Cardiovascular Research

Cardiovascular Research 2000 47(2):394-403; doi:10.1016/S0008-6363(00)00108-5
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Mechanism of Ca²⁺ overload in endothelial cells exposed to simulated ischemia

Yury Ladilov^*, Claudia Schäfer, Alexander Held, Matthias Schäfer, Thomas Noll and H.Michael Piper

Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany

^* Corresponding author. Tel.: +49-641-9947-215; fax: +49-641-9947-239 yury.ladilov{at}physiologie.med.uni-giessen.de

Received 17 December 1999; accepted 12 April 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Several studies have shown that myocardial ischemia leads to functional failure of endothelial cells (EC) whereby disturbance of Ca²⁺ homeostasis may play an important role. The mechanisms leading to Ca²⁺ disbalance in ischemic EC are not fully understood. The aim of this study was to test effects of different components of simulated ischemia (glucose deprivation, anoxia, low extracellular pH (pH_o) and lactate) on Ca²⁺ homeostasis in EC. Methods: Cytosolic Ca²⁺ (Ca_i), cytosolic pH (pH_i) and ATP content were measured in cultured rat coronary EC. Results: In normoxic cells 60 min glucose deprivation at pH_o 7.4 had no effect on pH_i. It only slightly increased Ca_i and decreased ATP content. Reduction of pH_o to 6.5 under these conditions led to marked cytosolic acidosis and Ca_i overload, but had no effect on ATP content. Anoxia at pH_o 6.5 had no additional effect on Ca_i overload, but significantly reduced cellular ATP. Addition of 20 mmol/l lactate to anoxia at pH_o 6.5 accelerated Ca_i overload due to faster cytosolic acidification. Acidosis-induced Ca_i overload was prevented by inhibition of Ca²⁺ release channels of endoplasmic reticulum (ER) with 3 µmol/l ryanodine or by pre-emptying the ER with thapsigargin. Re-normalisation of pH_o for 30 min led to recovery of pH_i, but not of Ca_i. Conclusion: The ischemic factors leading to cytosolic acidosis (low pH_o and lactate) cause Ca_i overload in endothelial cells, while anoxia and glucose deprivation play only a minor role. The ER is the main source for this Ca_i rise. Ca_i overload is not readily reversible.

KEYWORDS Acidosis; Ca-channel; Calcium (cellular); Endothelial function; Ischemia; Reperfusion

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The physiological functions of the coronary endothelium can be disturbed during myocardial ischemia and reperfusion. Loss of the cytosolic Ca²⁺ control in endothelial cells (EC) could be responsible for their functional failure. Elevation of cytosolic Ca²⁺ can reduce endothelial barrier function [1] and may be a cause of the ischemia-induced endothelial dysfunction [2]. The mechanisms leading to elevation of the cytosolic Ca²⁺ concentration in EC under ischemic conditions are still only partly understood.

Metabolic inhibition is an important cause of ionic disbalance in many types of cells. However, EC are exquisitely robust to metabolic stress like anoxia and glucose deprivation and can keep their ATP content under control for extended periods of time [3–5]. Apart from oxygen and glucose deprivation, EC may also be affected in ischemic myocardium by the release of protons and lactate from the surrounding cardiomyocytes which rapidly switch to anaerobic metabolism. These factors, exogenous to the EC, may interfere with their normal function, e.g. lactate may lead to ATP depletion through inhibition of glycolysis [6]. Additionally, extracellular acidosis and lactate may cause acidification of the cytosol, which has been shown to increase reversibly the cytosolic Ca²⁺ concentration in normoxic EC [7]. It is not known to which extent extracellular acidosis and lactate affect Ca²⁺ homeostasis in EC under anoxic conditions and which mechanisms are involved.

The aim of this study was to investigate how extracellular acidosis and lactate influence cytosolic Ca²⁺ homeostasis in coronary EC during simulated ischemia and reoxygenation. For this purpose, we used coronary EC isolated from adult rat heart as a model system. This model has been characterised in respect to normoxic substrate metabolism and metabolic inhibition in several studies before [4,5]. In the experiments of the present study EC were exposed to glucose-free anoxia and the effects of extracellular acidosis (pH_o 6.5) or lactate (20 mM) on cytosolic free Ca²⁺ concentration were investigated. We found that acidification of the cytosol triggers cytosolic Ca²⁺ overload in anoxic EC. The release of Ca²⁺ from the endoplasmic reticulum is the main source of this Ca²⁺ overload.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Cell cultures
Coronary endothelial cells were isolated from 200 to 250 g male Wistar rats and grown in culture as previously described [8]. Experiments were performed with confluent monolayers, 2 days after seeding. As previously reported [1] the purity of these cultures was >95% endothelial cells as determined by uptake of DiI-ac-LDL, contrasted by cells positive for smooth muscle actin <5%.

