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Discordant effects of nicotine on endothelial cell proliferation, migration, and the inward rectifier potassium current
Journal of Molecular and Cellular Cardiology 38 (2005) 315–322 www.elsevier.com/locate/yjmcc
Original Article
Discordant effects of nicotine on endothelial cell proliferation, migration, and the inward rectifier potassium current Christoph Rüdiger Wolfram Kuhlmann a,*, Wolfram Scharbrodt a, Christian Alexander Schaefer a, Astrid Kerstin Most a, Ulrich Backenköhler a, Thomas Neumann a, Harald Tillmanns a, Bernd Waldecker a, Ali Erdogan a, Johannes Wiecha b a
Department of Cardiology and Angiology, Justus-Liebig-University of Giessen, Klinikstr. 36, 35392 Giessen, Germany b Department of Internal Medicine, Hospital Bad Orb, Germany Received 9 July 2004; received in revised form 9 November 2004; accepted 12 November 2004 Available online 20 January 2005
Abstract The inward rectifier K+ current (Kir) determines the resting membrane potential of endothelial cells. Basic fibroblast growth factor (bFGF) has been shown to activate Kir and acts as angiogenic factor and vasodilator. In contrast, nicotine has been demonstrated to reduce endotheliumdependent vasorelaxation by increasing radical formation. Aim of the present study was to investigate whether nicotine modulates Kir and if this plays a role in bFGF-mediated proliferation, migration and nitric oxide (NO)-formation of endothelial cells. Using the patch-clamp technique in cultured endothelial cells of human umbilical cord veins (HUVEC), we found characteristic Kir, which were blocked by extracellular barium (100 µmol/l). Perfusion with nicotine (1 nmol/l–10 µmol/l) revealed a dose-dependent reduction of Kir. The simultaneous perfusion with bFGF (50 ng/ml) and nicotine (10 µmol/l) still significantly reduced Kir (n = 8; P < 0.01). Cell counts revealed that bFGFmediated proliferation of HUVEC was significantly inhibited when using 1–10 µmol/l nicotine (n = 8, P < 0.01). The bFGF-induced endothelial cell migration—examined using the “Fences-Migration-Assay”—was significantly reduced by 10 µmol/l nicotine (n = 12; P < 0.05). NO-production was examined using a cGMP-Radioimmunoassay. The significant bFGF-induced increase of cGMP-levels was reduced by nicotine (n = 10; P < 0.05). Our data indicate that the modulation of Kir seems to be an essential pathway in the antagonistic effects of nicotine on bFGF-mediated endothelial cell growth, migration and NO-formation. © 2004 Published by Elsevier Ltd. Keywords: Basic fibroblast growth factor; Nicotine; Inward rectifier potassium current; Endothelium; Angiogenesis; Nitric oxide
1. Introduction Vascular endothelial cells play an essential role in the process of angiogenesis and vessel repair. Basic fibroblast growth factor (bFGF), which is released from endothelial cells and macrophages during hypoxia or vascular injury, takes part in this process by influencing endothelial proliferation and migration [1–4]. Nicotine is a major component of cigarette smoke. Studies have shown that nicotine increases the production of prostacyclin and enhances macromolecular trans-
* Corresponding author. Tel.: +49-641-994-7266; fax: +49-641-994-7219. E-mail address: [email protected] (C.R.W. Kuhlmann). 0022-2828/$ - see front matter © 2004 Published by Elsevier Ltd. doi:10.1016/j.yjmcc.2004.11.017
port, furthermore modulation of DNA synthesis and proliferation of vascular endothelial cells were reported [5–7]. It is well documented that many of the endothelial functions such as the synthesis and release of nitric oxide (NO), the regulation of permeability, or the control of the cell cycle are initiated by Ca2+-dependent mechanisms [8–11]. The membrane potential, which modulates the driving force for transmembrane Ca2+ influx, is an important regulator of intracellular Ca2+ signaling and thereby regulates intracellular signaling cascades in endothelial cells [12–14]. Inward rectifier K+ currents (Kir) are thought to determine the resting membrane potential [11]. For this reason, changes of the Kir through vasoactive substances may be of importance to the endothelial regulatory functions. Evidence is growing that ion channels are involved in the process of cell proliferation and NO-generation. Prolifera-
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tion of human melanoma cells was inhibited by blockers of delayed rectifier potassium channels, and tamoxifen has been shown to block K+ channel-dependent proliferation in neuroblastoma cells [15,16]. Furthermore, voltage-dependent gating of the inward-rectifying K+ current was associated to the regulation of the cell cycle [17]. In addition to these findings in cancer cells, mitogenic peptides like platelet-derived growth factor (PDGF), and bFGF have been shown to activate ion channels, thereby regulating the cell growth of fibroblasts and endothelial cells. Activation of endothelial Ca2+-activated K+ channels by bFGF is linked to the bFGF-induced endothelial cell growth [18,19]. K+ channels have been shown to influence endotheliumdependent vasodilatation, since intracellular calcium has been shown to be essential for agonist-induced NO-formation [20]. Furthermore, it has been demonstrated that Ca2+-activated K+ channels of large conductance (BKCa) are associated with the regulation of NO synthesis [21–24]. The aim of our study, therefore, was to determine whether Kir is modulated by nicotine and to assess the role of nicotineinduced Kir modulation in bFGF-mediated proliferation, migration and NO-production of human endothelial cells.
2. Methods 2.1. Isolation and culture of endothelial cells Endothelial cells were isolated from human umbilical cord veins (HUVEC) and cultured in endothelial basal medium (EBM; Promo Cell, Heidelberg, Germany) as described recently [23]. HUVEC of passage three to eight were used in all experiments. 2.2. Electrophysiology Whole-cell recordings using the patch-clamp technique were performed as described by Hamill et al. [25]. Recording protocols and solutions were used as specified before [26]. In some experiments 100 µmol/l barium (Sigma, Deisenhofen, Germany), 50 ng/ml human bFGF (PeproTech, London, UK), or 1 nmol/l–10 µmol/l nicotine (Sigma) were added to the bath solution. 2.3. Endothelial proliferation To examine cell proliferation, HUVEC of confluent primary cultures were trypsinized (0.05% (w/v) trypsin and 5 mmol/l EDTA containing Ca2+ free solution) and seeded at a density of 20,000 cells/well. On the first day (day 0) the cells were incubated in above mentioned EBM. The following days the incubation medium was modified by adding 50 ng/ml bFGF and/or barium (100 µmol/l), and/or various concentrations of nicotine (0.001–10 µmol/l). The modified medium was replaced every 2 days and counting was done on day 7 using a neubauer chamber. The number of HUVEC
