Impairment of endothelial nitric oxide production by acute glucose overload

Chiwaka Kimura, Masahiro Oike, Tetsuya Koyama, and Yushi Ito

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the effects of acute glucose overload (pretreatment for 3 h with 23 mM D-glucose) on the cellular productivity of nitric oxide (NO) in bovine aortic endothelial cells (BAEC). We had previously reported (Kimura C, Oike M, and Ito Y. Circ Res, 82: 677-685, 1998) that glucose overload impairs Ca2+ mobilization due to an accumulation of superoxide anions (O2-) in BAEC. In control cells, ATP induced an increase in NO production, assessed by diaminofluorescein 2 (DAF-2), an NO-sensitive fluorescent dye, mainly due to Ca2+ entry. In contrast, ATP-induced increase in DAF-2 fluorescence was impaired by glucose overload, which was restored by superoxide dismutase, but not by catalase or deferoxamine. Furthermore, pyrogallol, an O2- donor, also attenuated ATP-induced increase in DAF-2 fluorescence. In contrast, a nonspecific intracellular Ca2+ concentration increase induced by the Ca2+ ionophore A-23187, which depletes the intracellular store sites, elevated DAF-2 fluorescence in both control and high D-glucose-treated cells in Ca2+-free solution. These results indicate that glucose overload impairs NO production by the O2--mediated attenuation of Ca2+ entry.

calcium; superoxide; diaminofluorescein 2


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIOVASCULAR DISEASE, including coronary heart disease, stroke, and peripheral vascular disease, is the most important cause of mortality and morbidity among patients with diabetes mellitus (1, 15, 31), in which impairment of endothelial functions may be involved (19). As vascular endothelium plays its physiological roles by producing various mediators, including nitric oxide (NO) (23), the production of these endothelium-derived substances might be impaired in hyperglycemic conditions. This idea is supported in a previous report, showing that the aortic ring from diabetic rabbit failed to induce endothelium-derived relaxation in response to acetylcholine (34).

Accumulation of superoxide anion (O2-) is one of the reported candidates for the pathogenesis of vascular and/or endothelial damage in a diabetic environment. Excess glucose causes autooxidation of glucose and nonenzymatic protein glycation, both of which generate O2- (40). Metabolism of excess D-glucose via its collateral pathway, the polyol pathway, increases the NADH/NAD+ ratio, which results in the activation of protein kinase C and phospholipase A2. Prostaglandins are then synthesized by cyclooxygenase, which is capable of generating O2- as an intermediate (34, 39). Fructose, a product of the polyol pathway, also generates O2-, because it induces protein glycation more efficiently than glucose (33). Furthermore, it has been reported that the activity of endogenous superoxide dismutase (SOD) is suppressed by hyperglycemia due to glycation (2). We have previously shown (12) that acute glucose overload abolishes ATP-induced Ca2+ oscillations by inhibiting Ca2+ release-activated Ca2+ entry (CRAC) and Ca2+ extrusion and by accelerating Ca2+ leak from the intracellular Ca2+ store sites in bovine aortic endothelial cells (BAEC). Normally, vascular endothelium produces NO in response to the elevation of intracellular Ca2+ concentration ([Ca2+]i), which complexes with calmodulin (CaM), and the Ca2+-CaM complex stimulates NO synthase (NOS) to produce NO (24). Therefore, our previous findings raise a question that needs to be clarified, i.e., whether these impairments of Ca2+ homeostasis are responsible for the attenuation of endothelium-derived vasodilatation in the hyperglycemic condition.

For the measurement of cellular NO production, direct measurement of released NO by porphyrinic microsensor (20) or measurement of its metabolite, nitrite, by the Griess method (38) has been used. Diaminofluorescein 2 (DAF-2) has been developed recently as an NO-sensitive fluorescent dye, whose fluorescence was shown to correlate with the NO concentration from submicromolar levels (14). Furthermore, intracellular production of NO has been measured successfully from living cells by use of a membrane-permeable form of DAF-2, diacetylated DAF-2 (DAF-2/DA), in rat aortic smooth muscle cells (14) and BAEC (11). The major advantage of using DAF-2 is that it enables the measurement of intracellular NO production from living cells after any arbitrary pretreatment of the cell, including glucose overload.

