal air for 45 min before measurement. Rotenone/TTFA was added to the culture well only during reoxygenation period. **P < 0.01, compared with control cells, which were not exposed to hypoxia/reoxygenation. B: time course of reoxygenation-induced O2- generation. Cells were exposed to hypoxia for 6 h and reoxygenated for the indicated period.

We then examined the time course of reoxygenation-induced O2- generation. After the reoxygenation period of 0, 20, 45, and 90 min, the amounts of O2- generation in 10 min were equivalent to 0.064 ± 0.003 (n = 6), 0.087 ± 0.003 (n = 6), 0.076 ± 0.006 (n = 6), and 0.022 ± 0.004 (n = 6) mU/ml of xanthine oxidase, respectively (Fig. 7B). The value of 45 min of reoxygenation of Fig. 7B was smaller to that in Fig. 7A, because these series of experiments were performed independently.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Alterations of Ca2+ mobilization by hypoxia/reoxygenation. In the present study we found several hypoxia/reoxygenation-induced alterations in Ca2+ mobilization in BBEC. Namely, in hypoxia/reoxygenation-treated BBEC 1) basal [Ca2+]i was elevated, 2) ATP-induced Ca2+ oscillations were attenuated, 3) the peak amplitude of thapsigargin-induced Ca2+ leak was decreased, and 4) CCE was inhibited. The hypoxic environment was obtained by using a commercial kit, which provides a strict hypoxic condition of <0.1% but also reduces CO2 concentration. The concomitant reduction of CO2 concentration seems not to be involved in these alterations of [Ca2+]i, because the pH of the HEPES-buffered culture medium was not changed during the incubation period. Though the reduced CO2 concentration itself may have influenced Ca2+-mobilizing properties in hypoxia/reoxygenation-treated cells by changing ionic strength or intracellular pH, we consider that the influence was limited because SOD and rotenone/TTFA restored many Ca2+ mobilizations as discussed in Involvement of O2- in hypoxia/reoxygenation-induced alterations of Ca2+ mobilization.

ATP elicited almost no Ca2+ oscillations in hypoxia/reoxygenation-treated cells (Fig. 2, B and C). It has been speculated that Ca2+ oscillations have a functional advantage to induce cellular functions at low levels of stimulation (9). We have also observed in aortic endothelium that the oscillatory increase in [Ca2+]i induces efficient production of nitric oxide (T. Koyama, C. Kimura, and M. Oike, unpublished observations). Therefore, the abolishment of Ca2+ oscillations in hypoxic/reoxygenation environment would attenuate endothelial functions significantly. Because Ca2+ oscillations are formed as the balance of Ca2+ release, extrusion, and entry (13, 28), the attenuation of any of these components would result in the abolishment of Ca2+ oscillations. The threshold of ATP-induced Ca2+ release was shifted to a higher concentration in hypoxia/reoxygenation-treated cells (Fig. 2C). Therefore, the cellular responsiveness to ATP was supposedly affected by hypoxia/reoxygenation, and this would be one of the reasons for the disappearance of Ca2+ oscillations.

Furthermore, we have observed that the amplitude of thapsigargin-induced Ca2+ leak was significantly smaller in hypoxia/reoxygenation-treated cells (Fig. 3C). Because thapsigargin induces complete store depletion (30), the amplitude of thapsigargin-induced Ca2+ leak would reflect the amount of stored Ca2+ in the intracellular Ca2+ storage sites. Therefore, this probably implies that the total amount of stored Ca2+ is decreased in hypoxia/reoxygenation-treated cells. Stored Ca2+ is continuously leaked out of the storage sites, and SERCA is normally pumping back the leaked Ca2+ into the storage sites (6), thereby playing a significant role in maintaining the Ca2+ sequestration and the basal level of [Ca2+]i. Therefore, we consider that the increase of the basal level of [Ca2+]i in hypoxia/reoxygenation-treated cells was also related to the reduction of stored Ca2+.

