Acute Glucose Overload Abolishes Ca²⁺ Oscillation in Cultured Endothelial Cells From Bovine Aorta

A Possible Role of Superoxide Anion

From the Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.

Correspondence to Masahiro Oike, MD, PhD, Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan. E-mail moike{at}pharmaco.med.kyushu-u.ac.jp

	Abstract

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Abstract—Effects of acute glucose overload on [Ca²⁺]_i were investigated in cultured endothelial cells from bovine aorta. Application of 0.1 µmol/L ATP elicited an oscillatory increase in [Ca²⁺]_i (Ca²⁺ oscillation) in Krebs solution containing 11.5 mmol/L glucose. The frequency of Ca²⁺ oscillation induced by ATP increased in a concentration-dependent manner, ranging between 0.03 and 1 µmol/L. When cells were preincubated with 23 mmol/L glucose–containing Krebs solution (high glucose solution) for 3 hours, 0.1 µmol/L ATP failed to induce Ca²⁺ oscillation but evoked only a phasic followed by sustained increase in [Ca²⁺]_i. Application of a higher concentration of ATP (10 µmol/L) evoked a transient increase in [Ca²⁺]_i both in control and high glucose–treated cells. However, the falling phase of [Ca²⁺]_i was prolonged in high glucose–treated cells. Thapsigargin (1 µmol/L), an inhibitor of endoplasmic Ca²⁺-ATPase, induced a transient followed by a sustained increase in [Ca²⁺]_i in control cells. Preincubation with high glucose solution increased the rate of rise of the thapsigargin-induced increase in [Ca²⁺]_i and abolished the sustained increase, suggesting that glucose overload accelerates Ca²⁺ leak from intracellular store sites and impairs Ca²⁺ release–activated Ca²⁺ entry. We found that all of the glucose overload–induced changes in Ca²⁺ mobilization could be mimicked by xanthine with xanthine oxidase and abolished by superoxide dismutase. These results indicate that acute glucose overload accumulates superoxide anion in bovine aortic endothelial cells, thereby diminishing ATP-induced Ca²⁺ oscillation through the impairment of Ca²⁺ homeostasis.

Key Words: endothelium • Ca²⁺ • oscillation • superoxide • glucose overload

	Introduction

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Oscillatory increase in [Ca²⁺]_i (Ca²⁺ oscillation) has been reported in nonexcitable cell types, including endothelial and pancreatic acinar cells.¹ In vascular endothelial cells, generation of Ca²⁺ oscillation occurs in response to histamine,² fluid flow,³ and cell adhesion⁴ or occurs spontaneously,⁵ and the frequency of histamine-induced Ca²⁺ oscillation was reported to increase in a dose-dependent manner.² Thus, Ca²⁺ oscillation has been considered to correlate directly to cellular functions, such as secretion of EDRFs.^{1 3}

Elevation of the plasma glucose level, on the other hand, has been known to impair vascular functions, including endothelium-dependent vascular relaxation.^{6 7 8} It results in vascular complications, which are commonly accompanied by hyperglycemia.⁹ Therefore, investigation of the effect of glucose overload on endothelial function would have significant importance in pathophysiological properties of endothelial cells. However, only a few studies have examined the effects of glucose overload on Ca²⁺/EDRF signaling in endothelial cells. It was reported that incubation of porcine aortic endothelial cells for 24 hours with high glucose solution (44 mmol/L) increased resting [Ca²⁺]_i and enhanced agonist-stimulated Ca²⁺/EDRF signaling.^{10 11} By contrast, it is also known that acute treatment (30 or 60 minutes) of vascular tissue with high glucose solution impairs endothelium-dependent vasodilatation in arterioles.^{12 13 14} However, no studies have examined the effect of acute glucose overload on Ca²⁺ transients in the endothelial cells in response to vasoactive agents.

The aim of the present study was to examine the effects of acute glucose overload on Ca²⁺ transients in the endothelial cells of bovine aorta in response to ATP. ATP has been known to be released from endothelium itself in response to the increased blood flow.^{15 16} During the course of the experiments, we found that ATP (0.01 to 1 µmol/L) evokes Ca²⁺ oscillation in endothelial cells and that glucose overload impairs the Ca²⁺ transient, which could be overcome by coincubation with SOD, thereby indicating the possible role of superoxide anion (O₂^-).

	Materials and Methods

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Cell Culture
Bovine thoracic aortas of 1-year-old calves were obtained from the local slaughterhouse. Endothelial cells were cultured as previously described.¹⁷ Identification of endothelial cells was confirmed by the specific uptake of acetylated LDL. Cells were grown on coverslips, and single nonconfluent cells were used. In order to compare the data from control and high glucose–treated cells, we incubated the cells with normal Krebs or high glucose solution for 3 hours before each experiment.