2.2 Ca²⁺, pH and cell length measurements
To measure cytosolic Ca²⁺ or H⁺ concentrations, endothelial cells were loaded in HEPES-buffered medium 199 with addition of 3 mM probenecid and 4% of newborn calf serum at 33°C for 60 min with acetoxymethyl esters of fura-2 (5 µmol/l) or 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) (1 µmol/l), respectively, followed by 30 min incubation without dyes. The fluorescence from dye-loaded cells was eight to 15 times higher than background fluorescence from unloaded cells. The background fluorescence was subtracted from the fluorescence of the loaded cells.

The cover-slip with loaded cells was introduced into a gas-tight, temperature-controlled (33°C), transparent perfusion chamber positioned in the light path of an inverted microscope. Alternating excitation of the fluorescent dye was performed at wavelength of 340 and 380 nm for fura-2 or 450 and 490 nm for BCECF. Emitted light (490–510 nm for fura-2 and 520–560 nm for BCECF) from a 100x100-µm area within the endothelial monolayer was collected by the photomultiplier. This allowed to collect light from six to ten cells simultaneously, which was represented as integrated signal from a single area. During experiment two to four areas were investigated simultaneously. To avoid the dye bleaching during long-lasting anoxia–reoxygenation experiments, excitation of fura-2 and BCECF was performed at indicated points not longer than 20 s.

2.3 Dye compartmentation and calibration
The loading protocols were selected from a number of variations because they provided the highest yield in fluorescence and minimal dye compartmentation. To assess the extent of intracellular dye compartmentation cells were chemically ‘skinned’ with digitonin as described previously [9]. It was found that the fluorescent signal from intracellular stores did not exceed 20% for fura-2 and 15% for BCECF compared with the signal from the intact cell.

The fura-2 signal was calibrated as described previously [9]. Since the affinity of fura-2 to Ca²⁺ inside a cell is dependent on cytosolic pH (pH_i), the K_d for fura-2 was defined at different pH_i. For this purpose cells loaded with fura-2 were exposed to 5 µmol/l ionomycin and 10 mg/ml nigericin in solution (pH 6.5 or 7.2) containing (in mmol/l): 10 NaCl, 2 NaCN, 125 KCl, 1 MgSO₄, 25 HEPES and different concentrations of Ca²⁺. The Ca²⁺ titration curve for the fura-2 ratio was constructed. K_d was calculated according to the equation of Grynkiewicz [10]:

We found that at pH_i 7.2 the K_d was 309±8 nmol/l (n=6) and for pH_i 6.5 the K_d was 347±7 nmol/l (n=5). The conversion of fura-2 ratios into absolute values of intracellular Ca²⁺ was done using these K_d values. Calibration of the BCECF ratio signal was performed according to Koop et al. [11], with 10 µg/ml nigericin, a K⁺/H⁺ ionophore, and incubation media with various pH values.

2.4 Media
Endothelial cells on glass cover slips were introduced into a perfusion chamber (0.5 ml filling volume) and were superfused at a flow-rate of 0.5 ml/min with modified Tyrode's solution containing (in mmol/l): 140.0 NaCl, 2.6 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.0 CaCl₂, 3.0 glucose and 25.0 HEPES. pH was 7.4 or 6.5 at 33°C. Normoxic medium was equilibrated with air. Medium was made anoxic by autoclaving as described previously [15]. The anoxic medium was glucose-free and equilibrated before and during experiments with 100% N₂. pO₂ in anoxic medium was less than 22 µPa as determined with resazurin [15]. The medium was transferred into the perfusion chamber through gas-tight steel capillaries.

2.5 Detection of Mn²⁺ influx
Since Mn²⁺ can share common entry pathways with Ca²⁺ [12], its influx may be used as a reporter for Ca²⁺ entry. Therefore, we used Mn²⁺ influx as indirect method for evaluation of extracellular Ca²⁺ influx. Measurements of Mn²⁺ influx were made by addition in medium of 1 mmol/l MnCl₂. Entering the cells, Mn²⁺ quenches fura-2 fluorescence, causing a decrease in emission of fura-2 at the Ca²⁺-insensitive isosbestic point (360 nm). Therefore, the rate of decline of the fura-2 fluorescence was used as indicator of the rate of Mn²⁺ influx.