is expressed per well. To examine possible cytotoxic effects, cell viability was tested using trypan blue staining of HUVEC treated as described above. 2.4. Endothelial migration The migration of HUVEC was investigated using the “Fences-Migration-Assay” as described before [27]. Briefly, the wells of a 24-well plate were precoated with 0.2% gelatin. The Fences Migration inserts were put into the wells, and the plate was placed in a 37 °C incubator for 1 h. The center of each insert was charged with 30,000 cells in 150 µl EBM to obtain a confluent cell layer. After allowing HUVEC to attach (5 h/37 °C/5% CO2), the inserts were removed and the cell layer was washed twice in order to remove nonadherent cells. Subsequently, EBM containing variations of the following substances was applied: control (no drugs), bFGF (50 ng/ml), nicotine (0.001–10 µmol/l). The endothelial cell monolayers were allowed to expand for 4 days and the culture medium containing the stimulation substances was replaced every 24 h. At the end of the incubation the cells were fixed in 1:1 methanol/ethanol solution for 20 min and stained in Giemsa solution. Four diameters (45° angle) of the cell covered area were measured. Results are demonstrated as mean values of the measured diameters in millimeter. 2.5. cGMP-Radioimmunoassay Endothelial NO-production was examined using a cGMPRadioimmunoassay-Kit (cGMP-RIA; Amersham, Freiburg, Germany). HUVEC were stimulated for 30 min with combinations of the following substances: bFGF (50 ng/ml), and nicotine (10 µmol/l). Incubation was stopped by the addition of ice cold ethanol. Afterwards cGMP-levels and protein contents (Bradford assay; Bio Rad; Heidelberg; Germany) were analyzed according to the manufacturer’s instructions. Data are expressed as means of cGMP in pmol/mg protein ± S.E.M. 2.6. Statistical analysis Statistical significance for repeated measurements of Kir was determined by using a Friedman test (P < 0.05; SPSSWindows; version 5.0.2) and for the following multiple comparisons by means of the Nemenyi test. The dose–response curve to describe the effect of nicotine on Kir was achieved by fitting the data using a single sigmoidal function. Data of cell proliferation, cell viability, migration and cGMPmeasurements were analyzed by ANOVA (SPSS for Windows; version 5.0.2). Results are expressed as mean value ± S.E.M. 3. Results 3.1. Inward rectifier K+ current in HUVEC To measure inward rectifier K+ currents hyperpolarizing voltage steps were applied from a holding potential of –20 mV
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to test potentials ranging from –45 to –120 mV (steps: –15 mV) to single endothelial cells in the whole-cell configuration of the patch-clamp technique. At –120 mV the current showed a fast inactivation. The reversal potential of the currents in these cells was –78 ± 8 mV (n = 10), which is close to the expected K+ equilibrium potential (EK: –83 mV). Typically inward rectifier K+ currents show a high-affinity block by extracellular barium. To proof for the existence of Kir in our HUVEC, we added 100 µmol/l barium to the bath solution and elicited inward currents in the whole-cell patchclamp mode. As described by other working groups inward rectifier K+ currents in endothelial cells were completely and reversibly blocked by 100 µmol/l barium as demonstrated in a representative recording in Fig. 1 [14,28–30]. 3.2. Effects of nicotine on the inward rectifier K+ current To test whether nicotine influences endothelial Kir activity, we applied various concentrations of nicotine to the endothelial cells. When using concentrations between 0.001 and 10 µmol/l we observed a significant reduction of Kir at nicotine concentrations higher than 0.1 µmol/l, which became obvious after a perfusion time of 1 min at test potentials between –90 and –120 mV. A characteristic recording and the summarized data for the concentration of 1 µmol/l is demonstrated in Fig. 2A, B. At the highest concentration of nicotine (10 µmol/l) Kir was reduced by 56.5% (n = 8; P < 0.01; test potential: –90 mV) and the concentration of 0.1 µmol/l nicotine also caused a significant reduction of Kir by 32.5% (n = 8; P < 0.01; test potential: –90 mV). Nicotine at a concentration of 0.001 µmol/l only caused a small, not significant reduction of Kir by 3% (n = 8; n.s.). The dose–response relationship is shown in Fig. 2C.
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3.3. Effect of nicotine on bFGF-induced Kir We have shown previously that the vasoactive substance bFGF that plays an important role in the process of angiogenesis and vascular remodeling, has an effect on the inward rectifier K+-current [1,4,26]. Application of 50 ng/ml bFGF caused an increase of Kir, which is demonstrated by a typical recording as shown in Fig. 3A. Since we observed a significant increase of endothelial Kir by bFGF and nicotine caused a reduction of this current, we were interested in the effect of a simultaneous application of these two substances. For this purpose we simultaneously perfused endothelial cells with 50 ng/ml bFGF and 10 µmol/l nicotine. After 1 min we observed a significant reduction of Kir, which lasted up to 5 min despite the presence of bFGF. An original recording is shown in Fig. 3B. The current was reduced by 40.3% (n = 8; P < 0.01, test potential: –90 mV) as shown in Fig. 3C. 3.4. Bimodal effect of nicotine on endothelial proliferation The dose-dependent effect of nicotine (0.001–10 µmol/l) on the proliferation of HUVEC was examined using cell counts. As demonstrated in Fig. 4, nicotine significantly increased endothelial proliferation (n = 12; P < 0.05). In detail, cell numbers were increased by: 34.2% (0.001 µmol/l), 58.1% (0.01 µmol/l), and 70% (0.1 µmol/l). Nicotine concentrations of 1 and 10 µmol/l did not cause significant changes of proliferation compared to the control group. 3.5. Blockade of bFGF-mediated endothelial cell proliferation by nicotine Our previously published study revealed a significant increase of Kir in endothelial cells by bFGF that was in part
Fig. 1. Inward rectifier K+ currents in HUVEC. Characteristic sample of whole-cell currents evoked by 250 ms voltage steps from a holding potential of –20 mV to test potentials starting from –45 up to –120 mV (15 mV spaced). Recording samples of Kir before and after 3 min of external perfusion with 100 µmol/l barium and a wash out period of 2 min in endothelial cells using a holding potential of –20 mV and test pulses ranging from –45 to –120 mV (steps: 15 mV, duration: 250 ms).
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added nicotine (0.1–10 µmol/l) every 2 days to the standard culture medium. Basic FGF alone caused an expected increase of cell proliferation as demonstrated recently [26]. When nicotine was supplemented, a significant reduction of bFGFmediated cell growth was observed after 7 days, namely –23% at 1 µmol/l and –27% at 10 µmol/l (n = 6; P < 0.01; Fig. 5). To exclude a cytotoxic effect of nicotine on HUVEC, trypan blue staining was performed. Compared to the control group (standard medium, without nicotine) there was no significant increase (n = 16; nicotine vs. control n.s.; H2O2 vs. control P < 0.01) of cell death (viable cells: 91% (control), 87.3% (nicotine 10 µmol/l), 39.6% (H2O2 0.01%). 3.6. Effect of nicotine on endothelial migration In analogy to the results of the cell proliferation experiments we observed a bimodal effect of nicotine on HUVEC migration. As shown in Fig. 6A, nicotine at concentrations from 0.01 to 1 µmol/l caused a significant increase of endothelial migration (n = 12; P < 0.05). Since 10 µmol/l nicotine did not cause significant changes, we examined the effect of this concentration on bFGF-induced migration. As expected, bFGF (50 ng/ml) caused a significant increase of HUVEC migration by 50.6%, which was blocked by 10 µmol/l nicotine. These data are demonstrated in Fig. 6B. 3.7. Inhibition of bFGF-induce cGMP-levels by nicotine The effect of bFGF on endothelial NO synthesis was measured by means of [3H]-cGMP-RIA. Endothelial cGMPlevels were significantly increased from 0.999 ± 0.119 pmol/mg protein (control) to 4.678 ± 0.329 pmol/mg protein (bFGF) when 50 ng/ml bFGF were added. In coherence with the results of the proliferation-, migration- and electrophysiolgical-experiments, nicotine (10 µmol/l) significantly reduced the effect of bFGF on cGMP-levels. The results are summarized in Fig. 7.