Measurement of [Ca²⁺]_i
For the measurement of [Ca²⁺]_i from a single bovine aortic endothelial cell, cells were loaded with 1 µmol/L of the acetoxymethylester form of the Ca²⁺ fluorescent dye fura 2 (fura 2-AM, Wako Corp) as previously described.¹⁵ The coverslip with fura 2–loaded cells was placed on a chamber of 0.5-mL volume and mounted on an inverted microscope (Diaphot TMD, Nikon). The cell was excited with two excitation wavelengths, 340 and 380 nm (each slit, 5 nm), applied by a spectrometer (Spex). The obtained fluorescence intensity data, after subtraction of the background fluorescence (F₃₄₀ and F₃₈₀, respectively), were used to obtain the fluorescence ratio (R), F₃₄₀/F₃₈₀. We calculated apparent [Ca²⁺]_i using the following equation:

where K_eff is the effective binding constant, R_min is the fluorescence ratio at zero Ca²⁺, and R_max is the fluorescence ratio at high Ca²⁺. Because precise in vivo calibration of [Ca²⁺]_i was difficult to perform, it should be noted that the calculated value is not an accurate intracellular concentration of Ca²⁺.

In some experiments, we calculated the net Ca²⁺ mobilized by ATP by integrating the elevated component of [Ca²⁺]_i ([Ca²⁺]_i): [Ca²⁺]_idt. In other experiments, we estimated the maximum rate of Ca²⁺ leak induced by thapsigargin by differentiating [Ca²⁺]_i: d[Ca²⁺]_i/dt. Both calculations were performed by Microsoft Excel.

All experiments were performed at room temperature.

Materials
Modified Krebs solution, used as the standard extracellular solution, contained (mmol/L) NaCl 132, KCl 5.9, MgCl₂ 1.2, CaCl₂ 1.5, glucose 11.5, and HEPES 11.5; pH was adjusted to 7.3 with NaOH. High glucose solution was made by increasing glucose to 23 mmol/L and reducing NaCl to 126 mmol/L so that its osmolarity calculated by their ionic strengths was equal to normal Krebs solution. The bath was perfused continuously with these solutions at a rate of 1.5 mL/min.

ATP (Sigma Chemical Co) and thapsigargin (Sigma) were used to release Ca²⁺ from the intracellular Ca²⁺ store sites. Ruthenium red (Sigma) was used to inhibit mitochondrial Ca²⁺ uniporter. Stock solutions of SOD (Sigma), catalase (Wako), and deferoxamine mesylate (Sigma) were made and diluted 1000 times to make the final concentration. Xanthine and xanthine oxidase (XO) were obtained from Sigma.

Data Analysis
Pooled data are given as mean±SEM, and statistical significance was determined using Student's unpaired t test, except for the rate of Ca²⁺ leak, for which we used the Mann-Whitney U test because of the marked difference of each group's variance. Values of P<.05 were regarded as significant.

	Results

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Effects of ATP on [Ca²⁺]_i of Bovine Aortic Endothelial Cells
The pattern of ATP-induced Ca²⁺ transient differed during the early and late stage of the culture. ATP (0.1 to 0.3 µmol/L) evoked only a phasic increase in [Ca²⁺]_i (Fig 1A) in most cells cultured for <3 days after seeding, whereas Ca²⁺ oscillation was the dominant response in cells cultured for >3 days (Fig 1B). The Ca²⁺ oscillation generally showed a decrease in the amplitude of each peak and a gradual increase in the basal level of [Ca²⁺]_i ([Ca²⁺]_i at 5 minutes after the start of the ATP application was 71.3±11.3 nmol/L; n=11). However, in 2 of 10 examined cells, the gradual increase in [Ca²⁺]_i during 0.1 µmol/L ATP–induced Ca²⁺ oscillation was not observed (Fig 1C). Fig 1D shows the relationship between the duration of culture and the appearance of Ca²⁺ oscillation. At 4 days of culture, Ca²⁺ oscillation in response to ATP (0.03 to 1 µmol/L) was observed in all examined cells (n=14). However, the frequency of Ca²⁺ oscillation or degree of elevated [Ca²⁺]_i did not depend on the culture period after the fourth day (data not shown). Thus, we used cells cultured at least 4 days for all subsequent experiments.

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of low concentration of ATP (0.1 µmol/L) on [Ca2+]i in bovine aortic endothelial cells in normal Krebs solution containing 11.5 mmol/L glucose. A, A cell cultured for 1 day after seeding showed only a single peak, followed by plateau elevation of [Ca2+]i in response to 0.1 µmol/L ATP. B, A cell cultured for 4 days after seeding shows a typical Ca2+ oscillation accompanied by a gradual elevation of basal level of [Ca2+]i. C, Two of 10 cells showed such Ca2+ oscillation, in which each peak returned to the basal level. D, Maturation dependence of the appearance of Ca2+ oscillation is plotted. ATP (0.1 and 0.3 µmol/L) was applied to cells for 5 minutes, and the percentage of cells showing more than two [Ca2+]i spikes in 5 minutes was calculated. Five to 14 cells were examined for each evaluation.

