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Cardiovascular Research 2003 60(3):626-634; doi:10.1016/j.cardiores.2003.08.010
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Copyright © 2003, European Society of Cardiology

Modulation of endothelial Ca2+-activated K+ channels by oxidized LDL and its contribution to endothelial proliferation

Christoph Rüdiger Wolfram Kuhlmanna, Matthias Schäferb, Fang Lia, Tatsuya Sawamurac, Harald Tillmannsa, Bernd Waldeckera and Johannes Wiecha*,d

aDepartment of Cardiology and Angiology, Justus-Liebig-University of Giessen, Germany
bDepartment of Physiology, Justus-Liebig-University of Giessen, Germany
cDepartment of Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan
dDepartment of Internal Medicine, Hospital Bad Orb, Frankfurter Strasse 2, 63619 Bad Orb, Germany

*Corresponding author. Tel.: +49-6052-918370; fax: +49-6052-918371. Email address: dr.johannes.wiecha{at}telemed.de

Received 27 March 2003; revised 7 August 2003; accepted 21 August 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Oxidized low-density lipoprotein (oxLDL) plays an important role in causing endothelial dysfunction and initiating atherosclerosis. Some of the endothelial functions have been shown to be modulated by changes in cellular electrophysiological properties. Therefore, we analysed the effect of oxLDL on endothelial Ca2+-activated K+ channels (BKCa) and its contribution to oxLDL-mediated changes of proliferation and syntheses of nitric oxide (NO). Methods: The patch-clamp technique was used to study the behavior of BKCa in human endothelial cells of umbilical cord veins (HUVEC). Changes of intracellular Ca2+ were measured by means of Fura-2 imaging. Cell counts and [3H]-thymidine incorporation were used to analyse endothelial proliferation. Synthesis of NO was measured by means of [3H]-cGMP radioimmunoassay. Results: oxLDL (10 µg/ml) caused a significant increase of BKCa activity, whereas preincubation of HUVEC with an antibody against the lectin-like-oxLDL-receptor-1 (LOX-1) abolished BKCa activation. Fura-2 measurements revealed a biphasic increase of intracellular Ca2+ after application of the atherogenic lipid. Endothelial proliferation was significantly increased by oxLDL. The highly selective BKCa inhibitor iberiotoxin (100 nmol/l IBX) blocked this proliferative response. Acetylcholine-induced NO synthesis was significantly decreased by IBX. Interestingly, oxLDL significantly decreased acetylcholine-induced NO synthesis if the production of superoxide was not blocked by antisense oligonucleotides against the NAD(P)H-oxidase. Conclusions: Our data demonstrate that oxLDL activates BKCa, which plays an important role in oxLDL-mediated endothelial proliferation. Acetylcholine-induced NO synthesis is modulated by BKCa, whereas the reduction of acetylcholine-induced NO-synthesis by oxLDL is related to an increase in superoxide production.

KEYWORDS Lipoproteins; K-channel; Endothelial function; Nitric oxide; Atherosclerosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Endothelial dysfunction caused by oxidized low density lipoprotein (oxLDL) has been implicated in the initiation and progression of atherosclerosis [1]. Some of the mechanisms by which this atherogenic lipid is involved in the process of atherosclerosis have already been described. oxLDL has been shown to transcriptionally induce endothelial genes relevant to atherogenesis, such as the expression of monocyte adhesion molecules and the regulation of nitric oxide synthase expression [2,3]. Furthermore, oxLDL stimulates the production of hydrogen peroxide, which causes an increase in endothelial proliferation and reduces NO synthesis [4,5]. Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity [6]. The vascular release of superoxide (O2–) radicals is strongly increased in atherosclerotic arteries [7] and O2– radicals are known to inactivate NO in a chemical reaction resulting in the formation of the cytotoxic radical peroxynitrite [8]. The presence of peroxynitrite-derived nitrotyrosines has been demonstrated in human atherosclerotic lesions [9]. The molecular mechanisms which are involved in endothelial dysfunction induced by oxLDL have not been fully clarified. As far as it is known, various endothelial functions are modulated by membrane potential [10]. Potassium channels have been shown to regulate the membrane potential and thereby influence intracellular calcium levels [11]. It seems to be possible that pathophysiological changes of endothelial functions, which are an early step within the process of atherosclerosis, are in part due to a modulation of potassium channels by oxLDL. Recently, an endothelial receptor for oxLDL, named lectin-like-oxLDL-receptor-1 (LOX-1), was cloned from cultured endothelial cells [12]. It has been suggested that oxLDL uptake through this receptor is an initial step in the signaling of endothelial dysfunction and atherogenesis.