4. Discussion
Fig. 2. Effect of nicotine on inward rectifier K+ currents A: Recording samples of Kir 1 min after applying 1 µmol/l nicotine. B: Summarized data of the amplitude changes of Kir during continuous nicotine (1 µmol/l) application (n = 8; *P < 0.01 vs. control). C: Dose–response relationship of the effect of nicotine (1 min application) on Kir amplitude. Data represent mean value ± S.E.M. (n = 8).
responsible for the proliferative response of HUVEC to this growth factor [26]. To examine whether nicotine affects this bFGF-induced Kir modulation-dependent endothelial proliferation, we investigated whether a blockade of Kir by nicotine, influences bFGF-mediated endothelial cell growth. HUVEC initially seeded at a density of 20,000 cells/well were counted on day 7 while being exposed to different culture media. In analogy to our electrophysiological studies we
The aim of this study was to prove whether endothelial Kir modulation by the angiogenic growth factor bFGF is affected by the alkaloid nicotine. If this is the case, it will be of further interest whether a modulation of this ion current by nicotine may have effects on bFGF-mediated proliferation, migration and NO-production of endothelial cells. We found characteristic inward rectifier K+ currents, that have already been identified and characterized in more detail by other working groups [13,28–31]. The reversal potential of these currents was –78 mV, which is close to the expected K+ equilibrium potential. For further validation of Kir in our HUVEC, we applied barium, which is known to cause a highaffinity block of inward rectifier K+ current. As described previously we found a voltage-dependent block of Kir by application of 100 µmol/l barium [26]. Barium completely blocked
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Fig. 3. Effect of nicotine on bFGF-induced inward rectifier K+ currents. A: Traces of whole-cell currents, elicited from voltage pulses ranging from –45 to –120 mV (steps: 15 mV; holding potential: –20 mV; duration: 250 ms) before (control) and after 3 min of application of 50 ng/ml bFGF. B: Recording samples of Kir after 5 min external perfusion with 10 µmol/l nicotine (nic) and 50 ng/ml bFGF. C: Amplitude of Kir measured 175 ms after the beginning of the test potential of –90 mV during simultaneous perfusion with 10 µmol/l nicotine and 50 ng/ml bFGF (n = 8; *P < 0.01 vs. bFGF; mean ± S.E.M.).
Kir and this effect was reversible after a short wash out period, which is in line with the findings of other working groups [12,26,30]. Since nicotine is known to adversely alter endothelial function, we were interested in a possible modulation of Kir by various concentrations of nicotine [7,32]. Indeed, we observed
a dose-dependent reduction of Kir with an ED50 of 10–7.2 mol/l nicotine. As demonstrated recently, the activation of Kir by bFGF in endothelial cells contributes to bFGF-mediated effects on endothelial cells [26]. In detail, we were able to demonstrate that bFGF-induced proliferation and synthesis of NO was
Fig. 4. Effect of nicotine on endothelial proliferation. Endothelial cell number after the treatment with nicotine (nic; 0.001–10 µmol/l). Statistical significance levels (n = 12; *P < 0.05 vs. control) are indicated in culture medium supplemented with various concentrations of nicotine compared to the culture medium without any supplements.
Fig. 5. Inhibition of bFGF-mediated HUVEC proliferation by nicotine. Endothelial cell number after the treatment with bFGF (50 ng/ml) and bFGF supplemented with various concentrations of nicotine (nic). Statistical significance levels (n = 8; *P < 0.01 vs. control; #P < 0.01 vs. bFGF) are indicated in culture medium supplemented with bFGF and various concentrations of nicotine (0.1–10 µmol/l) compared to the culture medium supplemented with bFGF alone.
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Fig. 6. Effect of nicotine and bFGF on endothelial cell migration. Endothelial cell migration is presented as mean values of diameter-measurements in millimeter. The starting diameter was 6.7 mm. HUVEC were stimulated with nicotine (nic; 0.001–10 µmol/l) and bFGF (50 ng/ml). Data represent mean value ± S.E.M. and statistical differences are indicated (n = 12; *P < 0.05 vs. control; #P < 0.05 vs. bFGF).
inhibited by blocking the Kir using a low dose of barium, which is relatively selective for Kir [33,34]. To test whether nicotine influences the bFGF-induced effects, we performed proliferation studies using the same concentrations of nicotine used in the electrophysiological studies. It was shown previously that activation of endothelial inward rectifier K+ current is an important early step in the bFGF-mediated endothelial cell proliferation [26]. It was demonstrated several times
Fig. 7. Nicotine blocks bFGF-induced cGMP-levels. Endothelial cGMPlevels are significantly increased by bFGF (50 ng/ml) compared to the control group (n = 10; *P < 0.05 vs. control). Addition of nicotine (nic; 10 µmol/l) reduced bFGF-induced cGMP-levels significantly (n = 10; #P < 0.05 vs. bFGF). cGMP-levels shown in pmol/mg protein as mean ± S.E.M.
that ionic currents are involved in the regulation of cell proliferation: endothelial proliferation was inhibited by the application of blockers of volume-sensitive Cl– channels and Ca2+activated K+ channels; the proliferation of human melanoma cells was inhibited in the presence of blockers of delayed rectifier potassium channels or by inhibitors of chloride channels [15,19,23,35]. In analogy to the electrophysiological studies, high concentrations (1 and 10 µmol/l) of nicotine caused a dosedependent reduction of bFGF-induced cell proliferation. This reduction of HUVEC proliferation was not due to a direct cytotoxic effect, since nicotine at a concentration of 10 µmol/l did not cause a significant increase of cell death compared to the control group as demonstrated with the trypan blue staining. Furthermore, we observed a bimodal mitogenic effect of nicotine alone. This finding is in line with the results of Heeschen et al. [36]. Interestingly, we did not observe a further increase of bFGF-induced proliferation if nicotine was added at a concentration of 0.1 µmol/l, despite the observation that this dose caused the largest effect if only nicotine was applied. A possible explanation for this discrepancy could be that there are two opposing mechansims of proliferation induction used by nicotine and bFGF. Nicotine induced mitogenesis might depend on prostacyclin generated by the cyclooxygenase (COX), whereas bFGF-induced proliferation is at least partly due to K+ channel activation [36]. In endothelial cells the COX is a possible source of free radicals [37]. It has been demonstrated by Brakemeier et al. [38] that radicals inhibit the activity of K+ channels, which could be an explanation for the effects of nicotine on Kir-mediated bFGF-induced endothelial proliferation. The influence of nicotine on endothelial cell proliferation has been described before controversially [7,36]. Nicotine significantly stimulated endothelial cell proliferation at concentrations lower than 10 nmol/l. In contrast, nicotine concentrations higher than 1 µmol/l decreased endothelial DNA synthesis and proliferation. Since angiogenesis does not only rely on cell proliferation but also on endothelial cell migration, HUVEC migration was examined using the “Fences-Migration-Assay”. In coherence with the results of the proliferation experiments we observed a bimodal effect of nicotine on endothelial proliferation, which is in line with the angiogenesis data presented by Heeschen et al. [36]. As expected, bFGF increased HUVEC migration and again this effect was significantly decreased by the 10 µmol/l nicotine. This result further confirms the hypothesis that high concentrations of nicotine have antiangiogenic effects. Previous studies have provided evidence that besides being a potent angiogenic agent, bFGF works as a vasorelaxing factor [39,40,41,42]. Previously, our working group was able to show that K+ channels play an important role in acetylcholineinduced NO-synthesis [23,24]. Very recently, we were able to demonstrate that barium-induced inhibition of Kir results in decreased bFGF-dependent NO response of endothelial cells [26]. In the present study we have shown that nicotine strongly reduces cGMP-levels in both control- and bFGF-
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stimulated HUVEC. It is tempting to conclude that this effect of nicotine on bFGF-dependent cGMP-levels is due to inhibition of Kir. Although there is no direct evidence for this hypothesis, it seems to be a possible explanation, since our electrophysiological data demonstrate an inhibition of basal and bFGF-induced Kir-activity by high concentrations of nicotine. In addition, it is not clear whether the effects of nicotine are mediated by nicotinic acetylcholine receptors (nACh). Further studies have to be performed to elucidate the role of nACh in nicotine-induced inhibition of the Kir. In conclusion the results of our study show that Kir plays an important role in endothelial proliferation, migration and synthesis of NO caused by bFGF and nicotine inhibits these growth factor-induced effects by blocking the Kir most likely.