We examined the characteristics of cellular NO production in hyperglycemic conditions in BAEC by using DAF-2. In the present study, we used ATP to induce NO production in BAEC. We recently reported (27) that ATP is released from BAEC in response to mechanical stress. Because vascular endothelium shows its various physiological functions autonomically in response to mechanical stress (5), we supposed that ATP-induced cellular responses play an important role in endothelial physiology. The results obtained indicate that glucose overload attenuates endothelial NO production, and that this can be attributed mainly to the O2--mediated impairment of Ca2+ mobilization that we previously clarified.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Culture of BAEC. Bovine thoracic aorta of 1-yr-old calf was obtained from the local slaughterhouse. Endothelial cells were scraped off from the intima with the edge of a razor. Collected endothelial cells were cultured in DMEM (Life Technologies, Rockville, MD) containing 10% fetal calf serum under 5% CO2-95% air at 37°C. Cells of a second subculture were used in the present experiment. Cells were seeded, either on coverslips for measuring [Ca2+]i and NO production or on a 96-well culture plate for measuring O2- generation, and were cultured for >= 4 days after seeding. Identification of endothelial cells was confirmed by the specific uptake of acetylated low-density lipoprotein.

Measurement of [Ca2+]i. [Ca2+]i was measured from nonconfluent single cells by use of an Attofluor digital fluorescence microscopy system (Atto Instruments, Rockville, MD). Cells were loaded with fura 2 by incubation with 1 µM fura 2-AM (Dojindo, Kumamoto, Japan) for 20 min at 37°C. Fura 2 was excited at two alternative wavelengths (340 and 380 nm), and the emitted fura 2 fluorescence images of 510-nm wavelength were recorded into a rewritable optical disc recorder (LQ-4100A, Panasonic, Osaka, Japan) at a rate of ~1 Hz. For each cell, fluorescence intensities excited at 340 and 380 nm (F340 and F380, respectively) were calculated from each image, and these data were converted to fluorescence ratio (F340/F380) and apparent [Ca2+]i as previously reported (27).

Measurement of O2- by MCLA. We measured the generation of O2- in BAEC by using an O2--sensitive luciferin derivative, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA; Tokyo Kasei, Tokyo, Japan) (26). Cells were cultured for 4 days on a 96-well culture plate, and the culture medium was replaced with Krebs or high-glucose Krebs solutions and incubated for 3 h. Then the solution was replaced with 50 µl of 1µM MCLA-containing solutions, and illuminated photons were counted for 10 min with a luminescence detection system (Argus-50/2D luminometer, Hamamatsu Photonics, Hamamatsu, Japan). It has been reported that MCLA chemiluminescence is highly specific for O2- and singlet molecular oxygen (1O2) (26). We used superoxide dismutase (SOD) to distinguish between chemiluminescence of O2- and 1O2, because SOD does not scavenge 1O2 (8). Because it is difficult to calibrate MCLA chemiluminescence into absolute values of O2- (25), we expressed the amount of O2- as an equivalent concentration of xanthine oxidase, which reacts with xanthine and generates O2- in a concentration-dependent manner (see inset of Fig. 2).

Measurement of intracellular production of NO. NO was measured by use of DAF-2, an NO-sensitive fluorescent dye (14). Cells were loaded with 10 µM DAF-2/DA (Daiichi Pure Chemicals, Tokyo, Japan) for 30 min at 37°C. The DAF-2 fluorescence was measured by use of the same equipment as for [Ca2+]i measurements but with a different filter set, i.e., excitation at 490 nm and emission at 515 nm. Because we found in the preliminary experiment that the frequent application of the excitation wavelength quenches DAF-2 fluorescence, we applied the excitation wavelength with an interval of 30 s. Therefore, temporal resolution of DAF-2 fluorescence was inevitably lower than that of [Ca2+]i in the present study. It has been reported that DAF-2 fluorescence increases almost linearly with the NO concentration (14); therefore, we expressed the intracellular NO production as the net increment of DAF-2 fluorescence in 15 min relative to its basal value. Because NO synthase (NOS) produces O2- instead of NO in the absence of a sufficient concentration of L-arginine (30), we added a maximal concentration of L-arginine (10 mM) to all solutions used for NO measurement, except for the experiment with Nomega -nitro-L-arginine methyl ester (L-NAME)-treated cells. The solutions were alkalized by the addition of a high concentration of L-arginine, and we therefore readjusted the pH to 7.3 with HCl.