In Fig 2, panels A and B show the relationships between the concentrations of ATP and the frequency of Ca²⁺ oscillation, the mean amplitude of Ca²⁺ oscillation, and the elevated [Ca²⁺]_i 5 minutes after starting the ATP application (elevated [Ca²⁺]_i). Although ATP evoked only a phasic increase in [Ca²⁺]_i at an extremely high concentration (10 µmol/L, Fig 2D), the frequency of Ca²⁺ oscillation induced by ATP at 0.001 to 0.3 µmol/L increased in a concentration-dependent manner, with a half-maximal concentration of 0.037 µmol/L. In contrast, mean peak amplitude of Ca²⁺ oscillation or the elevated [Ca²⁺]_i induced by ATP did not show any concentration dependence to ATP between 0.001 and 0.3 µmol/L. Furthermore, the [Ca²⁺]_i integral for 5 minutes also did not show strict concentration dependence to ATP (Fig 2C). These results indicate that the frequency of Ca²⁺ oscillation, but not the amplitude of [Ca²⁺]_i elevation, is correlated with ATP concentration in bovine aortic endothelial cells.

View larger version (23K): [in this window] [in a new window]   Figure 2. Concentration-response relationships of ATP-induced [Ca2+]i elevation. A, Numbers of [Ca2+]i peaks in 5 minutes plotted against ATP concentration were calculated as oscillation frequency (Hz). Each symbol indicates mean±SEM value of 5 to 24 cells. A continuous line was drawn by the logistical equation with a half-maximal concentration of 0.037 µmol/L. Values at 1 and 10 µmol/L ATP were not included in the fit. B, Net increment of [Ca2+]i ([Ca2+]i) after application of ATP is shown. Solid circles indicate elevated [Ca2+]i level measured at 5 minutes after starting the application of ATP. Inset shows how this value was measured. Open circles indicate mean peak elevation of [Ca2+]i, ie, the average [Ca2+]i for peaks 1 to 5 (P1 to P5) in the cell illustrated. C, [Ca2+]i integral for 5 minutes of ATP-induced Ca2+ transient is shown. Inset shows how this value was calculated. D, Supramaximal concentration of ATP (10 µmol/L) induced a single steep transient elevation of [Ca2+]i.

Effects of Acute Glucose Overload on Ca²⁺ Transients
We then examined the effect of acute glucose overload on Ca²⁺ transients in response to ATP. Three hours after incubation with high D-glucose solution (23 mmol/L), resting [Ca²⁺]_i (control, 30.7±1.6 nmol/L [n=36]; high glucose–treated cells, 36.6±2.6 nmol/L [n=30]; P>.05) and the threshold concentration of ATP (0.01 µmol/L) required to induce Ca²⁺ transients were not different from the control values. However, application of ATP (0.1 µmol/L) did not induce Ca²⁺ oscillation but induced only a phasic followed by a sustained increase in [Ca²⁺]_i. The amplitude of the elevated [Ca²⁺]_i was similar to that in normal Krebs solution (74.6±15.4 nmol/L, n=9, P>.05; Fig 3Aa). On the other hand, the [Ca²⁺]_i integral for 5 minutes was significantly decreased in high glucose conditions (control, 39 756±4728 nmol · L^-1 · s [n=7]; high glucose, 24 798±3083 nmol · L^-1 · s [n=10]; P<.05). We performed similar experiments with L-glucose. To avoid the energy deprivation of the cell, we added 11.5 mmol/L L-glucose to the Krebs solution containing 11.5 mmol/L D-glucose and adjusted the osmolarity as described in "Materials and Methods." As shown in Fig 3Ab, however, glucose overload with L-glucose did not abolish ATP-induced Ca²⁺ oscillation. The [Ca²⁺]_i integral was also not significantly different between control and glucose overload with L-glucose (high glucose with L-glucose, 34 045±5905 nmol · L^-1 · s [n=5]).

View larger version (20K): [in this window] [in a new window]   Figure 3. Inhibitory effect of glucose overload on low ATP–induced Ca2+ oscillation and high ATP–induced [Ca2+]i transient. The cell was incubated either with 23 mmol/L D-glucose solution or a solution containing 11.5 mmol/L D-glucose and 11.5 mmol/L L-glucose for 3 hours. Aa, A representative trace shows that the oscillatory increase in [Ca2+]i was not observed in response to 0.1 µmol/L ATP in a high D-glucose–treated cell. Ab, Ca2+ oscillation was normally observed in high L-glucose solution. Ba, The decreasing phase of the 10 µmol/L ATP–induced [Ca2+]i transient was prolonged in a high D-glucose–treated cell. The dotted line is the same peak-matched trace as in Fig 2B, showing the [Ca2+]i transient in 11.5 mmol/L D-glucose. Bb, Elevation of glucose concentration with L-glucose did not prolong the decreasing phase of the Ca2+ transient.