References [1]
[2]
[3]
[4]
[5]
[6]
[7] [8]
[9] [10]
[11] [12]
[13]
[14] [15] [16]
Lindner V, Majack RA, Reidy MA. Basic fibroblast growth factor stimulates endothelial regrowth and proliferation in denuded arteries. J Clin Invest 1990;85:2004–8. Michiels C, De Leener F, Arnould T, Dieu M, Remacle F. Hypoxia stimulates human endothelial cells to release smooth muscle cell mitogens: role of prostaglandins and bFGF. Exp Cell Res 1994;213: 43–54. Gajdusek CM, Carbon S. Injury-induced release of basic fibroblast growth factor from bovine aortic endothelium. J Cell Physiol 1989; 139:570–9. Waltenberger J. Modulation of growth factor action: implications for the treatment of cardiovascular diseases. Circulation 1997;96:4083– 94. Boutherin-Falson O, Blaes N. Nicotine increases basal prostacyclin production and DNA synthesis of human endothelial cells in primary cultures. Nouv Rev Fr Hematol 1990;32:253–9. Allen DR, Browse NL, Rutt DL, Butler L, Fletcher D. The effect of cigarette smoke, nicotine and carbon monoxide on the permeability of the arterial wall. J Vasc Surg 1988;7:139–52. Villablanca AC. Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro. J Appl Phyiol 1998;84:2089–98. Lückhoff A, Pohl U, Mülsch A, Busse R. Differential role of extra and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br J Pharmacol 1988;95:189–96. Imagami T, Naruse M, Hoover R. Endothelium as an endocrine organ. Annu Rev Physiol 1995;57:171–89. He P, Curry FE. Endothelial cell hyperpolarization increases [Ca2+]i and venular microvessel permeability. J Appl Physiol 1994;76:2288– 97. Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium. Phys Rev 2001;81:1415–59. Voets T, Droogmans G, Nilius B. Membrane currents and the resting membrane potential in cultured bovine pulmonary artery endothelial cells. J Physiol 1996;497:95–107. Himmel HM, Rasmusson RL, Strauss HC. Agonist-induced changes of [Ca2+]i and membrane currents in single bovine aortic endothelial cells. Am J Physiol 1994;267:C1338–C1350. Nilius B, Viana F, Droogmans G. Ion channels in vascular endothelium. Annu Rev Physiol 1997;59:145–70. Nilius B, Wohlrab W. Potassium channels and regulation of proliferation of human melanoma cells. J Physiol 1992;445:537–48. Rouzaire-Dubois B, Dubois JM. Tamoxifen blocks both proliferation and voltage-dependent K+ channels of neuroblastoma cells. Cell Signal 1990;2:387–93.
321
[17] Arcangeli A, Bianchi L, Becchetti A, Faravelli L, Coronnello M, Mini E, et al. A novel inward-rectifying K+ current with a cell-cycle dependence governs the resting potential of mammalian neuroblastoma cells. J Physiol 1995;489:455–71. [18] Wiecha J, Reineker K, Reitmayer M, Voisard R, Hannekum A, Mattfeldt T, et al. Modulation of Ca2+-activated K+ channels in human vascular cells by insulin and basic fibroblast growth factor. Growth Horm IGF Res 1998;8:175–81. [19] Wiecha J, Münz B, Wu Y, Noll T, Tillmanns H, Waldecker B. Blockade of Ca2+-activated K+ channels inhibits bFGF-induced proliferation of human endothelial cells. J Vasc Res 1998;35:363–71. [20] Luckhoff A, Pohl U, Mulsch A, Busse R. Differential role of extraand intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br J Pharmacol 1988;95:189–96. [21] Jaggar JH, Wellman GC, Heppner TJ, Porter VA, Perez GJ, Gollasch M, et al. Ca2+ channels, ryanodine receptors and Ca(2+)activated K+ channels: a functional unit for regulating arterial tone. Acta Physiol Scand 1998;164:577–87. [22] Stoen R, Lossius K, Persson AA, Karlsson JO. Relative significance of the nitric oxide (NO)/cGMP pathway and K+ channel activation in endothelium-dependent vasodilation in the femoral artery of developing piglets. Acta Physiol Scand 2001;171:29–35. [23] Kuhlmann CRW, Schäfer M, Li F, Sawamura T, Tillmanns H, Waldecker B, et al. Modulation of endothelial Ca(2+)-activated K(+) channels by oxidized LDL and its contribution to endothelial proliferation. Cardiovasc Res 2003;60:626–34. [24] Kuhlmann CRW, Gast C, Li F, Schäfer M, Tillmanns H, Waldecker B, et al. Cerivastatin activates calcium-activated potassium channels and thereby modulates endothelial NO production and cell proliferation. J Am Soc Nephrol 2004;15:868–75. [25] Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp technique for high resolution current recording from cells and cell-free membrane patches. Pflüg Arch 1981;391:85–100. [26] Scharbrodt W, Kuhlmann CRW, Wu Y, Schaefer CA, Most AK, Backenköhler U, et al. bFGF-induced endothelial proliferation and NO-synthesis involves inward rectifier K+ current. Arterioscler Thromb Vasc Biol 2004;24:1229. [27] Fischer EG, Stingl A, Kirkpatrick CJ. Migration assay for endothelial cells in multiwells application to studies on the effects of opioids. J Immunol Methods 1990;128:135–239. [28] Elam TR, Lansman JB. The role of Mg2+ in the inactivation of inwardly rectifying K+ channels in aortic endothelial cells. J Gen Physiol 1995;105:463–84. [29] Pennefather PS, Decoursey TE. A scheme to account for the effects of Rb+ and K+ on inward rectifier K channels of bovine artery endothelial cells. J Gen Physiol 1994;103:549–81. [30] Von Beckerath M, Dittrich M, Klieber H-G, Daut J. Inwardly rectifying K+ channels in freshly dissociated coronary endothelial cells from guinea-pig heart. J Physiol 1996;491:357–65. [31] Mehrke G, Pohl U, Daut J. Effects of vasoactive agonists on the membrane potential of cultured bovine aortic and guinea-pig coronary endothelium. J Physiol 1991;439:277–99. [32] Zidovetzki R, Chen PJ, Fisher M, Hofman FM. Nicotine increases plasminogen activator inhibitor-1 production by human brain endothelial cells via protein kinase C-associated pathway. Stroke 1999;30: 651–5. [33] Katnik C, Adams DJ. An ATP-sensitive potassium conductance in rabbit arterial endothelial cells. J Physiol 1995;485:595–606. [34] Rusko J, Tanzi F, Van Breemen C, Adams DJ. Calcium-activated potassium channels in native endothelial cells from rabbit aorta: conductance, Ca2+ sensitivity and block. J Physiol 1992;455:601–21. [35] Voets T, Szücs G, Droogmans G, Nilius B. Blockers of volumeactivated Cl– currents inhibit endothelial cell proliferation. Pflüg Arch 1995;431:132–4. [36] Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, et al. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nature 2001;7:833–9.
322
C.R.W. Kuhlmann et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 315–322
[37] Lander HM. An essential role for free radicals and derived species in signal transduction. FASEB 1997;11:118–24. [38] Brakemeier S, Eichler I, Knorr A, Fassheber T, Köhler R, Hoyer J. Modulation of Ca2+-activated K+ channel in renal artery endothelium in situ by nitric oxide and reactive oxygen species. Kidney Int 2003; 64:199–207. [39] Meurice T, Bauters C, Vallet B, Corseaux D, Van Belle E, Hamon M, et al. bFGF restores endothelium-dependent responses of hypercholesterolemic rabbit thoracic aorta. Am J Physiol 1997;13:613–7.
[40] Meurice T, Bauters C, Auffray JL, Vallet B, Hamon M, Valero F, et al. Basic fibroblast growth factor restores endothelium dependent reponses after balloon injury of rabbit arteries. Circulation 1996;93: 18–22. [41] Tiefenbacher CP, Chilian WM. Basic fibroblast growth factor and heparin influence coronary arteriolar tone by causing endotheliumdependent dilatation. Cardiovasc Res 1997;34:411–7. [42] Babaei S, Teichert-Kuliszewska K, Monge JC, Mohamed F, Bendecke MP, Stewart DJ. Role of nitric oxide in the angiogenic response in vitro to basic fibroblast growth factor. Circ Res 1998;82:1007–15.