Drugs and solutions. The standard extracellular solution was a modified Krebs solution (1.5 mM Ca2+ solution) containing (in mM): 132 NaCl, 5.9 KCl, 1.2 MgCl2, 1.5 CaCl2, 11.5 glucose, 11.5 HEPES, with pH adjusted to 7.3 with NaOH. High-glucose solution was made by replacing 6 mM NaCl with 11.5 mM D- or L-glucose, and Ca2+-free solution was made by replacing 1.5 mM CaCl2 with 1 mM EGTA. The bath was perfused continuously with these solutions at a rate of 1.5 ml/min.

Thapsigargin and cyclopiazonic acid (CPA), inhibitors of endoplasmic Ca2+-ATPase, were used to deplete intracellular Ca2+ store sites. Neomycin was used to inhibit phospholipase C. We used SOD, a scavenger of O2-, catalase, a scavenger of hydrogen peroxide, and deferoxamine, an inhibitor of hydroxyl radical production. A-23187, a Ca2+ ionophore, was used to deplete intracellular Ca2+ store sites in a nonspecific manner. Pyrogallol was used as a spontaneous generator of O2- due to autooxidation (21). L-NAME was used to inhibit NOS. All of these drugs were obtained from Sigma (St. Louis, MO). All experiments were performed at room temperature (25°C).

Exposure of cells to high-glucose environment. In this study, BAEC were pretreated for 3 h with either 11.5 mM D-glucose solution (normal glucose), 23 mM D-glucose solution (high D-glucose) or 11.5 mM D-glucose + 11.5 mM L-glucose solution (high L-glucose). Because a lower D-glucose concentration (5.8 mM) did not show any difference in Ca2+ mobilizing properties and DAF-2 fluorescence from those treated with 11.5 mM glucose, we used 11.5 mM glucose, employed in our laboratory as normal Krebs, as "normal glucose" solution. We did not examine the chronic effects of glucose overload, because culture medium, which contains sufficient concentration of antioxidative amino acids and vitamins, would have had to be used for pretreating the cells for days, and this would have artificially reduced the impairing effects of high-glucose-induced O2- accumulation.

Data analysis. Pooled data are given as means ± SE, and statistical significance was determined with Student's unpaired t-test and one-way ANOVA for comparing two groups and more than three groups, respectively. Probabilities of <5% (P < 0.05) were regarded as significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of glucose overload on Ca2+ mobilization in BAEC. First, we confirmed our previous findings, i.e., that D-glucose overload impairs Ca2+ mobilization due to the accumulation of O2- (12). As shown in Fig. 1A, ATP (1 µM) induced Ca2+ oscillations in Ca2+-containing Krebs solution in control cells treated with normal glucose. Ca2+ oscillations were not spontaneous, since perfusion of normal Krebs solution alone without ATP did not induce any Ca2+ responses. In high D-glucose-treated cells (Fig. 1B), but not in high L-glucose-treated cells (not shown), ATP (1 µM)-induced Ca2+ oscillations were abolished. Impairment of ATP-induced Ca2+ oscillations in high D-glucose-treated cells was restored by SOD (100 U/ml). However, catalase (1,200 U/ml) and deferoxamine (1 mM) did not reverse the impairment of Ca2+ oscillation in high D-glucose-treated cells (not shown).