When a supramaximal concentration of ATP (10 µmol/L) was applied to cells preincubated with high D-glucose solution, a single steep elevation of [Ca²⁺]_I was observed as in control cells. However, the falling phase of the Ca²⁺ transient was markedly prolonged in high glucose–treated cells. The time required to decrease [Ca²⁺]_i to one third of the peak (t_1/3) in the control and high glucose–treated cells was 61.2±5.7 and 134.8±16.2 seconds, respectively (n=6, P<.01; Fig 3Ba). Glucose overload with L-glucose solution, on the other hand, did not affect t_1/3 (72.1±8.7 seconds, n=5, P>.05; Fig 3Bb).

To examine the effect of glucose overload on Ca²⁺ leak from intracellular store sites and the following CRAC, we used thapsigargin, an inhibitor of endoplasmic Ca²⁺-ATPase. Thapsigargin (1 µmol/L) induced a transient followed by sustained increase in [Ca²⁺]_i in control cells (Fig 4Aa). The mean [Ca²⁺]_i elevation from the basal level 15 minutes after the application of thapsigargin ([Ca²⁺]_i at 15 minutes) was 43.3±8.1 nmol/L (n=6). When thapsigargin was applied in Ca²⁺-free solution, elevated [Ca²⁺]_i declined to the basal level in 15 minutes ([Ca²⁺]_i at 15 minutes, 0 nmol/L [n=5]; Fig 4Ab), thereby indicating that [Ca²⁺]_i at 15 minutes in Ca²⁺-containing Krebs solution was due to Ca²⁺ that entered from outside the cell.

View larger version (19K): [in this window] [in a new window]   Figure 4. CRAC after store depletion by thapsigargin (1 µmol/L), an inhibitor of endoplasmic Ca2+ pump, and the effect of glucose overload. Thapsigargin was present throughout the experiment from the time indicated by the arrow. Aa, Thapsigargin-induced transient increase followed by a sustained increase in [Ca2+]i in normal Krebs solution. Dotted line indicates the basal level of [Ca2+]i before application of thapsigargin. Ab, Thapsigargin-induced [Ca2+]i transient in Ca2+-free solution. Note that [Ca2+]i declined to the basal level in 15 minutes. B, After incubation of a cell for 3 hours with 23 mmol/L D-glucose solution, application of thapsigargin produced a steeper transient elevation of [Ca2+]i and a decline to the basal level (shown by dotted line), although Ca2+ was present extracellularly. On the other hand, L-glucose did not alter the thapsigargin-induced [Ca2+]i transient (dotted trace).

In cells preincubated with high D-glucose solution for 3 hours, thapsigargin evoked an initial transient elevation of [Ca²⁺]_i, but it was not followed by elevated [Ca²⁺]_i in the presence of extracellular Ca²⁺ ([Ca²⁺]_i at 15 minutes, 4.1±2.4 nmol/L [n=6]; P<.01; Fig 4B). In addition, the maximum rate of rise of [Ca²⁺]_i increase induced by thapsigargin was significantly increased after the glucose overload (control, 0.9±0.1 nmol · L^-1 · s^-1 [n=6]; high glucose solution, 12.1±7.1 nmol · L^-1 · s^-1 [n=7]; P<.01). When the glucose concentration was elevated with L-glucose (11.5 mmol/L of each D-glucose and L-glucose), on the other hand, thapsigargin-induced [Ca²⁺]_i responses were almost the same as those observed in the control condition ([Ca²⁺]_i at 15 minutes, 37.5±3.6 nmol/L [n=5, P>.05]; the maximum rate of rise of [Ca²⁺]_i, 1.1±0.2 nmol · L^-1 · s^-1 [n=5, P>.05]; Fig 4B).

Effects of La³⁺ and Ruthenium Red on ATP- or Thapsigargin-Induced Ca²⁺ Mobilization
To understand the mechanisms involved in the effects of glucose overload on Ca²⁺ transient, we used La³⁺, since La³⁺ is known to inhibit Ca²⁺ passage through plasma membrane nonselectively, including plasmalemmal Ca²⁺ pump¹⁸ and CRAC channel.¹⁹

In the presence of 1 mmol/L La³⁺, 0.1 µmol/L ATP did not induce Ca²⁺ oscillation but a phasic followed by sustained increase in [Ca²⁺]_i (Fig 5A). Furthermore La³⁺ (1 mmol/L) prolonged the falling phase of the Ca²⁺ transient evoked by a high concentration of ATP (10 µmol/L) as in the case of glucose overload (Fig 5B). However, La³⁺ did not affect the maximum rate of rise in [Ca²⁺]_i evoked by thapsigargin in Ca²⁺-free solution (Fig 5C), indicating that La³⁺ does not influence the Ca²⁺ leak. Therefore, we observed the effects of glucose overload on the thapsigargin-induced Ca²⁺ transient in the presence of 1 mmol/L La³⁺. As shown in Fig 5D, the maximum rate of rise in [Ca²⁺]_i induced by thapsigargin was increased in high glucose–treated cells in the presence of La³⁺, indicating that glucose overload accelerates Ca²⁺ leak itself even when Ca²⁺ movements through the plasma membrane were completely inhibited by La³⁺.