Considering clinical aspects, endothelial dysfunction is characterized by an inadequate vascular response to a vasorelaxant stimulus (i.e. acetylcholine), because of an imbalance between relaxing and contracting factors. Up to now, the evaluation of endothelium-dependent regulation of vascular tone in patients with cardiovascular disorders mainly focused on the role of NO.

Intimal angiogenesis, which is closely related to endothelial proliferation, is another feature of atherosclerotic lesions [13,14]. These new blood vessels are inherently weak and are therefore responsible for the development of intraplaque haemorrhage, sudden increase in plaque volume and the development of plaque instability.

Recently, endothelial Ca2+-activated K+ channels of large conductance have been associated with the regulation of proliferation [15,16] and synthesis of NO [17,18]. Therefore, the aim of our study was to investigate the effect of oxLDL on the Ca2+-activated K+ channel and to prove whether the modulation of this ion channel plays a role in the regulation of endothelial proliferation and synthesis of NO mediated by oxLDL.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Isolation and culture of HUVEC
Endothelial cells were isolated from human umbilical cord veins by a collagenase digestion procedure and grown in culture as described previously [19]. Cells were grown in endothelial cell basal medium (EBM; PromoCell, Heidelberg, Germany) with the addition of 10% fetal calf serum (FCS; PAA, Linz, Austria). The following substances were added to the culture medium: 0.4% endothelial cell growth supplement/heparin (ECGS/H), epidermal growth factor 0.1 ng/ml, hydrocortison 1 µg/ml, basic fibroblast factor 1 ng/ml, gentamicin 50 µg/ml (PromoCell). Culture medium was changed every 48 h. Cell proliferation studies were carried out using endothelial cells from subcultures four to eight.

2.2. Electrophysiological recordings
Single-channel membrane currents were measured by means of the patch-clamp technique [20] in cell-attached and cell-free patches. Patch pipettes of borosilicate glass (Hilgenberg, Malsfeld, Germany) with a final resistance of 5–8 M{Omega} when filled with pipette solution were used. For the recording, a model M-EPC-7 L/M-PC patch-clamp amplifier (List, Darmstadt, Germany) was used. Data were low-pass filtered at 1 kHz (six-pole Bessel filter), and digitalized (sample rate: 10 kHz) using a Digidata 1200A (Axon Instruments, Foster City, CA, USA) A/D converter, and captured on the hard disk of an IBM-compatible personal computer. Analysis of the unitary currents was performed with pClamp 6.0.3. software (Axon Instruments). Open-state probability (NPo) was calculated from the ratio between the channel open time and the total recording time. The mean amplitude of the unitary currents was obtained for individual patches by fitting simple Gaussian distributions to the all-points histogram. Single-channel slope conductance was calculated by linear regression using mean values of the current amplitudes at different voltages.

In case of oxLDL (10 µg/ml) and acetylcholine (1 µmol/l) application, intermittent recordings of BKCa were made up to 30 min. Indirectly, the uptake of oxLDL by LOX-1 was influenced by preincubating cells with 10 µg/ml anti-LOX-1 antibody JTX-20 (a kindly gift of Prof. Sawamura) for 2 h and then performing patch-clamp experiments as mentioned above. After the application of 100 nmol/l iberiotoxin (IBX; Sigma, Daisenhofen, Germany) to outside-out patches, records were obtained after 1 min of exposure to IBX. For all electrophysiological experiments, HUVEC were maintained in an extracellular (bath) solution containing (in mmol/l): NaCl 140; KCl 5; MgCl2 0.5; D-glucose 5.5; HEPES 10; CaCl2 1.5; pH 7.3 (with NaOH). In order to measure single-channel slope conductance a symmetrical 140 mmol/l K+ solution was used. Solutions with different free calcium concentrations were obtained by adding different amounts of EGTA to the bath solution according to the calculations of Fabiato [21]. The standard pipette solution contained (in mmol/l): K+ aspartate 110; KCl 30; HEPES 5; MgCl2 1; pH 7.3 (with KOH). All experiments were conducted at room temperature (20–22 °C).