Original Article
Discordant effects of nicotine on endothelial cell proliferation, migration, and the inward rectifier potassium current Christoph Rüdiger Wolfram Kuhlmann a,*, Wolfram Scharbrodt a, Christian Alexander Schaefer a, Astrid Kerstin Most a, Ulrich Backenköhler a, Thomas Neumann a, Harald Tillmanns a, Bernd Waldecker a, Ali Erdogan a, Johannes Wiecha b a
Department of Cardiology and Angiology, Justus-Liebig-University of Giessen, Klinikstr. 36, 35392 Giessen, Germany b Department of Internal Medicine, Hospital Bad Orb, Germany Received 9 July 2004; received in revised form 9 November 2004; accepted 12 November 2004 Available online 20 January 2005
Abstract The inward rectifier K+ current (Kir) determines the resting membrane potential of endothelial cells. Basic fibroblast growth factor (bFGF) has been shown to activate Kir and acts as angiogenic factor and vasodilator. In contrast, nicotine has been demonstrated to reduce endotheliumdependent vasorelaxation by increasing radical formation. Aim of the present study was to investigate whether nicotine modulates Kir and if this plays a role in bFGF-mediated proliferation, migration and nitric oxide (NO)-formation of endothelial cells. Using the patch-clamp technique in cultured endothelial cells of human umbilical cord veins (HUVEC), we found characteristic Kir, which were blocked by extracellular barium (100 µmol/l). Perfusion with nicotine (1 nmol/l–10 µmol/l) revealed a dose-dependent reduction of Kir. The simultaneous perfusion with bFGF (50 ng/ml) and nicotine (10 µmol/l) still significantly reduced Kir (n = 8; P < 0.01). Cell counts revealed that bFGFmediated proliferation of HUVEC was significantly inhibited when using 1–10 µmol/l nicotine (n = 8, P < 0.01). The bFGF-induced endothelial cell migration—examined using the “Fences-Migration-Assay”—was significantly reduced by 10 µmol/l nicotine (n = 12; P < 0.05). NO-production was examined using a cGMP-Radioimmunoassay. The significant bFGF-induced increase of cGMP-levels was reduced by nicotine (n = 10; P < 0.05). Our data indicate that the modulation of Kir seems to be an essential pathway in the antagonistic effects of nicotine on bFGF-mediated endothelial cell growth, migration and NO-formation. © 2004 Published by Elsevier Ltd. Keywords: Basic fibroblast growth factor; Nicotine; Inward rectifier potassium current; Endothelium; Angiogenesis; Nitric oxide
1. Introduction Vascular endothelial cells play an essential role in the process of angiogenesis and vessel repair. Basic fibroblast growth factor (bFGF), which is released from endothelial cells and macrophages during hypoxia or vascular injury, takes part in this process by influencing endothelial proliferation and migration [1–4]. Nicotine is a major component of cigarette smoke. Studies have shown that nicotine increases the production of prostacyclin and enhances macromolecular trans-
* Corresponding author. Tel.: +49-641-994-7266; fax: +49-641-994-7219. E-mail address: [email protected] (C.R.W. Kuhlmann). 0022-2828/$ - see front matter © 2004 Published by Elsevier Ltd. doi:10.1016/j.yjmcc.2004.11.017
port, furthermore modulation of DNA synthesis and proliferation of vascular endothelial cells were reported [5–7]. It is well documented that many of the endothelial functions such as the synthesis and release of nitric oxide (NO), the regulation of permeability, or the control of the cell cycle are initiated by Ca2+-dependent mechanisms [8–11]. The membrane potential, which modulates the driving force for transmembrane Ca2+ influx, is an important regulator of intracellular Ca2+ signaling and thereby regulates intracellular signaling cascades in endothelial cells [12–14]. Inward rectifier K+ currents (Kir) are thought to determine the resting membrane potential [11]. For this reason, changes of the Kir through vasoactive substances may be of importance to the endothelial regulatory functions. Evidence is growing that ion channels are involved in the process of cell proliferation and NO-generation. Prolifera-
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tion of human melanoma cells was inhibited by blockers of delayed rectifier potassium channels, and tamoxifen has been shown to block K+ channel-dependent proliferation in neuroblastoma cells [15,16]. Furthermore, voltage-dependent gating of the inward-rectifying K+ current was associated to the regulation of the cell cycle [17]. In addition to these findings in cancer cells, mitogenic peptides like platelet-derived growth factor (PDGF), and bFGF have been shown to activate ion channels, thereby regulating the cell growth of fibroblasts and endothelial cells. Activation of endothelial Ca2+-activated K+ channels by bFGF is linked to the bFGF-induced endothelial cell growth [18,19]. K+ channels have been shown to influence endotheliumdependent vasodilatation, since intracellular calcium has been shown to be essential for agonist-induced NO-formation [20]. Furthermore, it has been demonstrated that Ca2+-activated K+ channels of large conductance (BKCa) are associated with the regulation of NO synthesis [21–24]. The aim of our study, therefore, was to determine whether Kir is modulated by nicotine and to assess the role of nicotineinduced Kir modulation in bFGF-mediated proliferation, migration and NO-production of human endothelial cells.
2. Methods 2.1. Isolation and culture of endothelial cells Endothelial cells were isolated from human umbilical cord veins (HUVEC) and cultured in endothelial basal medium (EBM; Promo Cell, Heidelberg, Germany) as described recently [23]. HUVEC of passage three to eight were used in all experiments. 2.2. Electrophysiology Whole-cell recordings using the patch-clamp technique were performed as described by Hamill et al. [25]. Recording protocols and solutions were used as specified before [26]. In some experiments 100 µmol/l barium (Sigma, Deisenhofen, Germany), 50 ng/ml human bFGF (PeproTech, London, UK), or 1 nmol/l–10 µmol/l nicotine (Sigma) were added to the bath solution. 2.3. Endothelial proliferation To examine cell proliferation, HUVEC of confluent primary cultures were trypsinized (0.05% (w/v) trypsin and 5 mmol/l EDTA containing Ca2+ free solution) and seeded at a density of 20,000 cells/well. On the first day (day 0) the cells were incubated in above mentioned EBM. The following days the incubation medium was modified by adding 50 ng/ml bFGF and/or barium (100 µmol/l), and/or various concentrations of nicotine (0.001–10 µmol/l). The modified medium was replaced every 2 days and counting was done on day 7 using a neubauer chamber. The number of HUVEC