View larger version (17K): [in this window] [in a new window]   Figure 5. Effect of LaCl3, a nonspecific inhibitor of plasma membrane Ca2+ fluxes, on ATP-induced [Ca2+]i increase and thapsigargin-induced [Ca2+]i elevation. A, Effect of 0.1 µmol/L ATP on [Ca2+]i in the presence of 1 mmol/L LaCl3 is shown. The cell was cultured for 4 days after seeding. Ca2+ oscillation was not observed. B, A high concentration of ATP (10 µmol/L) in the presence of 1 mmol/L LaCl3 prolonged the decreasing phase, especially its later part, of the ATP-induced Ca2+. The difference of [Ca2+]i elevation compared with that in Fig 3Ba is not significant. C, Thapsigargin (1 µmol/L) was applied to the cell in the presence of 1 mmol/L LaCl3. Ca2+-free solution was used to exclude the possible involvement of CRAC on Ca2+ leak pathway. D, Effect of glucose overload on thapsigargin-induced Ca2+ transient in the presence of LaCl3 is shown. The cell was preincubated with 23 mmol/L glucose for 3 hours. Note that the increase in [Ca2+]i induced by thapsigargin is markedly faster than that shown in panel C.

In an attempt to study the possible involvement of other intracellular organelles such as mitochondria, we examined the effect of ruthenium red, an inhibitor of mitochondrial Ca²⁺ uniporter, on thapsigargin-induced Ca²⁺ transient in high glucose–treated cells. When a relatively high concentration of ruthenium red (100 µmol/L) was applied to the cell, this agent induced gradual elevation of [Ca²⁺]_i by itself. In the presence of a relatively low concentration of ruthenium red (30 µmol/L), on the other hand, thapsigargin induced a rapid increase in [Ca²⁺]_i, and the rate of rise was not significantly different from that observed in high glucose–treated cells without ruthenium red (11.7±6.7 nmol · L^-1 · s^-1, n=5, P>.05). Similarly, the time course of Ca²⁺ removal, t_1/3, was not affected at all by ruthenium red (high glucose alone, 138.6±28.4 seconds [n=6]; high glucose with ruthenium red, 169.9±34.2 seconds [n=6]; P>.05; data not shown).

Effects of Free Radical Scavengers on Glucose Overload–Induced Changes in Ca²⁺ Transient
It has been suggested that the overproduction and/or reduced scavenging of free radicals is one of the reasons for high glucose–induced impairment of vascular functions (for a review, see Reference 13¹³ ). Therefore, we examined the effects of active oxygen scavengers on the high glucose–induced effects of the Ca²⁺ transient.

After cells were coincubated with high glucose solution and SOD (150 IU/mL), an O₂^- scavenger, ATP (0.1 µmol/L) did evoke Ca²⁺ oscillation (Fig 6A). This observation indicates that glucose overload–induced suppression of Ca²⁺ oscillation could be restored by SOD. In addition, coincubation with high glucose solution and SOD abolished other changes in Ca²⁺ mobilization, such as prolongation of the falling phase of the Ca²⁺ transient evoked by 10 µmol/L ATP (t_1/3, 70.0±7.9 seconds [n=5]; P>.05 compared with control data in Krebs solution; Fig 6B) and the inhibition of CRAC ([Ca²⁺]_i at 15 minutes, 30.9±1.5 nmol/L [n=7]; P>.05 compared with control data in Krebs solution; Fig 6C). Furthermore, the maximum rate of rise in [Ca²⁺]_i induced by thapsigargin was not different from the control value after the coincubation with SOD and high glucose solution (maximum rate of rise of [Ca²⁺]_i, 1.0±0.2 nmol · L^-1 · s^-1 [n=7]; P>.05; Fig 6C). These results indicate the possible role of O₂^- in the impairment of Ca²⁺ transients induced by glucose overload.

View larger version (17K): [in this window] [in a new window]   Figure 6. Reversal by SOD of the high glucose–induced impairment of [Ca2+]i oscillation, decreasing phase of [Ca2+]i, CRAC, and Ca2+ leak. The cells were pretreated with 23 mmol/L glucose Krebs solution containing 150 IU/mL SOD. A, Application of 0.1 µmol/L ATP produced [Ca2+]i oscillation. B, Decreasing phase of 10 µmol/L ATP–induced [Ca2+]i transient was restored. C, Ca2+ entry after depletion by 1 µmol/L thapsigargin was restored. The initial increase of [Ca2+]i by Ca2+ leak became slower as in 11.5 mmol/L glucose Krebs solution. Dotted line indicates basal level of [Ca2+]i.