Changes in quench rate were analysed as follows. The initial (before Mn²⁺ addition) intensity of fura-2 fluorescence was taken as 100% of fura-2 fluorescence. Then, 15 min after Mn²⁺ addition, EC were treated with 10 µmol/l ionomycin to determine the background fluorescence, which was taken as 0% of fura-2 fluorescence. The maximal rate of decline of fluorescence after Mn²⁺ addition was expressed as percentage per minute, using this relative scale.

2.6 ATP measurement
To measure intracellular ATP concentration EC were plated on 35 mm diameter Petri dishes. After washing with glucose-free Tyrode's solution (pH 7.4), EC were incubated for 60 min in this solution at pH_o 7.4 or 6.5 with or without 1.5 mmol/l NaCN at 33°C. Experiments were terminated by addition of 1 ml of 0.6 mol/l HClO₄ per dish. Protein was determined according to Bradford [13] using bovine serum albumin as standard. After neutralisation of perchloric acid, extracts of cultures were analyzed for ATP [14].

2.7 Ammonium prepulse
Prepulse with NH₄Cl was performed in EC under normoxic conditions in order to define an effective concentration of HOE642 to inhibit the Na⁺/H⁺ exchanger. For this purpose cells were incubated for 5 min with 20 mmol/l NH₄Cl. The osmolality of NH₄Cl containing medium was corrected by appropriate reduction of NaCl. Wash-out of NH₄Cl led to rapid acidification of the cytosol (pH_i 6.64±0.04) followed by recovery of pH_i to the control value (pH_i 7.11±0.05, n=5) within 10 min. Treatment with 3 µmol/l HOE642 during the wash-out of NH₄Cl did not affect the initial acidification (pH_i 6.61±0.03), but blocked the recovery of pH_i (6,68±0.04, n=6, P<0.05 vs. contol). Thus 3 µmol/l of HOE642 is an appropriate concentration for inhibition of the Na⁺/H⁺ exchanger in EC.

2.8 Experimental protocols
Eight groups of experiments were designed (Fig. 1). In the control group (protocol 1) cells were superfused for 60 min with normoxic glucose-free modified Tyrode's solution at pH_o 7.4. Subsequently, superfusion was continued in presence of 3 mM glucose for another 30 min.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Experimental protocols. The numbers under the bars represent the duration of each period. Black bar=anoxic superfusion. pH 7.4 or 6.5=extracellular pH 7.4 or 6.5. pH 6.9–6.5=extracellular pH 6.9 during first 10 min of anoxia and pH 6.5 during the following anoxia. Lactate 0–20 mM=gradual increase of extracellular lactate (1 mmol/l per min) to 20 mmol/l during initial 20 min of anoxia. THG 20 nM and THG 100 nM=treatment with 20 or 100 nmol/l thapsigargin. Ryanodine 3 µM=treatment with 3 µmol/l ryanodine.

 
In the second group (protocol 2) cells were exposed to 60 min of glucose-free anoxia at pH_o 7.4 and 30 min of reoxygenation at pH_o 7.4 with glucose.

In the third group (protocol 3), cells were exposed to 60 min of glucose-free normoxia at pH_o 6.5 and 30 min further normoxic incubation at pH_o 7.4 with glucose.

In the fourth group (protocol 4) glucose-free anoxia was performed at pH_o 6.5 and reoxygenation at pH_o 7.4 with glucose.

The fifth group (protocol 5) was identical to protocol 4 with exception that 20 mmol/l sodium lactate was present throughout 60 min glucose-free anoxia. The osmolality was corrected by appropriate reduction of NaCl.

In the sixth group (protocol 6), lactate concentration was gradually increased to 20 mmol/l (1 mmol/l per min) in glucose-free anoxic medium with pH_o 6.9 during initial 10 min of anoxia and with pH_o 6.5 during the following 10 min. This protocol was selected from a number of variations because it provided the similar time course of cytosolic acidification as protocol 4.

The seventh group (protocol 7) was identical to the fourth group with exception that cells were treated before anoxia for 20 min with 20 nmol/l thapsigargin, a specific inhibitor of the Ca²⁺-ATPase of the endoplasmic reticulum, in nominally Ca²⁺-free medium. This protocol was found to deplete the intracellular store of Ca²⁺ (see below, Fig. 6A). During anoxia thapsigargin was present at 100 nmol/l.