is expressed per well. To examine possible cytotoxic effects, cell viability was tested using trypan blue staining of HUVEC treated as described above. 2.4. Endothelial migration The migration of HUVEC was investigated using the “Fences-Migration-Assay” as described before [27]. Briefly, the wells of a 24-well plate were precoated with 0.2% gelatin. The Fences Migration inserts were put into the wells, and the plate was placed in a 37 °C incubator for 1 h. The center of each insert was charged with 30,000 cells in 150 µl EBM to obtain a confluent cell layer. After allowing HUVEC to attach (5 h/37 °C/5% CO2), the inserts were removed and the cell layer was washed twice in order to remove nonadherent cells. Subsequently, EBM containing variations of the following substances was applied: control (no drugs), bFGF (50 ng/ml), nicotine (0.001–10 µmol/l). The endothelial cell monolayers were allowed to expand for 4 days and the culture medium containing the stimulation substances was replaced every 24 h. At the end of the incubation the cells were fixed in 1:1 methanol/ethanol solution for 20 min and stained in Giemsa solution. Four diameters (45° angle) of the cell covered area were measured. Results are demonstrated as mean values of the measured diameters in millimeter. 2.5. cGMP-Radioimmunoassay Endothelial NO-production was examined using a cGMPRadioimmunoassay-Kit (cGMP-RIA; Amersham, Freiburg, Germany). HUVEC were stimulated for 30 min with combinations of the following substances: bFGF (50 ng/ml), and nicotine (10 µmol/l). Incubation was stopped by the addition of ice cold ethanol. Afterwards cGMP-levels and protein contents (Bradford assay; Bio Rad; Heidelberg; Germany) were analyzed according to the manufacturer’s instructions. Data are expressed as means of cGMP in pmol/mg protein ± S.E.M. 2.6. Statistical analysis Statistical significance for repeated measurements of Kir was determined by using a Friedman test (P < 0.05; SPSSWindows; version 5.0.2) and for the following multiple comparisons by means of the Nemenyi test. The dose–response curve to describe the effect of nicotine on Kir was achieved by fitting the data using a single sigmoidal function. Data of cell proliferation, cell viability, migration and cGMPmeasurements were analyzed by ANOVA (SPSS for Windows; version 5.0.2). Results are expressed as mean value ± S.E.M. 3. Results 3.1. Inward rectifier K+ current in HUVEC To measure inward rectifier K+ currents hyperpolarizing voltage steps were applied from a holding potential of –20 mV
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to test potentials ranging from –45 to –120 mV (steps: –15 mV) to single endothelial cells in the whole-cell configuration of the patch-clamp technique. At –120 mV the current showed a fast inactivation. The reversal potential of the currents in these cells was –78 ± 8 mV (n = 10), which is close to the expected K+ equilibrium potential (EK: –83 mV). Typically inward rectifier K+ currents show a high-affinity block by extracellular barium. To proof for the existence of Kir in our HUVEC, we added 100 µmol/l barium to the bath solution and elicited inward currents in the whole-cell patchclamp mode. As described by other working groups inward rectifier K+ currents in endothelial cells were completely and reversibly blocked by 100 µmol/l barium as demonstrated in a representative recording in Fig. 1 [14,28–30]. 3.2. Effects of nicotine on the inward rectifier K+ current To test whether nicotine influences endothelial Kir activity, we applied various concentrations of nicotine to the endothelial cells. When using concentrations between 0.001 and 10 µmol/l we observed a significant reduction of Kir at nicotine concentrations higher than 0.1 µmol/l, which became obvious after a perfusion time of 1 min at test potentials between –90 and –120 mV. A characteristic recording and the summarized data for the concentration of 1 µmol/l is demonstrated in Fig. 2A, B. At the highest concentration of nicotine (10 µmol/l) Kir was reduced by 56.5% (n = 8; P < 0.01; test potential: –90 mV) and the concentration of 0.1 µmol/l nicotine also caused a significant reduction of Kir by 32.5% (n = 8; P < 0.01; test potential: –90 mV). Nicotine at a concentration of 0.001 µmol/l only caused a small, not significant reduction of Kir by 3% (n = 8; n.s.). The dose–response relationship is shown in Fig. 2C.
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3.3. Effect of nicotine on bFGF-induced Kir We have shown previously that the vasoactive substance bFGF that plays an important role in the process of angiogenesis and vascular remodeling, has an effect on the inward rectifier K+-current [1,4,26]. Application of 50 ng/ml bFGF caused an increase of Kir, which is demonstrated by a typical recording as shown in Fig. 3A. Since we observed a significant increase of endothelial Kir by bFGF and nicotine caused a reduction of this current, we were interested in the effect of a simultaneous application of these two substances. For this purpose we simultaneously perfused endothelial cells with 50 ng/ml bFGF and 10 µmol/l nicotine. After 1 min we observed a significant reduction of Kir, which lasted up to 5 min despite the presence of bFGF. An original recording is shown in Fig. 3B. The current was reduced by 40.3% (n = 8; P < 0.01, test potential: –90 mV) as shown in Fig. 3C. 3.4. Bimodal effect of nicotine on endothelial proliferation The dose-dependent effect of nicotine (0.001–10 µmol/l) on the proliferation of HUVEC was examined using cell counts. As demonstrated in Fig. 4, nicotine significantly increased endothelial proliferation (n = 12; P < 0.05). In detail, cell numbers were increased by: 34.2% (0.001 µmol/l), 58.1% (0.01 µmol/l), and 70% (0.1 µmol/l). Nicotine concentrations of 1 and 10 µmol/l did not cause significant changes of proliferation compared to the control group. 3.5. Blockade of bFGF-mediated endothelial cell proliferation by nicotine Our previously published study revealed a significant increase of Kir in endothelial cells by bFGF that was in part
Fig. 1. Inward rectifier K+ currents in HUVEC. Characteristic sample of whole-cell currents evoked by 250 ms voltage steps from a holding potential of –20 mV to test potentials starting from –45 up to –120 mV (15 mV spaced). Recording samples of Kir before and after 3 min of external perfusion with 100 µmol/l barium and a wash out period of 2 min in endothelial cells using a holding potential of –20 mV and test pulses ranging from –45 to –120 mV (steps: 15 mV, duration: 250 ms).
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added nicotine (0.1–10 µmol/l) every 2 days to the standard culture medium. Basic FGF alone caused an expected increase of cell proliferation as demonstrated recently [26]. When nicotine was supplemented, a significant reduction of bFGFmediated cell growth was observed after 7 days, namely –23% at 1 µmol/l and –27% at 10 µmol/l (n = 6; P < 0.01; Fig. 5). To exclude a cytotoxic effect of nicotine on HUVEC, trypan blue staining was performed. Compared to the control group (standard medium, without nicotine) there was no significant increase (n = 16; nicotine vs. control n.s.; H2O2 vs. control P < 0.01) of cell death (viable cells: 91% (control), 87.3% (nicotine 10 µmol/l), 39.6% (H2O2 0.01%). 3.6. Effect of nicotine on endothelial migration In analogy to the results of the cell proliferation experiments we observed a bimodal effect of nicotine on HUVEC migration. As shown in Fig. 6A, nicotine at concentrations from 0.01 to 1 µmol/l caused a significant increase of endothelial migration (n = 12; P < 0.05). Since 10 µmol/l nicotine did not cause significant changes, we examined the effect of this concentration on bFGF-induced migration. As expected, bFGF (50 ng/ml) caused a significant increase of HUVEC migration by 50.6%, which was blocked by 10 µmol/l nicotine. These data are demonstrated in Fig. 6B. 3.7. Inhibition of bFGF-induce cGMP-levels by nicotine The effect of bFGF on endothelial NO synthesis was measured by means of [3H]-cGMP-RIA. Endothelial cGMPlevels were significantly increased from 0.999 ± 0.119 pmol/mg protein (control) to 4.678 ± 0.329 pmol/mg protein (bFGF) when 50 ng/ml bFGF were added. In coherence with the results of the proliferation-, migration- and electrophysiolgical-experiments, nicotine (10 µmol/l) significantly reduced the effect of bFGF on cGMP-levels. The results are summarized in Fig. 7.