2.3 Measurements of intracellular Ca2+
Intracellular [Ca2+] was determined using the fluorescent Ca2+ indicator fura-2. HUVEC were loaded with 2.5 µmol/l fura-2-acetoxymethyl ester (AM) (Molecular Probes, Leiden, Netherlands) at 20 °C in the dark, after they had reached confluency. After an incubation period of 45 min, extracellular fura-2-AM was removed and the medium was exchanged with HEPES buffer composed of 25 mmol/l HEPES, 125 mmol/l NaCl, 1.0 mmol/l CaCl2, 2.6 mmol/l KCl, and 1.2 mmol/l MgCl2, 1.2 mmol/l KH2PO4 at pH 7.4. Coverslips were mounted into a temperature-controlled incubation chamber adapted to the fluorescence microscope (IX 70, Olympus, Hamburg, Germany). Fluorescence intensities for both excitation wavelengths were acquired during intervals of 6 s and averaged over 1 min. During incubation, the excitation wavelength alternated between 340 and 380 nm. Emitted light was detected at 510 nm and the background was corrected. Measurements were performed at 32 °C. Ca2+ changes in endothelial cells were analyzed with the TILL Photonics imaging system (Martinsried, Germany) adapted to an upright fluorescence microscope (BX 50 WI, Olympus). Data were expressed as ratio values of fura-2 fluorescence.

2.4. Cell proliferation
Proliferation of HUVEC was measured by means of cell counts with the help of a Neubauer chamber and by using a [3H]-thymidine incorporation assay (Amersham, Freiburg, Germany). For cell counts, HUVEC of confluent primary cultures were seeded at a density of 10,000 cells/well. On the first day (day 0), the cells were incubated in abovementioned basal medium. After 24 h (day 1), medium was exchanged to serum-free EBM with the following supplements: hydrocortison 1 µg/ml and gentamicin 50 µg/ml. On the next day (day 2), medium was exchanged to normal EBM containing only 2% FCS and oxidized low density lipoprotein (oxLDL, 10 µg/ml; Biotrend, Köln, Germany), and/or iberiotoxin (100 nmol/l; Sigma). Cell counts were carried out on day 3 using a Neubauer chamber. For each sample, the mean values of four counts were used for the statistical analysis. [3H]-Thymidine incorporation assay was performed using 10,000 cells/well which were cultured for 24 h in serum-free EBM. Thereafter, HUVEC were incubated with the various supplements as described in the cell count procedure for 22 h before adding 2 µl/ml [3H]-thymidine (0.25 MBq; Amersham) to the medium. After 7 h, incorporation was stopped by adding 10% trichloracetic acid (Sigma). Afterwards, HUVEC were lysed in 250 µl NaOH (1 N) containing 10% SDS (Sigma) for 90 min at 37 °C without CO2. The lysate was transferred to scintivials and the content of [3H]-thymidine was measured using a β-counter (Canberra-Packard, Dreieich, Germany). Because the final activity of tritium-labeled thymidine is strongly influenced by the length of time passed after the initial labeling, it is not useful to calculate thymidine incorporation on the basis of absolute counts per minute, in particular when several experiments are compared. Therefore, we defined the activity of control cells as 100%, and the activity of treated cells was set in relation to the activity of the control cells.

2.5 [3H]-cGMP radioimmunoassay
A [3H]-cGMP radioimmunoassay (cGMP-RIA; Amersham) was used to analyse NO production. HUVEC were seeded at a density of 5000 cells/cm2. After 48 h culture medium was exchanged to serum-free medium for 24 h. Transfection of cells with oligonucleotides against the p22phox subunit of the NAD(P)H-oxidase (Calbiochem, Bad Soden, Germany) was performed referring to the protocol of Heinloth et al. [5]. HUVEC were cultured in serum free EBM for 24 h after the addition of 10 µmol/l antisense oligonucleotides 5'-phosphorothioate-GAT-CTG-CCC-CAT-GGT-GAG-GAC-C-phosphorothioate-3' and 10 µmol/l nonsense oligonucleotides 5'-phosphorothioate-CCA-GGA-GTG-GTA-CCC-CGT-CTA-G-phosphorothioate-3'. Cells were then stimulated for 30 min by replacing the culture medium with bath solution and the addition of the following substances: 1 mmol/l arginine (Arg; Sigma), 1 µmol/l acetylcholine (Ach; Sigma), 10 µg/ml oxLDL and 100 nmol/l IBX. The stimulation was stopped by supplementing 98% ethanol (Riedel-de Haen, Seelze, Germany). The cell lysate was centrifuged, and measurements of cGMP levels of the supernatant were done using a cGMP-RIA.