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Fig. 1.   Effects of glucose overload on intracellular Ca2+ concentration ([Ca2+]i) in bovine aortic endothelial cells (BAEC). Normal glucose, 11.5 mM; high D-glucose, 23 mM; high L-glucose, 11.5 mM D- plus 11.5 mM L-glucose. A: ATP (1 µM) induced Ca2+ oscillations in control cells. B: ATP (1 µM) did not induce Ca2+ oscillations in high D-glucose-treated cells. C: thapsigargin (1 µM) induced initial Ca2+ transient in Ca2+-free solution, and the subsequent application of Ca2+ induced a sustained increase in [Ca2+]i due to Ca2+ release-activated Ca2+ entry (CRAC) in control cells. D: CRAC was inhibited in high D-glucose-treated cells. E: statistical analysis of Ca2+ reapplication-induced [Ca2+]i increase (Delta [Ca2+]i-CRAC) in control, high D-glucose-treated, high L-glucose-treated, and high D-glucose treated with superoxide dismutase (SOD), catalase, or deferoxamine cells. **P < 0.01 vs. control cells.

In our previous report (12), we also showed that the abolition of Ca2+ oscillations was partially due to the impairment of CRAC. Thapsigargin (1 µM) induced transient [Ca2+]i elevation in Ca2+-free solution due to store depletion (Fig. 1C). Subsequent application of Ca2+-containing solution induced further [Ca2+]i increase due to CRAC in control cells (Fig. 1C). On the other hand, the CRAC phenomenon was not observed in high D-glucose-treated cells (Fig. 1D). This was also restored by SOD but not by catalase or deferoxamine (Fig. 1E). The same results were also obtained by use of CPA (not shown).

Generation of O2- in high-glucose-treated cells. We then measured the amount of O2- released from BAEC into extracellular space by the use of MCLA (26). As shown in Fig. 2, the amount of O2- generated from BAEC after the incubation with normal glucose for 3 h was equivalent to 0.018 ± 0.005 mU/ml of xanthine oxidase (n = 10). In contrast, the value was significantly higher in high D-glucose (0.139 ± 0.003 mU/ml; n = 10) but similar in high L-glucose solution (0.020 ± 0.008 mU/ml; n = 10). This was completely scavenged by SOD (below the detection limit) but not by catalase or deferoxamine (Fig. 2), thereby suggesting that glucose overload-induced MCLA chemiluminescence was due to the generation of O2- but not 1O2. On the other hand, O2- was not detected in normal Krebs or high D-glucose solutions without endothelium. These suggest that O2- is actually generated and released from BAEC in high D-glucose condition and that SOD completely scavenges the released O2-.


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Fig. 2.   Chemiluminescence measurement of superoxide anion (O2-) with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) in bovine aortic endothelial cells (BAEC). Values are expressed by corresponding concentrations of xanthine oxidase, which reacts with xanthine (100 µM) to generate O2- in a concentration-dependent manner (inset). **P < 0.01; n.d., not significantly different, P > 0.05; dagger value below the detection limit.

High D-glucose attenuates NO production in BAEC. ATP (1 µM) induced a gradual increase in DAF-2 fluorescence in normal glucose-treated cells (Fig. 3A). It should be noted that the reaction between DAF-2 and NO is irreversible (14); therefore, the accumulated level of DAF-2 fluorescence corresponds to the total amount of cellular NO production of the period. The ATP-induced increase in DAF-2 fluorescence was suppressed in cells pretreated with 0.1 mM L-NAME for 30 min, suggesting that DAF-2 fluorescence is linked to the cellular NO production (Fig. 3A). ATP increased DAF-2 fluorescence in a concentration-dependent manner in both Ca2+-containing and Ca2+-free solutions (Fig. 3C). Pretreatment with 1 mM neomycin for 30 min suppressed the 10 µM ATP-induced increase in DAF-2 fluorescence in Ca2+-free solution (Fig. 3C), thereby indicating that the increase in DAF-2 fluorescence in Ca2+-free solution was due to Ca2+ release from intracellular store sites in BAEC. However, the increment of DAF-2 fluorescence was significantly smaller in Ca2+-free solution than in Krebs solution, and ATP <1 µM did not induce a DAF-2 increase in Ca2+-free solution (Fig. 3C).