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 (A) The effect of treatment with 5 µmol/l ionomycin (original recordings of the fura-2 ratio, arbitrary units) in control EC and in EC after 20 min pretreatment with 20 nmol/l thapsigargin (Thaps) in nominally Ca2+-free medium. No release of Ca2+ from endoplasmic reticulum is observed in pretreated EC. (B) Time course of fura-2 ratio (arbitrary units) during 60 min anoxic incubation of EC in glucose-free medium at pHo 6.5 without treatment (Control, protocol 4, ––, n=12) or after pretreatment with 20 nmol/l thapsigargin (Thaps, protocol 7, ––, n=12) or with presence of 3 µmol/l ryanodine during anoxia (Ryan, protocol 8, ––, n=8). Data are means±S.E. *P<0.05 vs. protocol 4.

 
In the eighth group (protocol 8), cells were treated during anoxia at pH_o 6.5 with 3 µmol/l ryanodine in order to inhibit Ca²⁺ release channels of the endoplasmic reticulum.

2.9 Materials
Medium 199 was purchased from Boehringer-Mannheim; newborn and fetal calf serum from Gibco; acetoxymethyl esters of fura-2 and BCECF from Paesel and Lorey; ionomycin, nigericin, thapsigargin and ryanodine from Calbiochem-Novabiochem. All other chemicals were from Merck and of highest purity.

2.10 Statistics
Data are given as mean values±S.E. For each experimental protocol, eight to 20 individual areas of endothelial monolayer were used. The comparison of means between the groups was performed by one-way analysis of variance (ANOVA) followed by a Bonferroni post-hoc test. Changes in parameters within the same group were assessed by multiple ANOVA analysis. Statistical significance was accepted when P<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Effect of extracellular acidosis in normoxia and anoxia on cytosolic Ca²⁺ and pH
Under control conditions the cytosolic free Ca²⁺ concentration in EC amounted to 74±5 nmol/l. 60 min incubation of EC in glucose-free medium at pH_o 7.4 led to a slight increase in cytosolic Ca²⁺ under normoxic conditions (protocol 1, 157±12 nmol/l, n=9, P<0.05 vs. initial control value). Similar Ca²⁺ overload was observed under anoxic incubation (protocol 2) (Fig. 2A).

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (A) Time course of fura-2 ratio (arbitrary units) during 60 min incubation of EC in glucose-free medium under normoxia at pHo 7.4 (protocol 1, ––, n=9) or pHo 6.5 (protocol 3, ––, n=12), or under anoxia at pHo 7.4 (protocol 2, ––, n=18) or pHo 6.5 (protocol 4, ––, n=20). Each protocol was followed by 30 min incubation in glucose-supplemented normoxic medium at pHo 7.4. *P<0.05 vs. protocol 1. Data are means±S.E. (B) Time course of cytosolic pH during 60 min incubation of EC in glucose-free medium under normoxia at pHo 7.4 (protocol 1, ––, n=8) or pHo 6.5 (protocol 3, ––, n=8), or under anoxia at pHo 7.4 (protocol 2, ––, n=7) or pHo 6.5 (protocol 4, ––, n=15). Each protocol was followed by 30 min incubation in glucose-supplemented normoxic medium at pHo 7.4. *P<0.05 vs. protocol 1. Data are means±S.E.

 
In protocols 3 and 4, EC were exposed to normoxia and anoxia at pH_o 6.5. Under these conditions the fura-2 ratio rose significantly faster than at pH_o 7.4. At the end of 60 min incubation the cytosolic Ca²⁺ concentration reached 480±16 nmol/l (n=12, P<0.05 vs. protocol 1) and 504±10 nmol/l (n=20, P<0.05 vs. protocol 1), respectively, and was not different between the groups (Fig. 2A). When cells of either protocol were then re-exposed to oxygenated medium with glucose at pH_o 7.4, the cytosolic Ca²⁺ concentration remained elevated and did not recover within a subsequent 30-min observation period.

In separate experiments anoxia in protocols 2 and 4 was substituted by a treatment with 1.5 mmol/l NaCN. After 60 min incubation, comparable Ca²⁺ accumulations at pH_o 7.4 (142±6 nmol/l, n=8, not significant vs. protocol 2) or at pH_o 6.5 (528±16 nmol/l, n=12, not significant vs. protocol 4) were observed.

Before interventions, cytosolic pH in EC was equal to 7.2 on average. Glucose-free normoxic incubation at pH_o 7.4 had no significant effect on cytosolic pH (Fig. 2B). During glucose-free anoxia at pH_o 7.4, a slight acidification of the cytosol was observed (pH_i after 60 min: 6.93±0.02, n=7, P<0.05 vs. normoxia). In contrast, a pronounced acidification of the cytosol was observed under anoxia at pH_o 6.5. At the end of anoxia cytosolic pH reached 6.44±0.05 (n=15). Exposure of EC to extracellular acidosis (pH_o 6.5) under normoxia led to a similar decline of cytosolic pH. When extracellular pH was restored to 7.4 after acidotic incubation, a rapid recovery of cytosolic pH was observed.