2.6. Statistical analysis
Statistical significance for repeated measurements of NPo after oxLDL application was determined using a Friedman test (p<0.05; SPSS for Windows; release 10.0) followed by multiple comparisons (Nemenyi test). Data obtained from proliferation studies and cGMP-RIA were analysed by means of ANOVA (p<0,01, SPSS for Windows; release 10.0) followed by post hoc Tukey Test. Results are expressed as mean values±S.E.M.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Identification of BKCa
Initial experiments were conducted to test for the presence of BKCa in our primary cultured human endothelial cells. The application of depolarizing voltage pulses to the patch pipette (cell-attached patches, symmetrical 140 mmol/l K+ solutions) from a holding potential of 0 mV to test potentials ranging from +20 to +120 mV (+20 mV increments) revealed a single-channel slope conductance of 170.3±2.1 pS (n = 7). To verify the Ca2+ dependence of this ion channel, different Ca2+ concentrations ranging from 10–7 to 10–4 mol/l were applied to the internal side of inside-out patches at a test potential of +60 mV. Fitting of channel open probabilities, derived from five excised membrane patches, for each Ca2+ concentration with the Boltzmann equation resulted in half-maximal activation of the channel at pCa 5.9 (n = 4). Further evidence that the unitary outward currents observed in cultured human endothelial cells are carried through Ca2+-activated K+ channels was provided by the effects of the highly specific BKCa blocker IBX. Applying 100 nmol/l IBX to outside-out patches caused a complete block of the unitary outward currents (further details about the identification of the BKCa in HUVEC and about the patch-clamp technique are given in Wiecha et al. [15] and Hamill et al. [20]). Our electrophysiological study to identify Ca2+-activated K+ channels in HUVEC is consistent with the description of BKCa in other endothelial cells [22].

3.2 Effect of oxLDL on BKCa
In order to test whether external oxLDL can modulate BKCa, we performed recordings in cell-attached patches. After a control measurement in an oxLDL-free bath solution, repetitive recordings of BKCa were elicited during a continuous perfusion with 10 µg/ml oxLDL. Since BKCa activity was very low at low depolarizing test potentials, we only studied the channel behavior at pipette potentials of +80 and +100 mV. A representative recording of BKCa activity after oxLDL perfusion is shown in Fig. 1A. The perfusion with oxLDL resulted in an increase of the single-channel open probability (NPo), which was significant after 15 min and lasted up the whole recording time of 30 min. In detail, using a pipette potential of +80 mV, the open-state probability was significantly increased from 0.001±0.001 at control conditions to 0.028±0.020 (n = 21; p<0.05) after 15 min of oxLDL treatment. When applying test potentials of +100 mV, NPo was significantly increased from 0.001±0.001 (control) to 0.036±0.008 after 15 min. of oxLDL perfusion. To test whether this effect is due to the binding of oxLDL to the LOX-1 receptor, recordings in cell-attached patches were performed after preincubating HUVEC with the selective antibody JTX-20 (10 µg/ml). Under this condition, perfusion with oxLDL did not result in an increase of BKCa open-state probability (n = 21). These data are summarized in Fig. 1B. Single-channel slope conductance was not affected by oxLDL (control: 170.3±2.1 pS vs. oxLDL: 166.8±4.1 pS; n = 7).


Figure 1
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  		Fig. 1 Effect of oxLDL on Ca2+-activated K+ channels in HUVEC. (A) Original recordings of BKCa in cell-attached patches after 20 min superfusion with oxLDL (10 µg/ml) at a pipette potential of +100 mV. The closed (c) and open (o) states of the channel are indicated. (B) Plot of the open-state probability (NPo) as a function of time prior to (0 min; control) and during continuous bath application of 10 µg/ml oxLDL with and without 10 µg/ml JTX-20 preincubation as indicated by the vertical bars (n = 21; *p<0.05 vs. control).

 
To verify that the highly selective BKCa blocker IBX is sufficient to block oxLDL-induced BKCa activation, we added 100 nmol/l IBX to the pipette solution and carried out cell-attached measurements of BKCa. Within 2 min after gigaseal formation, we observed a total block of initial BKCa openings which assures the existence of BKCa in the patch. The following application of oxLDL did not alter the blockade of BKCa by IBX (n = 10, data not shown).