To investigate whether other active forms of oxygen contribute to the changes in Ca²⁺ transients evoked by glucose overload, we used catalase, which reduces hydrogen peroxide to H₂O, and deferoxamine, which inhibits the conversion of H₂O₂ into hydroxy radical in the presence of Fe²⁺. Coincubation of the cells with catalase (1200 U/mL) or deferoxamine (1 mmol/L) and high glucose solution, however, did not prevent the glucose overload–induced impairments of Ca²⁺ mobilization. Namely, 0.1 µmol/L ATP did not evoke Ca²⁺ oscillation but a phasic followed by sustained increase in [Ca²⁺]_i (data not shown). The falling phase of 10 µmol/L ATP–induced Ca²⁺ transients was slightly shortened after incubation with catalase (t_1/3 was 90±4.4 seconds, which is significantly shorter than high glucose alone [P<.05] but significantly longer than control [P<.05]; n=5) but not with deferoxamine. In addition, catalase or deferoxamine did not restore the glucose overload–induced changes in Ca²⁺ leak and CRAC.

Fig 7 summarizes the effects of free radical scavengers on the falling phase of 10 µmol/L ATP–induced Ca²⁺ transients (panel A), CRAC (panel B), and Ca²⁺ leak (panel C). These results indicate that O₂^- may be responsible for all of the changes in Ca²⁺ mobilization induced by glucose overload.

View larger version (39K): [in this window] [in a new window]   Figure 7. Statistical analysis of the effects of active oxygen scavengers and O2- on Ca2+ extrusion (A), CRAC (B), and Ca2+ leak from intracellular store sites (C). A, To express Ca2+ extrusion after 10 µmol/L ATP application quantitatively, the time required to decrease elevated [Ca2+]i to one third was measured (t1/3). B, [Ca2+]i elevation by the opening of CRAC channel was measured 15 minutes after the application of thapsigargin. C, Changing velocity of Ca2+ was calculated by differentiating the [Ca2+]i curve. Inset in panel C shows an example of d[Ca2+]i/dt curve with an original [Ca2+]i curve superimposed. 23Glu indicates cells treated with 23 mmol/L glucose; Cat, catalase; and Def, deferoxamine. *P<.05 and **P<.01 vs control; §P<.05 vs control and 23Glu; and ¶P<.05 and ¶¶P<.01 vs cell treated with xanthine alone.

Effect of O₂^- on Ca²⁺ Mobilization
We incubated cells for 1 hour in the presence of xanthine (100 µmol/L) alone or xanthine (100 µmol/L) with XO (10 mU/mL), which donates O₂^-. ATP (0.1 µmol/L) evoked Ca²⁺ oscillation in the cells treated with xanthine alone (n=6) but not in cells treated with xanthine with XO (xanthine/XO, n=5; Fig 8A). When the high concentration of ATP (10 µmol/L) was applied, a transient elevation of [Ca²⁺]_i was observed in both groups, but the falling phase of the Ca²⁺ transient was prolonged in xanthine/XO-treated cells (t_1/3: xanthine alone, 72.5±3.9 seconds [n=4]; xanthine/XO, 146.0±20.1 seconds [n=5]; P<.05; Fig 8B). Treatment of cells with xanthine/XO also enhanced the maximum rate of rise in [Ca²⁺]_i (1.4±0.2 nmol · L^-1 · s^-1 [n=6] and 15.0±8.3 nmol · L^-1 · s^-1 [n=4] after xanthine alone and xanthine/XO pretreatment, respectively; P<.01; Fig 8C) and abolished the sustained increase in [Ca²⁺]_i evoked by thapsigargin ([Ca²⁺]_i at 15 minutes, 39.8±11.1 nmol/L [n=4] or 3.7±2.3 nmol/L [n=5] after xanthine alone or xanthine/XO pretreatment, respectively; P<.05; Fig 8C). The effects of xanthine alone or xanthine/XO on t_1/3, [Ca²⁺]_i at 15 minutes, and the maximum rate of rise in [Ca²⁺]_i induced by thapsigargin are also summarized in Fig 7.

View larger version (19K): [in this window] [in a new window]   Figure 8. Effect of superoxide anion (O2-) produced by incubation with xanthine (100 µmol/L) and XO (10 mU/mL) simultaneously. A, Effect on Ca2+ oscillation of the incubation with xanthine alone (a) or xanthine with XO (b) for 1 hour. Note that the cell treated with xanthine/XO shows a single peak elevation of [Ca2+]i, whereas that with xanthine alone shows Ca2+ oscillation. B, Prolongation of the decreasing phase of the high ATP–induced [Ca2+]i elevation by treatment with xanthine and XO. The control trace obtained from the cell treated with xanthine alone is superimposed (dotted trace). The difference of peak [Ca2+]i elevation from Fig 3Ba is not significant. C, Absence of [Ca2+]i elevation after thapsigargin-induced store depletion by treatment with xanthine and XO. A control trace obtained from another cell treated with xanthine alone is superimposed. On treatment with xanthine/XO, the initial increase of [Ca2+]i became steeper, and a sustained elevation of [Ca2+]i was not observed, although Krebs solution containing Ca2+ was used.