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Fig. 3.   Effects of glucose overload on ATP-induced nitric oxide (NO) production in BAEC. A: control cells showed an increase in diaminofluorescein 2 (DAF-2) fluorescence in response to 1 µM ATP. open circle , Actual values of DAF-2 fluorescence, and continuous line was drawn by averaging the adjacent 6 points. , Values from 0.1 mM Nomega - nitro-L-arginine (L-NAME)-treated cells. B: statistical analysis of 1 µM ATP-induced increase in DAF-2 fluorescence. Values were measured 15 min after the start of ATP application. **P < 0.01 vs. Krebs. C: concentration-response relationships of ATP-induced increase in DAF-2 fluorescence. open circle  and , 1.5 mM Ca2+-containing solution with normal glucose and high D-glucose, respectively;  and , Ca2+-free solution without and with treatment with neomycin, respectively. *P < 0.05, **P < 0.01.

On the other hand, the ATP (1 and 10 µM)-induced increase in DAF-2 fluorescence was significantly smaller in high D-glucose-treated cells (Fig. 3, B and C). As in the case of Ca2+ mobilization, high L-glucose-treated cells showed a normal increase in DAF-2 fluorescence (Fig. 3B). The basal level of NO production, estimated by perfusing L-arginine alone without Ca2+-mobilizing agents for 15 min, was not different between control and high-glucose-treated cells [control, 0.017 ± 0.006 (n = 20); high-glucose-treated, 0.022 ± 0.008 (n = 18); P > 0.05].

As shown in Fig. 1, D-glucose overload interferes with endothelial Ca2+ homeostasis. We therefore further examined whether the attenuation of ATP-induced NO production by D-glucose overload is due to impairment of Ca2+ homeostasis or to other factors such as the inhibition of NOS. For this purpose, we investigated the effects of D-glucose overload on NO production induced by the Ca2+ ionophore A-23187, which nonselectively induces Ca2+ entry from the extracellular space and Ca2+ release from the intracellular store sites (18). To avoid the involvement of CRAC, we applied A-23187 in Ca2+-free solution. Figure 4A shows that 1 µM A-23187 induced [Ca2+]i elevation in Ca2+-free solution and that the subsequent application of thapsigargin did not induce a further increase in [Ca2+]i, thereby suggesting that the intracellular Ca2+ store sites were depleted. The time integral of the [Ca2+]i response over 5 min was not different between control and high D-glucose conditions (Fig. 4B; P > 0.05), indicating that glucose overload does not affect the A-23187-induced store depletion. A-23187 also increased DAF-2 fluorescence in the absence of extracellular Ca2+ in both normal glucose- and high D-glucose-treated cells (Fig. 4C; P > 0.05). This was not because A-23187 scavenged O2-, because MCLA chemiluminescence was not restored by A-23187 (Fig. 4D). These observations suggest that NO production still occurs in high D-glucose-treated cells, provided there is a proper [Ca2+]i elevation. Thus it can be speculated that attenuation of NO production in the high D-glucose condition may be due to the impairments of Ca2+ mobilization.


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Fig. 4.   Ca2+ ionophore A-23187-induced [Ca2+]i increase and NO production in Ca2+-free solution. A: A-23187 (1 µM) induced a transient increase in [Ca2+]i in Ca2+-free solution in control cells. Subsequent application of 1 µM thapsigargin did not induce further [Ca2+]i elevation. B: time-integral of A-23187-induced [Ca2+]i increase for 5 min. n.d., P > 0.05. C: net increment of DAF-2 fluorescence induced by A-23187. n.d. P > 0.05, D: high D-glucose-induced generation of O2- was measured in the presence and absence of 1 µM A-23187. n.d. P > 0.05.

O2- is responsible for the attenuation of NO production in glucose-overloaded BAEC. We have shown that impairment of Ca2+ homeostasis by D-glucose overload is due to O2- (Fig. 1 and Ref. 12), which is markedly accumulated in the extracellular space in the high D-glucose condition (Fig. 2). Thus we pretreated the cells with high D-glucose solution in the presence of SOD, catalase, or deferoxamine, and we measured ATP-induced NO production. SOD-treated cells showed an increase in DAF-2 fluorescence in response to 1 µM ATP similar to that in control cells (Fig. 5). In contrast, catalase and deferoxamine did not restore the glucose overload-induced attenuation of NO production (Fig. 5).