3.3 Changes of intracellular [Ca2+] after treatment with oxLDL
Since changes of intracellular calcium by oxLDL have already been described [23], we were interested whether the effects of oxLDL on intracellular Ca2+ are due to the modulation of the BKCa. Administration of HUVEC with oxLDL caused a biphasic rise of cytosolic [Ca2+]. A fast intracellular [Ca2+] peak was observed after 1 min, which immediately declined and after 3 min cytosolic [Ca2+] started to rise slowly again (Fig. 2). The initial increase of the intracellular [Ca2+] was effectively blocked by the addition of 100 nmol/l IBX, indicating that the activation of BKCa contributes to oxLDL-mediated changes of intracellular [Ca2+]. To test whether IBX itself has an effect on endothelial calcium homeostasis, [Ca2+] measurements were carried out after an application of IBX. Compared to the untreated cells, intracellular [Ca2+] was not changed by IBX. The data are summarized in Fig. 2.


Figure 2
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  		Fig. 2 Changes of the cytosolic Ca2+ concentration induced by oxLDL. Measurements of [Ca2+]i after the addition of 10 µg/ml oxLDL. Data are means±S.E.M. (oxLDL, n = 74; oxLDL+IBX, n = 40; IBX, n = 52; control, n = 46).

 
3.4. Effect of oxLDL on cell proliferation
The addition of oxLDL (10 µg/ml) to the basal culture medium revealed a significant increase of the endothelial cell number, which was 131% (n = 12; p<0.01). Because it has already been shown that oxLDL promotes endothelial proliferation in a concentration-dependent manner [5], we did not use further concentrations to verify the proliferative effect of oxLDL. To assess the contribution of BKCa in oxLDL-mediated endothelial proliferation, IBX (100 nmol/l) was applied to the culture medium. Interestingly, the combination of oxLDL and IBX, which caused a total block of BKCa, completely prevented oxLDL-mediated proliferation. To exclude a direct cytotoxic effect of IBX on HUVEC, 100 nmol/l IBX was supplemented to the basal culture medium. Compared to the control group (basal medium, without IBX), no changes in cell number were observed.

To further confirm our results of the cell counts, we analysed endothelial proliferation on DNA level by the measurement of [3H]-thymidine incorporation. Again, treatment of HUVEC with 10 µg/ml oxLDL resulted in a significant increase of endothelial proliferation which was 132% (n = 12; p<0.01). This effect on proliferation was significantly reduced after the addition of 100 nmol/l IBX. No significant changes in [3H]-thymidine incorporation, compared to the control group, were observed when HUVEC were incubated with IBX alone.

Our data demonstrate that endothelial proliferation induced by oxLDL is in part mediated by the activation of BKCa. The summarized data of cell counts and [3H]-thymidine incorporations are shown in Fig. 3.


Figure 3
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  		Fig. 3 Effects of oxLDL on HUVEC proliferation. Cell growth was detected by cell counts (grey column) and 3H-thymidine incorporation assay (black column) when oxLDL (10 µg/ml) and/or iberiotoxin (100 nmol/l) were supplemented. The number of cells is expressed per well ±S.E.M. (n = 12, *p<0.01 vs. oxLDL), and as percentages of the proliferation for 3H-thymidine incorporation assay (n = 12, *p<0.01 vs. control/oxLDL+IBX/IBX).

 
3.5. Effect of oxLDL on acetylcholine-induced NO synthesis
The effect of oxLDL on endothelial NO synthesis was measured using a [3H]-cGMP radioimmunoassay [24]. First of all, HUVEC were stimulated with the combination of 1 mmol/l arginine and 1 µmol/l acetylcholine. Acetylcholine caused a significant increase of the cGMP levels when compared to the control group, which only contained arginine. It is known that acetylcholine increases the activity of BKCa in endothelial cells derived from the rabbit aorta [25]. For this reason, we carried out patch-clamp experiments in HUVEC. Indeed, BKCa open-state probability was increased from 0.001±0.001 at control conditions to 0.038±0.015 after 10 min of continuous perfusion with acetylcholine (n = 25; p<0.05; test potential 100 mV). To assess a possible contribution of the BKCa activation in NO synthesis, cGMP levels were measured after the addition of arginine, acetylcholine and 100 nmol/l IBX to the culture medium. Iberiotoxin significantly reduced the increase of cGMP induced by acetylcholine. In detail, cGMP levels under control conditions (1 mmol/l arginine) were significantly increased from 66±16.41 to 811±153.7 fmol/well when 1 µmol/l acetylcholine was supplemented. This effect was reduced to 168±11.83 fmol/l after the addition of 100 nmol/l iberiotoxin (n = 10; p<0.05). These results demonstrate that NO synthesis, measured via the second messenger cGMP, is modulated by endothelial BKCa activity. The data are summarized in Fig. 4A.