3.2 Effect of extracellular lactate in anoxia on cytosolic Ca²⁺ and pH
In protocol 5, EC were exposed to anoxia at pH_o 6.5 in the continuous presence of 20 mmol/l lactate. Addition of lactate accelerated significantly the fura-2 ratio rise during the initial 10 min of anoxia (Fig. 3A). The subsequent further rise of the fura-2 ratio proceeded in parallel to the control situation of acidotic anoxia. After 60 min of anoxia, Ca²⁺ overload in lactate treated EC was significantly higher than in EC exposed to acidotic anoxia without lactate (602±16 nmol/l, n=16, P<0.05 vs. protocol 4). As in protocol 4, cytosolic Ca²⁺ did not recover to initial level during 30 min reoxygenation at pH_o 7.4 without lactate.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A) Time course of fura-2 ratio (arbitrary units) during 60 min anoxic incubation of EC in glucose-free medium (pHo 6.5) under control condition (protocol 4, ––, n=18) and under lactate treatment. 20 mmol/l lactate was added with the beginning of anoxia (protocol 5, ––, n=16) or a gradual increase of extracellular lactate up to 20 mmol/l during initial 20 min anoxia was applied (protocol 6, ––, n=16). Each protocol was followed by 30 min incubation in glucose-supplemented normoxic medium at pHo 7.4. *P<0.05 vs. protocol 4. Data are means±S.E. (B) Time course of cytosolic pH during 60 min anoxic incubation of EC in glucose-free medium (pHo 6.5) under control condition (protocol 4, ––, n=10) and under lactate treatment. 20 mmol/l lactate was added with the beginning of anoxia (protocol 5, ––, n=11) or a gradual increase of extracellular lactate up to 20 mmol/l during initial 20 min anoxia was applied (protocol 6, ––, n=10). Each protocol was followed by 30 min incubation in glucose-supplemented normoxic medium at pHo 7.4. *P<0.05 vs. protocol 4. Data are means±S.E.

 
The addition of 20 mmol/l lactate during anoxia at pH_o 6.5 significantly accelerated the initial acidification of the cytosol. Already after 10 min cytosolic pH reached 6.57±0.05 (n=11, P<0.05 vs. protocol 4) (Fig. 3B). To prevent this lactate-induced initial drop of pH_i, lactate concentration in anoxic medium was gradually increased and medium pH was lowered in 2 steps during the initial 20 min of anoxia (protocol 6). These experimental conditions, selected from a number of variations, produced a course of cytosolic acidification identical to the one observed in anoxic EC at pH_o 6.5 without lactate (protocol 4). The time course of the fura-2 ratio rise under protocol 6 was the same as under protocol 4 (Fig. 3A), indicating an identical rise of cytosolic Ca²⁺.

3.3 Effect of acidosis in normoxia and simulated anoxia on the intracellular ATP content
To analyse whether acidosis-induced Ca²⁺ overload is caused by energy dysbalance, cellular ATP contents were measured in EC at the end of 60 min incubation in glucose-free media with pH_o of 7.4 or 6.5. In these experiments, anoxic inhibition of oxidative phosphorylation was simulated by treatment with 1.5 mmol/l NaCN. As reported above, we found that substitution of anoxia by NaCN treatment in 60 min glucose-free incubation led to comparable Ca²⁺ accumulation.

60 min of glucose-free normoxic incubation at pH_o 7.4 reduced slightly the cellular ATP content, while it had no effect at pH_o 6.5 (Fig. 4). In contrast, depletion of ATP content by more than 80% was found under treatment with NaCN, similarly at pH_o 7.4 and 6.5.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 ATP contents in EC after 60 min incubation in glucose-free medium at pHo 7.4 with (n=12) or without (n=8) 1.5 mmol/l NaCN treatment, or pHo 6.5 with (n=12) or without (n=10) 1.5 mmol/l NaCN treatment. Data are means±S.E. Dashed line indicate the ATP content before incubation. *P<0.05 vs. before incubation.