	Discussion

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A possible mechanism for encoding the agonist concentration sensed by endothelial cells is frequency or amplitude modulation of an intracellular Ca²⁺ signal. After the findings that concentration-dependent agonist-induced Ca²⁺ oscillation occurs in hepatocytes,²⁰ epithelial cells,²¹ and endothelial cells,² it is generally considered that frequency of Ca²⁺ oscillation may be the important factor in controlling the cellular response to agonists in nonexcitable cells. A frequency modulation system may have advantages for a high signal-to-noise ratio and in protecting cells from prolonged elevation of [Ca²⁺]_i (for example, see References 22 and 23^{22 23} ). However, it was reported that the frequency of Ca²⁺ oscillation is independent of agonist concentrations in parotid²⁴ and pancreatic acinar cells²⁵ stimulated with a cholinergic agonist and in bradykinin-stimulated endothelial cells.^{11 26} In those cases, the mean amplitude of the Ca²⁺ response, especially the plateau level of intracellular Ca²⁺, correlates closely with the agonist concentration.²⁷ In the present experiments, Ca²⁺ oscillation was normally accompanied by a gradual elevation of basal [Ca²⁺]_i (Fig 1B), and this observation raises the possibility that continuous elevation of [Ca²⁺]_i as well as Ca²⁺ oscillation may play a role in the intracellular signaling. However, the frequency of Ca²⁺ oscillation but not the amplitude of the plateau level of [Ca²⁺]_i, mean peak amplitude of the Ca²⁺ oscillation, or [Ca²⁺]_i integral showed concentration dependence to ATP (Fig 2). Therefore, it seems reasonable to assume that the frequency of Ca²⁺ oscillation may play an important role in controlling cellular response to ATP in the endothelial cells. We found that glucose overload inhibits the generation of Ca²⁺ oscillation, and this would result in the failure of endothelial function, such as the regulation of vascular tonus via production of nitric oxide.²⁸ Such an inhibition of endothelial Ca²⁺ oscillation by glucose overload was also observed in histamine-induced Ca²⁺ oscillation (C. Kimura and M. Oike, unpublished data, 1997).

Transient elevation of [Ca²⁺]_i in endothelial cells is mainly generated by inositol trisphosphate, and the Ca²⁺ fluxes that contribute to the generation of Ca²⁺ oscillation are (1) Ca²⁺ entry through channels or transporters on the plasma membrane down a concentration gradient, (2) active Ca²⁺ pumping by a plasmalemmal Ca²⁺ pump, (3) Ca²⁺ flow out of the intracellular store sites through the inositol trisphosphate receptor channel or passive Ca²⁺ leak pathway, and (4) active Ca²⁺ pumping back into the store sites (for a review, see References 23 and 29^{23 29} ). Therefore, the impairment of each one of these would potentially impair Ca²⁺ oscillation.

We found in the present study that acute glucose overload (1) prolongs the falling phase of a phasic Ca²⁺ transient evoked by a high concentration of ATP and (2) accelerates the maximum rate of rise in [Ca²⁺]_i elevation and abolishes the sustained increase evoked by thapsigargin. These effects were not observed when glucose concentration was increased with L-glucose. Therefore, it seems reasonable to assume that the effects of acute glucose overload on Ca²⁺ transients observed in the present experiment is due to the stereospecific action of D-glucose; ie, the metabolism of D-glucose is responsible for its impairing action. The experiments with thapsigargin suggest that acute glucose overload accelerates the Ca²⁺ leak from intracellular store sites through yet-unknown mechanisms and inhibits CRAC, under the assumption that the sustained increase in [Ca²⁺]_i evoked by thapsigargin is predominantly due to CRAC. It is known that thapsigargin specifically inhibits the endoplasmic Ca²⁺- ATPase, thereby depleting intracellular store sites by Ca²⁺ leak,³⁰ and in the control endothelial cells, application of thapsigargin activated the CRAC mechanism, as reported previously.³¹ CRAC is a major Ca²⁺ entry pathway for the endothelium, which does not possess voltage-dependent Ca²⁺ channels.³² Thus, inhibition of CRAC would severely deprive the endothelial cell of Ca²⁺. In vascular smooth muscle cells, it has also been reported that thapsigargin-induced Ca²⁺ entry was inhibited by incubation with 25 mmol/L glucose for 48 hours³³ .