Figure 4
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  		Fig. 4 Influence of BKCa activity on cGMP levels. Arginine (Arg; 1 mmol/l) was supplemented to ensure NOS substrate saturation. Effect of oxLDL (10 µg/ml) on acetylcholine (1 µmol/l)-induced cGMP levels untransfected (A) and NAD(P)H-antisense/nonsense transfected HUVEC (B). Data are means±S.E.M. (n = 10; *p<0.01).

 
To study the effects of oxLDL on acetylcholine-induced NO production, HUVEC were stimulated with 1 mmol/l arginine, 1 µmol/l acetylcholine and 10 µg/ml oxLDL. oxLDL significantly reduced acetylcholine-induced cGMP levels, although oxLDL activates the BKCa. To assess the role of endothelial BKCa within this process, 100 nmol/l IBX was supplemented to the culture medium. The addition of IBX revealed no significant changes compared to the stimulation with oxLDL alone. Fig. 4A summarizes these findings. These results are surprising, since our electrophysiological findings would suggest an increase in cGMP levels due to the strongly increased BKCa open-state probability.

Recently, the working group of Comanicini et al. [26] have shown that oxLDL decreases intracellular levels of nitric oxide by increasing the amount of reactive oxygen species (ROS) production. Their experiments lead to the conclusion that the most likely candidate as a source for oxLDL-induced ROS production is the NAD(P)H-oxidase. For this reason, we transfected HUVEC with antisense oligonucleotides against the NAD(P)H-oxidase subunit p22phox to study the effect of oxLDL on NO production without the interaction of NAD(P)H-oxidase. The successful transfection was monitored by measurements of intracellular free radicals using the dichlorofluorescein fluorescence (DCF) microscopy (data not shown). Production of free radicals was drastically reduced in antisense transfected HUVEC, whereas transfection with nonsense oligonucleotides did not show any differences compared to untreated cells. After HUVEC were successfully transfected, [3H]-cGMP RIA were performed under the same conditions as described above. The cGMP levels in antisense transfected cells were significantly increased when 10 µg/ml oxLDL was supplemented. Interestingly, this effect was abolished after the addition of 100 nmol/l IBX, demonstrating again the influence of BKCa activity on NOS regulation. In nonsense transfected cells, stimulation with oxLDL did not result in an increase of cGMP levels. The data are summarized in Fig. 4B.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The aim of our study was to show that the atherogenic lipid oxLDL modulates endothelial Ca2+-activated K+ channels and thereby influences endothelial cell proliferation and NO synthesis.

In our patch-clamp experiments, we could demonstrate a significant increase of the BKCa open-state probability by oxLDL. Although we did not measure binding, the activation of this ion channel was possibly due to the interaction of oxLDL with the lectin-like receptor for oxLDL (LOX-1). In HUVEC, activation of BKCa is associated with pertussis toxin-sensitive G proteins [15]. It has been shown that oxLDL-induced intracellular signaling is mediated by G proteins [27]. Therefore, we conclude that after interacting with the LOX-1 receptor oxLDL induces a G-protein-dependent signaling pathway resulting in the activation of the BKCa. Under physiological conditions, activation of endothelial BKCa hyperpolarizes the endothelial cell membrane and thereby increases the electrochemical gradient for a maintained Ca2+ entry [28,29]. Therefore, it was necessary to investigate the influence of oxLDL on intracellular Ca2+ levels and to assess the contribution of BKCa on oxLDL-mediated changes of the intracellular Ca2+. Previously, data of Zhao et al. [23] show that oxLDL at a concentration of 100 µg/ml causes an increase of intracellular Ca2+ in endothelial cells. In contrast to these results, we observed a biphasic increase of intracellular Ca2+ at oxLDL concentrations of 10 µg/ml. It is possible that the different findings resulted from different oxLDL preparation procedures. Surprisingly, the early increase of intracellular Ca2+ was significantly reduced when the highly selective BKCa blocker IBX was applied. One possible explanation could be that IBX is internalized by the cells and thereafter interacts with intracellular Ca2+ stores, since under physiological conditions, the early peak is due to Ca2+ release from these internal stores [29]. However, previous data indicate that the second transient Ca2+ increase after agonist stimulation of endothelial cells is related to a rise of the Ca2+-driving force induced by BKCa-dependent membrane hyperpolarisation [29,30].