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Fig. 5.   Effects of scavengers of reactive oxygen on glucose overload-induced attenuation of NO production. **P < 0.01 vs. control.

We then examined the effects of exogenously applied O2- on ATP-induced NO production. For this purpose, cells were treated for 30 min at 37°C with 200 µM pyrogallol. Control cells treated with Krebs solution for the same period showed Ca2+ oscillations in response to 1 µM ATP (Fig. 6A). However, in pyrogallol-treated cells, ATP showed a [Ca2+]i increase with a single peak but no Ca2+ oscillations (Fig. 6B), as in the case of high D-glucose-treated cells (Fig. 1B). Thapsigargin and CPA-induced [Ca2+]i increase (Delta [Ca2+]i-CRAC) were also suppressed in pyrogallol-treated cells (not shown). Furthermore, 1 µM ATP did not show any apparent increase in DAF-2 fluorescence (Fig. 6C).


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Fig. 6.   Effects of exogenously applied O2- on ATP-induced Ca2+ transient and NO production. ATP (1 µM) evoked Ca2+ oscillation in control cells (A) but not in 200 µM pyrogallol-treated cells (B). C: net increment of DAF-2 fluorescence in response to 1 µM ATP was significantly smaller in pyrogallol-treated cells. **P < 0.01 vs. control.

These results suggest that the generation of O2- is responsible for the impairment of NO production in the high D-glucose condition.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Many investigators have reported that the high-glucose condition attenuates endothelium-derived vasorelaxation (7, 22, 29, 35). Therefore, it has been speculated that glucose overload would cause the impairment of NO release and/or the increased destruction of released NO. Langenstroer and Pieper (16) reported that the resting level of NO release was rather increased in diabetic rat aorta but that the endothelial functions were nevertheless attenuated since the destruction of NO was increased by O2-. Their report is supported by the fact that NO easily reacts with O2- to generate peroxynitrite (9). The present results indicate that the production of NO is also impaired by acute glucose overload in BAEC due to the O2--induced attenuation of Ca2+ mobilization. The impairing effects of glucose overload are not due to the nonspecific action of glucose but to the metabolism of excess glucose, because nonmetabolizable L-glucose did not show an inhibitory effect on DAF-2 fluorescence (Fig. 3B). We measured the intracellular NO production induced by ATP and A-23187 with DAF-2; therefore, it should be noted that the present results did not take into account the possible destruction of released NO by O2-. It should also be noted that the present study was performed with the use of cultured endothelial cells, whose properties may not be identical to those in vivo, and DAF-2 fluorescence may have been influenced by dye quenching. Furthermore, the present study did not investigate the possible involvement of other cellular changes, such as the activation of protein kinase C (13) and the polyol pathway (3) in glucose overload-induced endothelial dysfunction.