4. Discussion
Fig. 2. Effect of nicotine on inward rectifier K+ currents A: Recording samples of Kir 1 min after applying 1 µmol/l nicotine. B: Summarized data of the amplitude changes of Kir during continuous nicotine (1 µmol/l) application (n = 8; *P < 0.01 vs. control). C: Dose–response relationship of the effect of nicotine (1 min application) on Kir amplitude. Data represent mean value ± S.E.M. (n = 8).
responsible for the proliferative response of HUVEC to this growth factor [26]. To examine whether nicotine affects this bFGF-induced Kir modulation-dependent endothelial proliferation, we investigated whether a blockade of Kir by nicotine, influences bFGF-mediated endothelial cell growth. HUVEC initially seeded at a density of 20,000 cells/well were counted on day 7 while being exposed to different culture media. In analogy to our electrophysiological studies we
The aim of this study was to prove whether endothelial Kir modulation by the angiogenic growth factor bFGF is affected by the alkaloid nicotine. If this is the case, it will be of further interest whether a modulation of this ion current by nicotine may have effects on bFGF-mediated proliferation, migration and NO-production of endothelial cells. We found characteristic inward rectifier K+ currents, that have already been identified and characterized in more detail by other working groups [13,28–31]. The reversal potential of these currents was –78 mV, which is close to the expected K+ equilibrium potential. For further validation of Kir in our HUVEC, we applied barium, which is known to cause a highaffinity block of inward rectifier K+ current. As described previously we found a voltage-dependent block of Kir by application of 100 µmol/l barium [26]. Barium completely blocked
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Fig. 3. Effect of nicotine on bFGF-induced inward rectifier K+ currents. A: Traces of whole-cell currents, elicited from voltage pulses ranging from –45 to –120 mV (steps: 15 mV; holding potential: –20 mV; duration: 250 ms) before (control) and after 3 min of application of 50 ng/ml bFGF. B: Recording samples of Kir after 5 min external perfusion with 10 µmol/l nicotine (nic) and 50 ng/ml bFGF. C: Amplitude of Kir measured 175 ms after the beginning of the test potential of –90 mV during simultaneous perfusion with 10 µmol/l nicotine and 50 ng/ml bFGF (n = 8; *P < 0.01 vs. bFGF; mean ± S.E.M.).
Kir and this effect was reversible after a short wash out period, which is in line with the findings of other working groups [12,26,30]. Since nicotine is known to adversely alter endothelial function, we were interested in a possible modulation of Kir by various concentrations of nicotine [7,32]. Indeed, we observed
a dose-dependent reduction of Kir with an ED50 of 10–7.2 mol/l nicotine. As demonstrated recently, the activation of Kir by bFGF in endothelial cells contributes to bFGF-mediated effects on endothelial cells [26]. In detail, we were able to demonstrate that bFGF-induced proliferation and synthesis of NO was
Fig. 4. Effect of nicotine on endothelial proliferation. Endothelial cell number after the treatment with nicotine (nic; 0.001–10 µmol/l). Statistical significance levels (n = 12; *P < 0.05 vs. control) are indicated in culture medium supplemented with various concentrations of nicotine compared to the culture medium without any supplements.
Fig. 5. Inhibition of bFGF-mediated HUVEC proliferation by nicotine. Endothelial cell number after the treatment with bFGF (50 ng/ml) and bFGF supplemented with various concentrations of nicotine (nic). Statistical significance levels (n = 8; *P < 0.01 vs. control; #P < 0.01 vs. bFGF) are indicated in culture medium supplemented with bFGF and various concentrations of nicotine (0.1–10 µmol/l) compared to the culture medium supplemented with bFGF alone.
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Fig. 6. Effect of nicotine and bFGF on endothelial cell migration. Endothelial cell migration is presented as mean values of diameter-measurements in millimeter. The starting diameter was 6.7 mm. HUVEC were stimulated with nicotine (nic; 0.001–10 µmol/l) and bFGF (50 ng/ml). Data represent mean value ± S.E.M. and statistical differences are indicated (n = 12; *P < 0.05 vs. control; #P < 0.05 vs. bFGF).
inhibited by blocking the Kir using a low dose of barium, which is relatively selective for Kir [33,34]. To test whether nicotine influences the bFGF-induced effects, we performed proliferation studies using the same concentrations of nicotine used in the electrophysiological studies. It was shown previously that activation of endothelial inward rectifier K+ current is an important early step in the bFGF-mediated endothelial cell proliferation [26]. It was demonstrated several times
Fig. 7. Nicotine blocks bFGF-induced cGMP-levels. Endothelial cGMPlevels are significantly increased by bFGF (50 ng/ml) compared to the control group (n = 10; *P < 0.05 vs. control). Addition of nicotine (nic; 10 µmol/l) reduced bFGF-induced cGMP-levels significantly (n = 10; #P < 0.05 vs. bFGF). cGMP-levels shown in pmol/mg protein as mean ± S.E.M.
that ionic currents are involved in the regulation of cell proliferation: endothelial proliferation was inhibited by the application of blockers of volume-sensitive Cl– channels and Ca2+activated K+ channels; the proliferation of human melanoma cells was inhibited in the presence of blockers of delayed rectifier potassium channels or by inhibitors of chloride channels [15,19,23,35]. In analogy to the electrophysiological studies, high concentrations (1 and 10 µmol/l) of nicotine caused a dosedependent reduction of bFGF-induced cell proliferation. This reduction of HUVEC proliferation was not due to a direct cytotoxic effect, since nicotine at a concentration of 10 µmol/l did not cause a significant increase of cell death compared to the control group as demonstrated with the trypan blue staining. Furthermore, we observed a bimodal mitogenic effect of nicotine alone. This finding is in line with the results of Heeschen et al. [36]. Interestingly, we did not observe a further increase of bFGF-induced proliferation if nicotine was added at a concentration of 0.1 µmol/l, despite the observation that this dose caused the largest effect if only nicotine was applied. A possible explanation for this discrepancy could be that there are two opposing mechansims of proliferation induction used by nicotine and bFGF. Nicotine induced mitogenesis might depend on prostacyclin generated by the cyclooxygenase (COX), whereas bFGF-induced proliferation is at least partly due to K+ channel activation [36]. In endothelial cells the COX is a possible source of free radicals [37]. It has been demonstrated by Brakemeier et al. [38] that radicals inhibit the activity of K+ channels, which could be an explanation for the effects of nicotine on Kir-mediated bFGF-induced endothelial proliferation. The influence of nicotine on endothelial cell proliferation has been described before controversially [7,36]. Nicotine significantly stimulated endothelial cell proliferation at concentrations lower than 10 nmol/l. In contrast, nicotine concentrations higher than 1 µmol/l decreased endothelial DNA synthesis and proliferation. Since angiogenesis does not only rely on cell proliferation but also on endothelial cell migration, HUVEC migration was examined using the “Fences-Migration-Assay”. In coherence with the results of the proliferation experiments we observed a bimodal effect of nicotine on endothelial proliferation, which is in line with the angiogenesis data presented by Heeschen et al. [36]. As expected, bFGF increased HUVEC migration and again this effect was significantly decreased by the 10 µmol/l nicotine. This result further confirms the hypothesis that high concentrations of nicotine have antiangiogenic effects. Previous studies have provided evidence that besides being a potent angiogenic agent, bFGF works as a vasorelaxing factor [39,40,41,42]. Previously, our working group was able to show that K+ channels play an important role in acetylcholineinduced NO-synthesis [23,24]. Very recently, we were able to demonstrate that barium-induced inhibition of Kir results in decreased bFGF-dependent NO response of endothelial cells [26]. In the present study we have shown that nicotine strongly reduces cGMP-levels in both control- and bFGF-
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stimulated HUVEC. It is tempting to conclude that this effect of nicotine on bFGF-dependent cGMP-levels is due to inhibition of Kir. Although there is no direct evidence for this hypothesis, it seems to be a possible explanation, since our electrophysiological data demonstrate an inhibition of basal and bFGF-induced Kir-activity by high concentrations of nicotine. In addition, it is not clear whether the effects of nicotine are mediated by nicotinic acetylcholine receptors (nACh). Further studies have to be performed to elucidate the role of nACh in nicotine-induced inhibition of the Kir. In conclusion the results of our study show that Kir plays an important role in endothelial proliferation, migration and synthesis of NO caused by bFGF and nicotine inhibits these growth factor-induced effects by blocking the Kir most likely.