Recent data indicate growing evidence that BKCa is involved in the regulation of endothelial cell proliferation [15,30]. Our proliferation studies demonstrate an important role of BKCa activation in oxLDL-induced endothelial cell growth. IBX in the concentration of 100 nmol/l caused a complete block of oxLDL-mediated BKCa activity, as well 100 nmol/l IBX did cause a significant inhibition of oxLDL-induced HUVEC proliferation. A direct cytotoxic effect of IBX was excluded, since 100 nmol/l IBX did not significantly change endothelial cell growth regarding cell counts and [3H]-thymidine incorporation. However, BKCa activation is not the only pathway involved in the mitogenic response to oxLDL. Heinloth et al. [5] were able to show that oxLDL-induced production of superoxide is also responsible for endothelial proliferation. On the other hand, nitric oxide inhibits endothelial proliferation and oxLDL has been shown to reduce nitric oxide bioavailability [26,31]. The proliferation of endothelial cells is an important step within the process of angiogenesis and the development of atherosclerotic plaques, because it contributes to intima thickening [13,14]. The weakness of the new blood vessels within the atherosclerotic plaques contributes to intraplaque haemorrhage, which has been related to a sudden increase in plaque volume and the development of plaque instability leading to plaque rupture [32].

Previously, a possible role of endothelial BKCa in the regulation of NO synthesis was described by Stoen et al. [18] and Demirel et al. [33]. Acetylcholine is known to activate BKCa in rabbit endothelial cells [25]. Our patch-clamp studies confirmed that this is also the fact in HUVEC. Furthermore, acetylcholine significantly increased cellular cGMP levels. This effect was blocked by 100 nmol/l IBX, which indicates a close interaction between BKCa activation and endothelial NO production. The reason for this mechanism could be seen in the BKCa-induced increase of intracellular Ca2+ which results in the activation of the NOS. Since oxLDL had been shown to increase BKCa activity, we were surprised that this atherogenic lipid caused a significant reduction of the acetylcholine-induced NO synthesis measured by [3H]-cGMP-RIA, because we have shown that BKCa activation contributes to acetylcholine-induced NO production.

We supposed that oxLDL may directly block the endothelial NOS. Interestingly, Comanicini et al. [26] have shown that oxLDL reduces intracellular levels of nitric oxide not by an inhibition of the NOS but through an increase in the production of ROS. The NAD(P)H-oxidase was identified as a possible source for ROS synthesis. Therefore, HUVEC were transfected with antisense oligonucleotides directed against the p22phox subunit of the NAD(P)H-oxidase to inhibit the synthesis of free radicals, as described by Heinloth et al. [5]. When antisense oligonucleotides were applied, acetylcholine-induced cGMP levels were significantly increased by oxLDL. This increase of cGMP levels was due to the activation of BKCa, since the addition of IBX significantly reduced this effect of oxLDL. Our results confirm the findings of Cominacini et al. and demonstrate the important role of endothelial BKCa in the modulation of the cGMP/nitric oxide pathway. The reduction of NO as a result of ROS generation may also have clinical implications, because it has been recognized that atherosclerotic blood vessels are very susceptible to the development of vasospasm [34,35]. There is growing evidence that endothelial dysfunction characterized by impaired endothelium-dependent vasodilatation is due to an impaired release of vasodilatory factors of endothelial cells. As a result, inadequate vasodilatation causes ischemic manifestations [36].

In summary, our study demonstrates that oxLDL activates calcium-activated potassium channels of large conductance in human endothelial cells via LOX-1 receptors. Interestingly, this ion channel plays an important role in oxLDL-mediated changes of endothelial proliferation. Endothelial NO synthesis is reduced after treatement with oxLDL, due to free radical production. In contrast, after inhibition of free radical synthesis, oxLDL increases endothelial NO synthesis, which involves the activation of calcium-activated potassium channels. These findings provide a new mechanism of oxLDL-induced signaling in endothelial cells.


   Acknowledgements
 
This study was supported by the Deutsche Forschungsgemeinschaft (DFG), Graduiertenkolleg 534.


   Notes
 
Time for primary review 34 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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