 
3.4 Effect of extracellular acidosis in anoxia on Mn²⁺ influx
To investigate the effect of acidosis in anoxic EC on the transplasmalemmal Ca²⁺ influx, Mn²⁺ was used as a reporter for Ca²⁺ entry. Mn²⁺ entering the cells quenches the cytosolic fura-2 fluorescence. The maximal rate of fura-2 fluorescence quenching after Mn²⁺ addition was used to estimate the rate of Ca²⁺ influx from the extracellular space (Fig. 5A). 1 mM Mn²⁺ was added to the anoxic medium after 30 min of anoxic incubation. Anoxia at pH_o 7.4 (protocol 2) did not influence significantly the quenching of fura-2 fluorescence as compared with normoxic control (protocol 1) (Fig. 5B). In contrast, a significant decrease of the fura-2 quenching was found under anoxia at pH_o 6.5 (protocol 4). The latter finding indicates that influx of divalent cations from extracellular medium is reduced under anoxia with acidosis.

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 (A) Original recordings of fura-2 fluorescence (excitation at 360 nm, arbitrary units) in EC incubated in glucose-supplemented normoxic medium at pHo 7.4 without (Control) and with addition of 1 mmol/l MnCl2 (Mn2+). (B) Maximal rate of Mn2+-induced quenching of fura-2 fluorescence in EC exposed for 30 min to glucose-free normoxia at pHo 7.4 (protocol 1, n=8) or glucose-free anoxia at pHo 7.4 (protocol 2, n=8) or glucose-free anoxia pHo 6.5 (protocol 4, n=6). Data are means±S.E. *P<0.05 vs. protocol 1.

 
3.5 Role of endoplasmic reticulum in acidosis-induced Ca²⁺ overload
To investigate the role of endoplasmic reticulum in cytosolic accumulation of Ca²⁺ under anoxia at pH_o 6.5, two approaches were used. First, the intracellular Ca²⁺ store was emptied by pretreatment with thapsigargin, a specific inhibitor of the Ca²⁺ ATPase of the endoplasmic reticulum, in nominally Ca²⁺-free normoxic medium (protocol 7). After this pretreatment the release of Ca²⁺ from endoplasmic reticulum, inducible by 5 µmol/l ionomycin in normoxic EC, was fully abolished (Fig. 6A). When pretreated with thapsigargin, EC were exposed to anoxia at pH_o 6.5 under continuing presence of thapsigargin. Under these conditions, Ca²⁺ overload did not occur. In fact the cytosolic Ca²⁺ concentration was significantly reduced and was lower than in normoxic cells (after 60 min anoxia: 66±6 nmol/l, n=12, P<0.05 vs. protocol 1 and 4) (Fig. 6B).

In a second approach, the inhibitor of ryanodine sensitive Ca²⁺ release channels ryanodine (3 µmol/l) was applied during anoxia (protocol 8). Under these conditions the Ca²⁺ overload, otherwise developing in anoxia at pH_o 6.5, was markedly reduced (after 60 min anoxia: 172±10 nmol/l, n=8, P<0.05 vs. protocol 4) (Fig. 6B).

3.6 Role of the Na⁺/H⁺ exchanger and extracellular Ca²⁺ influx in Ca²⁺ overload during reoxygenation
Since 30 min of reoxygenation of EC after 60 min of anoxia at pH_o 6.4 did not lead to the recovery of cytosolic Ca²⁺ control, we tested the possibility that activation of the Na⁺/H⁺ exchanger with following activation of the reverse mode of the Na⁺/Ca²⁺ exchanger leading to Ca²⁺ influx may contribute to the persistent Ca²⁺ overload. To test this hypothesis a specific inhibitor of the Na⁺/H⁺ exchanger, HOE642, was applied, which at 3 µmol/l blocks the Na⁺/H⁺ exchanger in EC (see Methods). Treatment with 3 µmol/l HOE642 during 30 min reoxygenation under protocol 4 had no effect on the recovery of cytosolic Ca²⁺ (560±18 nmol/l, n=9, vs. 545±12 nmol/l, n=10 in control).

In a second approach, reoxygenation under protocol 4 was performed with nominally Ca²⁺-free medium. Under this condition cytosolic Ca²⁺ also remained elevated (518±20 nmol/l, n=10, vs. 532±14 nmol/l, n=12, in control).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
4.1 Main findings
The aim of the present study was to investigate the effects of extracellular acidosis and lactate on cytosolic Ca²⁺ homeostasis in anoxic coronary EC. The model used in this study was designed to simulate some of the extracellular conditions for EC in ischemic-reperfused myocardium. These are the rapid deprivation of oxygen and glucose and accumulation of protons and lactate which occur in the interstitium as well as in the lumen of coronary vessels during ischemia [16]. The main findings are the following: (1) Extracellular acidosis and lactate promote cytosolic Ca²⁺ overload in anoxic EC due to cytosolic acidification, while anoxia alone does not. (2) Acidosis-induced Ca²⁺ overload is independent of general cellular ATP depletion. It is due to Ca²⁺ release from the endoplasmic reticulum. (3) Acidosis-induced Ca²⁺ overload is, in contrast to cytosolic acidification, not readily reversible.