References [1]
[2]
[3]
[4]
[5]
[6]
[7] [8]
[9] [10]
[11] [12]
[13]
[14] [15] [16]
Lindner V, Majack RA, Reidy MA. Basic fibroblast growth factor stimulates endothelial regrowth and proliferation in denuded arteries. J Clin Invest 1990;85:2004–8. Michiels C, De Leener F, Arnould T, Dieu M, Remacle F. Hypoxia stimulates human endothelial cells to release smooth muscle cell mitogens: role of prostaglandins and bFGF. Exp Cell Res 1994;213: 43–54. Gajdusek CM, Carbon S. Injury-induced release of basic fibroblast growth factor from bovine aortic endothelium. J Cell Physiol 1989; 139:570–9. Waltenberger J. Modulation of growth factor action: implications for the treatment of cardiovascular diseases. Circulation 1997;96:4083– 94. Boutherin-Falson O, Blaes N. Nicotine increases basal prostacyclin production and DNA synthesis of human endothelial cells in primary cultures. Nouv Rev Fr Hematol 1990;32:253–9. Allen DR, Browse NL, Rutt DL, Butler L, Fletcher D. The effect of cigarette smoke, nicotine and carbon monoxide on the permeability of the arterial wall. J Vasc Surg 1988;7:139–52. Villablanca AC. Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro. J Appl Phyiol 1998;84:2089–98. Lückhoff A, Pohl U, Mülsch A, Busse R. Differential role of extra and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br J Pharmacol 1988;95:189–96. Imagami T, Naruse M, Hoover R. Endothelium as an endocrine organ. Annu Rev Physiol 1995;57:171–89. He P, Curry FE. Endothelial cell hyperpolarization increases [Ca2+]i and venular microvessel permeability. J Appl Physiol 1994;76:2288– 97. Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium. Phys Rev 2001;81:1415–59. Voets T, Droogmans G, Nilius B. Membrane currents and the resting membrane potential in cultured bovine pulmonary artery endothelial cells. J Physiol 1996;497:95–107. Himmel HM, Rasmusson RL, Strauss HC. Agonist-induced changes of [Ca2+]i and membrane currents in single bovine aortic endothelial cells. Am J Physiol 1994;267:C1338–C1350. Nilius B, Viana F, Droogmans G. Ion channels in vascular endothelium. Annu Rev Physiol 1997;59:145–70. Nilius B, Wohlrab W. Potassium channels and regulation of proliferation of human melanoma cells. J Physiol 1992;445:537–48. Rouzaire-Dubois B, Dubois JM. Tamoxifen blocks both proliferation and voltage-dependent K+ channels of neuroblastoma cells. Cell Signal 1990;2:387–93.
321
[17] Arcangeli A, Bianchi L, Becchetti A, Faravelli L, Coronnello M, Mini E, et al. A novel inward-rectifying K+ current with a cell-cycle dependence governs the resting potential of mammalian neuroblastoma cells. J Physiol 1995;489:455–71. [18] Wiecha J, Reineker K, Reitmayer M, Voisard R, Hannekum A, Mattfeldt T, et al. Modulation of Ca2+-activated K+ channels in human vascular cells by insulin and basic fibroblast growth factor. Growth Horm IGF Res 1998;8:175–81. [19] Wiecha J, Münz B, Wu Y, Noll T, Tillmanns H, Waldecker B. Blockade of Ca2+-activated K+ channels inhibits bFGF-induced proliferation of human endothelial cells. J Vasc Res 1998;35:363–71. [20] Luckhoff A, Pohl U, Mulsch A, Busse R. Differential role of extraand intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br J Pharmacol 1988;95:189–96. [21] Jaggar JH, Wellman GC, Heppner TJ, Porter VA, Perez GJ, Gollasch M, et al. Ca2+ channels, ryanodine receptors and Ca(2+)activated K+ channels: a functional unit for regulating arterial tone. Acta Physiol Scand 1998;164:577–87. [22] Stoen R, Lossius K, Persson AA, Karlsson JO. Relative significance of the nitric oxide (NO)/cGMP pathway and K+ channel activation in endothelium-dependent vasodilation in the femoral artery of developing piglets. Acta Physiol Scand 2001;171:29–35. [23] Kuhlmann CRW, Schäfer M, Li F, Sawamura T, Tillmanns H, Waldecker B, et al. Modulation of endothelial Ca(2+)-activated K(+) channels by oxidized LDL and its contribution to endothelial proliferation. Cardiovasc Res 2003;60:626–34. [24] Kuhlmann CRW, Gast C, Li F, Schäfer M, Tillmanns H, Waldecker B, et al. Cerivastatin activates calcium-activated potassium channels and thereby modulates endothelial NO production and cell proliferation. J Am Soc Nephrol 2004;15:868–75. [25] Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp technique for high resolution current recording from cells and cell-free membrane patches. Pflüg Arch 1981;391:85–100. [26] Scharbrodt W, Kuhlmann CRW, Wu Y, Schaefer CA, Most AK, Backenköhler U, et al. bFGF-induced endothelial proliferation and NO-synthesis involves inward rectifier K+ current. Arterioscler Thromb Vasc Biol 2004;24:1229. [27] Fischer EG, Stingl A, Kirkpatrick CJ. Migration assay for endothelial cells in multiwells application to studies on the effects of opioids. J Immunol Methods 1990;128:135–239. [28] Elam TR, Lansman JB. The role of Mg2+ in the inactivation of inwardly rectifying K+ channels in aortic endothelial cells. J Gen Physiol 1995;105:463–84. [29] Pennefather PS, Decoursey TE. A scheme to account for the effects of Rb+ and K+ on inward rectifier K channels of bovine artery endothelial cells. J Gen Physiol 1994;103:549–81. [30] Von Beckerath M, Dittrich M, Klieber H-G, Daut J. Inwardly rectifying K+ channels in freshly dissociated coronary endothelial cells from guinea-pig heart. J Physiol 1996;491:357–65. [31] Mehrke G, Pohl U, Daut J. Effects of vasoactive agonists on the membrane potential of cultured bovine aortic and guinea-pig coronary endothelium. J Physiol 1991;439:277–99. [32] Zidovetzki R, Chen PJ, Fisher M, Hofman FM. Nicotine increases plasminogen activator inhibitor-1 production by human brain endothelial cells via protein kinase C-associated pathway. Stroke 1999;30: 651–5. [33] Katnik C, Adams DJ. An ATP-sensitive potassium conductance in rabbit arterial endothelial cells. J Physiol 1995;485:595–606. [34] Rusko J, Tanzi F, Van Breemen C, Adams DJ. Calcium-activated potassium channels in native endothelial cells from rabbit aorta: conductance, Ca2+ sensitivity and block. J Physiol 1992;455:601–21. [35] Voets T, Szücs G, Droogmans G, Nilius B. Blockers of volumeactivated Cl– currents inhibit endothelial cell proliferation. Pflüg Arch 1995;431:132–4. [36] Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, et al. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nature 2001;7:833–9.
322
C.R.W. Kuhlmann et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 315–322
[37] Lander HM. An essential role for free radicals and derived species in signal transduction. FASEB 1997;11:118–24. [38] Brakemeier S, Eichler I, Knorr A, Fassheber T, Köhler R, Hoyer J. Modulation of Ca2+-activated K+ channel in renal artery endothelium in situ by nitric oxide and reactive oxygen species. Kidney Int 2003; 64:199–207. [39] Meurice T, Bauters C, Vallet B, Corseaux D, Van Belle E, Hamon M, et al. bFGF restores endothelium-dependent responses of hypercholesterolemic rabbit thoracic aorta. Am J Physiol 1997;13:613–7.
[40] Meurice T, Bauters C, Auffray JL, Vallet B, Hamon M, Valero F, et al. Basic fibroblast growth factor restores endothelium dependent reponses after balloon injury of rabbit arteries. Circulation 1996;93: 18–22. [41] Tiefenbacher CP, Chilian WM. Basic fibroblast growth factor and heparin influence coronary arteriolar tone by causing endotheliumdependent dilatation. Cardiovasc Res 1997;34:411–7. [42] Babaei S, Teichert-Kuliszewska K, Monge JC, Mohamed F, Bendecke MP, Stewart DJ. Role of nitric oxide in the angiogenic response in vitro to basic fibroblast growth factor. Circ Res 1998;82:1007–15.