Cosentino et al. (4) reported that long-term incubation of human endothelium with 22.2 mM glucose for 5 days increased the expression level of endothelial NOS mRNA and ionomycin-induced nitrite production. However, we consider that this does not contradict the present results, because the agonist-induced Ca2+ mobilizing pathways were bypassed by use of the Ca2+ ionophore ionomycin in their report. Actually we observed that A-23187-induced NO production was not affected by glucose overload (Fig. 4). Therefore, although agonist-induced, Ca2+-dependent NO production requires CaM and NOS (24), we suppose that the attenuation of NO production by acute glucose overload can be attributed to the impairment of Ca2+ homeostasis but not to the alteration of these molecules. We have shown that glucose overload abolishes Ca2+ oscillations (Fig. 1 and Ref. 12). Generation of Ca2+ oscillations requires integrity of various Ca2+ pathways, i.e., Ca2+ release from the intracellular store sites, extrusion of released Ca2+ out of the cell, Ca2+ entry from the extracellular space, and reloading of the intracellular Ca2+ store sites (32). Therefore, although 1 µM ATP induced Ca2+ transient in high glucose-treated cells also (Fig. 1B), the fact that Ca2+ oscillations were abolished would indicate that some of the Ca2+ mobilizing pathways were impaired. Because we observed that CRAC was attenuated by glucose overload (Fig. 1, D and E), we suppose that at least the impairment of CRAC was involved in the disappearance of Ca2+ oscillations in high glucose-treated cells. Lantoine et al. (17) reported that, in human umbilical cord vein endothelial cells, NO production is triggered by Ca2+ entry from the extracellular space but not by Ca2+ release from store sites. The authors speculated that this was due to the submembrane localization of endothelial NOS. We also observed that ATP-induced NO production was much larger in the Ca2+-containing solution than in the Ca2+-free solution (Fig. 3C), suggesting the preferential production of NO by entered Ca2+, especially in the case of low levels of stimulation. However, considerable elevation of DAF-2 fluorescence was also induced by a high concentration of ATP or A-23187 in Ca2+-free solution, thereby indicating that depletion of the intracellular Ca2+ store sites leads to NO production in BAEC. As shown in Fig. 4C, store depletion-induced NO production was not inhibited by glucose overload. Therefore, we hypothesized that the impairment of CRAC plays a central role in the attenuation of ATP-induced NO production by acute D-glucose overload. This is not selective for ATP-induced NO production, since glucose overload also attenuated 0.3 µM ACh-induced NO production (C. Kimura and M. Oike, unpublished observation).

A high-glucose environment leads to the accumulation of O2-, as summarized in the introductory section. In the present experiment, SOD, but not catalase or deferoxamine, restored high-glucose-induced impairment of Ca2+ homeostasis (Fig. 1) and NO production (Fig. 5). Furthermore, we confirmed the impairing effects of O2- on Ca2+ homeostasis and NO production by pretreating the cell with pyrogallol (Fig. 6). Therefore, we conclude that O2-, which affects Ca2+ homeostasis, is responsible for the impairment of NO production in the hyperglycemic condition. Using MCLA chemiluminescence, we confirmed that the high D-glucose condition induces the increased generation and/or decreased scavenging of O2-, which was abolished by SOD (Fig. 2). MCLA is a water-soluble substance that does not permeate the cell membrane (25), and MCLA chemiluminescence was not detected in cell-free culture wells (Fig. 2). Therefore, although O2- is supposed to be generated inside the cell as a result of glucose metabolism, it seems that O2- permeates the membrane and is released into the extracellular space and then affects Ca2+ pathways of the cell and/or neighboring cells. We suppose that this is why SOD, a large molecule (31 kDa) that could not penetrate the cell membrane, scavenged the intracellularly generated O2- and restored Ca2+ mobilization and NO production. Graier et al. (10) reported that the high-glucose-induced generation of O2- enhanced endothelium-derived relaxing factor formation in porcine aortic endothelium. They incubated the cells for 24 h with a high-glucose culture medium and observed that bradykinin-induced Ca2+ transient and cGMP concentration were augmented in high-glucose-treated cells due to O2- production. However, the present study and many reports from other laboratories (7, 22, 28, 35) indicate that glucose overload attenuates endothelium-derived relaxation or NO production. Although it may be possible that this discrepancy was due to the differences in the period of glucose overload, species, and agonist, we could not identify the precise reason for the discrepancy.

It is well known that the impairment of endothelial constitutive NO production would result not only in the loss of local control of vascular tonus but also in the development of atherosclerosis (36), heart failure (6), or cerebrovascular events (37). Therefore, the mechanism shown in the present study is probably involved in the pathogenesis of some diabetic vascular complications. In conclusion, we have shown that acute glucose overload attenuates endothelial NO production by the impairment of Ca2+ homeostasis, especially CRAC. Our results also suggest that scavenging O2- might be a good therapeutic approach to diabetic vascular complications.


    ACKNOWLEDGEMENTS

The authors thank Dr. Guy Droogmans for critical reading of the manuscript.


    FOOTNOTES

This work was supported by a grant-in-aid from the Japan Society for the promotion of Science (JSPS, 11003235) and the Japan Heart Foundation (C. Kimura). C. Kimura is a research fellow of JSPS.

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 3 April 2000; accepted in final form 21 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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