4.2 Effects of extracellular acidosis and lactate on cytosolic Ca²⁺ and pH
In ischemic myocardium, anoxia occurs in conjunction with acidosis and lactate accumulation. In the cell culture model the relative importance of each of these ischemic factors for ionic disbalance can be evaluated separately. We therefore first investigated the effect of anoxia alone. At pH_o 7.4 glucose deprivation caused a small increase in cytosolic Ca²⁺ which was not higher when anoxia or NaCN were applied simultaneously. When otherwise identical experiments were carried out in media with pH 6.5, the increase in cytosolic Ca²⁺ was markedly augmented. The Ca²⁺ overload developing at pH_o 6.5 was the same under normoxic and anoxic conditions. Extracellular acidosis led to a reduction of cytosolic pH which was also comparable for normoxic and anoxic conditions. When 20 mmol/l lactate was applied with onset of anoxia in medium with pH 6.5, the acidification of the cytosol during the initial 10 min of anoxia was markedly accelerated. This effect of lactate is in agreement with the observation that lactate is transported across the sarcolemma in conjunction with protons [18]. In presence of lactate the accumulation of cytosolic Ca²⁺ was also significantly accelerated during the first 10 min of anoxia. To test whether the lactate-induced rapid initial acidification is the cause of the fast Ca²⁺ accumulation, medium lactate and extracellular acidosis were gradually incremented during initial 20 min of anoxia (protocol 6). Under this protocol, the lactate-induced acceleration of cytosolic acidification remained absent, apparently because sufficient time is given for extrusion of the protons entering the EC together with lactate. In the cytosol of EC treated with lactate in this manner, acceleration of Ca²⁺ overload was not found. This shows that extracellular lactate accelerates cytosolic Ca²⁺ accumulation in anoxic EC only if it enhances cytosolic acidosis.

Together these data indicate that in EC exposed to simulated ischemia cytosolic acidosis, induced by an extracellular rise of H⁺ and lactate, is a key determinant for development of cytosolic Ca²⁺ overload, while anoxia per se plays only a minor role. In agreement with this conclusion, it was reported previously that 60 min hypoxia [17] or 30 min chemical anoxia (NaCN treatment) [19] in glucose-free medium at pH_o 7.4 lead only to a small (<100 nmol/l) rise in cytosolic Ca²⁺ concentration of EC. In the latter study metabolic inhibition did not produce acidification of the cytosol. It was not, however, analysed in these studies whether cytosolic acidosis can enhance this small anoxic Ca²⁺ accumulation. A few studies investigated the effects of cytosolic acidosis on Ca²⁺ homeostasis in normoxic EC. The results are contradictory. Ziegelstein et al. [7] observed a cytosolic Ca²⁺ overload, while Dwyer et al. [20] did not find an effect of intracellular acidification on cytosolic Ca²⁺. The reason for this discrepancy is unknown. In the latter study cytosolic acidification was induced by short (2 min) application of 20 mmol/l sodium propionate at pH_o 7.4. The cytosolic pH was not monitored, however. It is, therefore, possible that the decline of cytosolic pH in this study was too small to induce a cytosolic Ca²⁺ rise.

4.3 The mechanism of acidosis-induced Ca²⁺ overload
It has been shown previously that depletion of the cellular ATP stores of EC by combined inhibition of oxidative phosphorylation and glycolysis is followed by rapid increase in the intracellular Ca²⁺ concentration of EC [1,19]. Since cytosolic acidosis may disturb energy production, we tested whether acidosis-induced Ca²⁺ overload is due to ATP depletion. No depletion of the cellular ATP content was found under normoxia at pH_o 6.5. This condition induced, however, accumulation of Ca²⁺ in the cytosol. We also investigated the effect of mitochondrial inhibition with 1.5 mmol/l NaCN in glucose-free medium. This treatment led to significant and similar loss of ATP at pH_o 7.4 and pH_o 6.5, while only under acidotic conditions cytosolic Ca²⁺ overload was observed. Taken together these results show that acidosis-induced Ca²⁺ overload in EC is not caused by a general loss of cellular ATP. They do not exclude the possibility, however, that loss of ATP from a small subcellular compartment elicits the cytosolic Ca²⁺ rise.

A previous study had indicated that the primary mechanism of Ca²⁺ accumulation induced by decrease in pH_i in EC is mobilisation of Ca²⁺ from endogenous stores rather than transplasmalemmal Ca²⁺ influx [7]. We analysed whether the same mechanism is responsible for the development of Ca²⁺ overload in our experiments.