On the other hand, it is also conceivable that glucose overload impairs the Ca²⁺ pumping into store sites or into the extracellular space through endoplasmic or plasmalemmal Ca²⁺ pumps, since the falling phase of the Ca²⁺ transient evoked by ATP was extremely prolonged after incubation with high glucose solution. Prolongation of t_1/3 of the 10 µmol/L ATP–induced Ca²⁺ transient (Fig 3B) may indicate the impairment of the plasmalemmal and/or endoplasmic Ca²⁺ pump or the acceleration of Ca²⁺ entry. Since La³⁺, which inhibits Ca²⁺ passage nonselectively through the plasmalemma, mimics the effects of glucose overload (Fig 5B), the impairment of the Ca²⁺ pump rather than the acceleration of Ca²⁺ entry may be involved in the prolongation of the time course.

However, the time course of Ca²⁺ removal after thapsigargin-induced [Ca²⁺]_i elevation was much faster in the high glucose condition than in control (Fig 4). These contradicting results suggest the possibility that the inhibition of the plasmalemmal Ca²⁺ pump may not be responsible for the dominant effect of glucose overload. Thapsigargin-induced [Ca²⁺]_i responses in control or high glucose were not substantially altered by La³⁺ (Fig 5C and 5D), thereby suggesting that the plasmalemmal Ca²⁺ pump does not contribute by removing the leaked Ca²⁺ induced by thapsigargin. Ruthenium red (30 µmol/L) also did not affect the thapsigargin-induced Ca²⁺ leak in the high glucose condition. Thus, assuming that ruthenium red (30 µmol/L) inhibits the mitochondrial uptake of intracellular Ca²⁺,³⁴ this indicates that the uptake by mitochondria may not be involved in the removal of leaked Ca²⁺ induced by thapsigargin. It has been reported that the cell has a huge Ca²⁺-buffering capacity, mainly due to cytosolic proteins.²⁷ Furthermore, it is generally considered that uptake of Ca²⁺ into the nucleus plays a role in the Ca²⁺ homeostasis.³⁵ In the present experiments, however, it was not technically feasible to evaluate the effect of glucose overload on the Ca²⁺-buffering capacity of cytosolic proteins or uptake by the nucleus. Further experiments are needed to understand the detailed effects of glucose overload on the intracellular Ca²⁺ mobilization in the endothelial cells.

The changes in [Ca²⁺]_i induced by glucose overload observed in the present experiments could be overcome with SOD or mimicked by coincubation with xanthine/XO. These observations strongly indicate the possible role of O₂^- in the action of acute glucose overload on the Ca²⁺ mobilization. Wesson and Elliott,³⁶ on the other hand, reported that hydrogen peroxide but not O₂^- affects Ca²⁺ entry and extrusion in endothelial cells of the calf pulmonary artery.³⁶ However, hydrogen peroxide is unlikely to play a role in the present experiments, since catalase as well as deferoxamine did not restore glucose overload impairment of Ca²⁺ mobilization.

Recently, it has been shown that high glucose treatment enhances bradykinin-induced Ca²⁺ transient via the generation of O₂^- in endothelial cells.¹¹ Furthermore, preincubation for 24 hours of porcine aortic endothelial cells with 3-O-methylglucopyranose, a nonmetabolizing D-glucose analogue, also increased the generation of O₂^-.¹¹ The effects of high glucose or 3-O-methylglucopyranose were abolished by coincubation with SOD, as in the present experiments. Therefore, the authors postulated that accumulation of O₂^- by glucose overload would result in pronounced Ca²⁺/EDRF signaling via a yet-unknown mechanism.¹¹ However, in the present experiments, acute glucose overload did not enhance but decreased the Ca²⁺ signaling, including Ca²⁺ oscillation. In this regard, it was reported that acute glucose overload impairs endothelium-dependent vasodilation in arterioles.^{12 14} The precise reason for the discrepancy between the present and previous observations^{10 11} is unknown. However, one possible explanation may be that the discrepancy is due to the difference in the pattern of endothelial Ca²⁺ signaling in response to bradykinin and ATP, since bradykinin induced a steep increase in [Ca²⁺]_i¹¹ and ATP induced an oscillatory increase (present study).

In conclusion, acute glucose overload impairs intracellular Ca²⁺ mobilization probably via accumulation of O₂^- in bovine aortic endothelial cells, thereby abolishing ATP-induced Ca²⁺ oscillation. The present observations suggest the possibility that the O₂^- scavenger could be used therapeutically to reduce acute hyperglycemic vascular complications.

	Selected Abbreviations and Acronyms

 

CRAC = Ca2+ release–activated Ca2+ entry EDRF = endothelium-derived relaxing factor SOD = superoxide dismutase t1/3 = time to decrease [Ca2+]i to 1/3 peak

	Acknowledgments

 
This study was supported in part by a grant-in-aid from the Ministry of Education of Japan. We thank Dr K. Creed for critical reading of the manuscript.

Received August 8, 1997; accepted December 16, 1997.

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