Supplement of Ca2+ from the extracellular space through CCE was also attenuated markedly in hypoxia/reoxygenation-treated cells (Fig. 3, B and D). CCE is the main Ca2+ entry pathway for vascular endothelium (16), therefore the inhibition of this pathway would impair the integrity of cellular Ca2+-mobilizing properties in hypoxia/reoxygenation-treated BBEC.

Taken together, Ca2+ oscillations were inhibited in hypoxia/reoxygenation-treated BBEC in the present experiment, and this was probably because of the mixture of the impaired responsiveness to ATP, decreased amount of stored Ca2+, and the inhibition of CCE.

Involvement of O2- in hypoxia/reoxygenation-induced alterations of Ca2+ mobilization. We have previously reported in BAEC that glucose overload abolishes Ca2+ oscillations in BAEC due to the accumulation of O2- (17). In this study, abolishment of Ca2+ oscillations induced by hypoxia/reoxygenation was also by the accumulation of O2-, because SOD restored Ca2+ oscillations and CCE (Fig. 4), and because O2- was actually generated by the reoxygenation after hypoxic challenge (Fig. 7). Overproduction of O2- by hypoxia/reoxygenation has been reported repeatedly (11, 27, 29). It has been speculated that the hypoxia/reoxygenation induces the generation of reactive oxygen species by mitochondria as a result of the decrease in maximal velocity (Vmax) of cytochrome oxidase (11). We also confirmed, by measuring O2- with MCLA, that hypoxia/reoxygenation-induced O2- generation was completely reversed by rotenone and TTFA (Fig. 7). In the present study, we exposed the cells to a hypoxic environment for 6 h, but the fura 2-loading step and [Ca2+]i measurement were performed under normal air containing 20% oxygen. Because the amount of O2- generation is expected to be very low in a hypoxic environment, we suppose that O2- was mainly generated during this reoxygenation period. We actually observed that rotenone and TTFA, when added only during the reoxygenation period, considerably but not completely restored hypoxia/reoxygenation-induced alterations in Ca2+ mobilization (Figs. 5 and 6A). Furthermore, maximal generation of O2- was obtained not immediately after the hypoxic period but after 20 min of reoxygenation (Fig. 7B). We therefore conclude that hypoxia/reoxygenation-induced impairments of Ca2+ mobilization are exclusively due to O2-, which is generated by a mitochondrial electron transport system during reoxygenation.

The hypoxia/reoxygenation-induced alterations of basal level of [Ca2+]i and thapsigargin-induced Delta [Ca2+]i-peak were not restored by SOD or rotenone/TTFA (Fig. 5). These Ca2+ mobilizations largely depend on the intracellular ATP level as described in the Introduction, and it has been reported that ATP production is markedly attenuated by hypoxia (2). So we suppose that the alterations of these Ca2+-mobilizing properties were not due to O2- generation but to the inhibition of ATP production. The reduction of stored Ca2+ would attenuate agonist-induced Ca2+ release, and the elevation of the resting [Ca2+]i itself would increase capillary permeability (23). Therefore, these alterations of Ca2+ mobilization induced by hypoxia/reoxygenation, which are induced by a mechanism other than O2-, will also attenuate endothelial homeostasis significantly.

In summary, the present study showed that Ca2+-mobilizing properties are impaired by a hypoxic environment partially due to the generation of O2- by mitochondrial electron transport. More importantly, these hypoxia/reoxygenation-induced alterations in Ca2+ mobilization were reversed by scavenging O2-, and this would provide a possibility for the novel therapeutic approach to ischemic cerebral diseases.


    ACKNOWLEDGEMENTS

This work was supported by Grant-In-Aid 12670089 from Japan Society for the Promotion of Science (JSPS) and Kaibara Morikazu Medical Science Promotion Foundation. C. Kimura is a research fellow of JSPS.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Oike, Dept. of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan (E-mail: moike{at}pharmaco.med.kyushu-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 March 2000; accepted in final form 13 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 279(5):H2310-H2318
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