First, we measured extracellular Mn²⁺ influx as a reporter for Ca²⁺ entry by monitoring the rate of fura-2 quenching with Mn²⁺. We found that acidosis reduced significantly the quenching rate of fura-2 indicating that influx of divalent cations is suppressed by acidosis. Albeit indirectly, the results indicate that acidosis does not activate transplasmalemmal Ca²⁺ influx into EC. Similar data were obtained by Wakabayashi and Groschner [21], who observed a suppression of Ca²⁺ entry by extracellular acidosis in vascular EC. The authors suggested a direct inhibitory effect of extracellular protons on the plasmalemmal Ca²⁺ channels. Extracellular alkalosis was found to promote Ca²⁺ entry into EC [22].

Thereafter, we investigated whether Ca²⁺ release from the endoplasmic reticulum may be responsible for the acidosis-induced Ca²⁺ overload. When, before exposure to acidosis, the endoplasmic reticulum was depleted from Ca²⁺ by thapsigargin pretreatment, no increase in cytosolic Ca²⁺ concentration was observed under acidotic anoxia. In contrast, the cytosolic Ca²⁺ concentration was slightly, but significantly reduced. This reduction is possibly due to the acidotic depression of extracellular Ca²⁺ influx as indicated by the experiments with Mn²⁺. In another set of experiments, the inhibitor of Ca²⁺ release channels of the endoplasmic reticulum, ryanodine, was applied throughout acidotic anoxia. The functional role of these channels in EC was demonstrated previously [23]. In the present study it is demonstrated for the first time that these Ca²⁺ release channels participate in Ca²⁺ overload of EC exposed to ischemic conditions. Taken together, the experiments with thapsigargin and ryanodine are in line with the measurements of Mn²⁺ influx by showing that Ca²⁺ release from the intracellular stores, particularly from the endoplasmic reticulum, rather than Ca²⁺ influx from extracellular medium, is responsible for Ca²⁺ overload in EC under ischemic conditions.

The time course of acidosis-mediated increase in cytosolic Ca²⁺ in this study is slower than that reported for agonist-mediated Ca²⁺ mobilization from intracellular stores by IP₃-dependent release mechanism [24]. The results do not exclude the possibility that acidosis also inhibits the Ca²⁺-ATPase of the endoplasmic reticulum [25]. Inhibition of Ca²⁺ sequestration into the endoplasmic reticulum could also participate in the cytosolic Ca²⁺ overload initiated by the slow Ca²⁺ release through ryanodine sensitive channels.

An important finding of the present study is that Ca²⁺ overload induced by prolonged acidosis is not readily reversible (i.e. within the 30 min observation period in this study) upon a re-normalisation of the cytosolic pH. The reasons for this persistent Ca²⁺ overload remain at present unknown. They do not include general cellular loss of energy since Ca²⁺ overload persists also in normoxic cells exposed to extracellular acidosis in which the cellular ATP content was not reduced. It is also not due to the rapid washout of extracellular protons which may cause a Na⁺ influx through the Na⁺/H⁺ exchanger and subsequent Ca²⁺ influx through the reverse mode of the Na⁺/Ca²⁺ exchanger [26]. Blockade of the Na⁺/H⁺ exchanger with HOE642 or removal of Ca²⁺ from extracellular medium had no effect on cytosolic Ca²⁺ balance during reoxygenation. The latter results exclude the possibility that the lack of recovery of Ca²⁺ balance in the reoxygenation period is due to Ca²⁺ influx from the extracellular medium. It must therefore be due to an inability of the cells to sequestrate intracellularly or extrude to the extracellular space an excess load of cytosolic Ca²⁺.

In conclusion, this study shows that extracellular acidosis and lactate are important factors leading to a disturbance of Ca²⁺ homeostasis in anoxic EC which is not readily reversed upon reoxygenation. The accumulation of cytosolic Ca²⁺ under these conditions is due to cytosolic acidification and does not require cellular ATP depletion. The main source of acidosis-induced Ca²⁺ overload is release of Ca²⁺ from the endoplasmic reticulum.

Time for primary review 25 days.

	Acknowledgements

 
The technical help of H. Holzträger and D. Schreiber is gratefully acknowledged. This work was supported by Grants A3 and A4 of the SFB 547 and LA 1159/2-1 of the Deutsche Forschungsgemeinschaft. Part of this study was part of the thesis of A. Held submitted in fulfillment of the requirements for the degree of Doctor of Medicine at the Justus-Liebig-Universität, Giessen, Germany.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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