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PHAREP-61; No. of Pages 6 Pharmacological Reports xxx (2014) xxx–xxx
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Short communication
HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes Itsuro Kazama *, Asuka Baba, Yoshio Maruyama Department of Physiology I, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan
A R T I C L E I N F O
Article history: Received 18 October 2013 Received in revised form 3 March 2014 Accepted 3 March 2014 Available online xxx Keywords: HMG-CoA reductase inhibitors (statins) Immunosuppressive effects Lymphocytes Kv1.3-channel Membrane capacitance (Cm)
A B S T R A C T
Background: Since lymphocytes predominantly express delayed rectifier K+-channels (Kv1.3) that trigger lymphocyte activation, statins, which exert immunosuppressive effects, would affect the channel currents. Methods: Employing the patch-clamp technique in murine thymocytes, we examined the effects of statins on Kv1.3-channel currents and the membrane capacitance (Cm). Results: Pravastatin significantly suppressed the pulse-end currents of the channels. Lovastatin and simvastatin also suppressed the peak currents, significantly decreasing the Cm. Conclusions: This study demonstrated for the first time that statins inhibit thymocyte Kv1.3-channels. The slow inactivation patterns induced by lovastatin and simvastatin may be associated with their accumulation in the plasma membranes. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (or statins), which potently inhibit cholesterol biosynthesis, are widely used in the treatment of hypercholesterolemia and atherosclerosis [1]. In concert with such lipid-lowering effects, statins have also been demonstrated to exert antiinflammatory or immunomodulatory properties that reduce the risk of cardiovascular events [2]. Regarding the mechanisms by which they exert immunosuppressive effects, Kwak et al. originally found the involvement of major histocompatibility complex class II (MHC-II) expressed on T-lymphocytes [3]. Then, later in vitro studies also revealed that statins, such as pravastatin, lovastatin and simvastatin, suppress the production of pro-inflammatory cytokines from leukocytes [4,5]. These cells, including macrophages and lymphocytes, predominantly express delayed rectifier K+-channels (Kv1.3) in their plasma membranes [6]. Since these channels play crucial roles in the initiation of the immune response [7], statins would affect the channel currents when they modulate the immune response. In our recent studies, we demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs), dihydropyridine Ca2+ channel blockers and macrolide antibiotics suppress the
* Corresponding author. E-mail address: [email protected] (I. Kazama).
Kv1.3-channel currents in thymocytes and thus exert immunomodulatory effects [8–10]. Among them, lipophilic drugs induced microscopic structural changes in the membrane surface, as detected by the increase in the membrane capacitance [9,10]. Since statins, such as lovastatin and simvastatin, are highly lipophilic [11], they would directly disturb the thymocyte plasma membranes and thus affect the Kv1.3-channel currents. To test this, employing the standard patch-clamp whole-cell recording technique in murine thymocytes, we examined the effects of statins on the channel currents and the membrane capacitance. Here, we show for the first time that statins, such as pravastatin, lovastatin and simvastatin, suppress thymocyte Kv1.3-channel currents. We also show that the current suppression patterns of lovastatin and simvastatin were associated with their accumulation in the plasma membranes, as detected by decreases in the membrane capacitance. Materials and methods Cell sources and preparation Male ddy mice (4–5 weeks old), supplied by Japan SLC Inc. (Shizuoka, Japan), were deeply anaesthetized with isoflurane and then sacrificed by cervical dislocation. The protocol for animal use was approved by the Animal Care and Use Committee of the Tohoku University Graduate School of Medicine. Single thymocytes, isolated
http://dx.doi.org/10.1016/j.pharep.2014.03.002 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
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from murine thymus as described previously [8,12], were resuspended in standard external (bathing) solution containing (in mM): NaCl, 145; KCl, 4.0; CaCl2, 1.0; MgCl2, 2.0; Hepes, 5.0; bovine serum albumin, 0.01% (pH 7.2 adjusted with NaOH). They were maintained at room temperature (22–24 8C) for use within 4 h. Electrical setup and patch-clamp recordings We conducted standard whole-cell patch-clamp recordings using an EPC-9 patch-clamp amplifier system (HEKA Electronics, Lambrecht, Germany) as described previously [8,12]. The patch pipette resistance was 4–6 MV when filled with internal (patch pipette) solution containing (in mM): KCl, 145; MgCl2, 1.0; EGTA, 10; Hepes, 5.0 (pH 7.2 adjusted with KOH). After the giga-seal formation, we applied suction briefly to the pipette to rupture the patch membrane. The series resistance of the whole-cell recordings was maintained below 10 MV during the experiments. Peak currents were normalized by the membrane capacitance and expressed as the current densities (pA/pF). All experiments were carried out at room temperature. Drug delivery Pravastatin and simvastatin, purchased from Sigma Chemical Co. (St. Louis, MO, USA) and lovastatin, from Cayman Chemical (Ann Arbor, MI, USA), were separately dissolved in the external solution at final concentrations of 1 mM, 10 mM and 10 mM, respectively. We delivered one of the drugs to the cells by the standing hydrostatic pressure of 3 cm H2O from a nearby pipette as described previously [8,12]. Briefly, using a three-dimensional hydraulic manipulator (M-103, Narishige, Tokyo, Japan), the application pipette initially positioned outside the bathing solution was brought to within 10 mm of the cell surface. Then, to wash out the reagents, the pipette was brought up outside the bathing solution. Whole-cell membrane currents were recorded before and after 30 s exposure to the drug and after a 2 min washout. To rule out the possibility that the observed effect resulted from the procedure of the reagent application, we simply applied the external solution to the cells and confirmed the absence of any significant changes in the channel currents. Membrane capacitance measurements To measure the membrane capacitance of the thymocytes, we employed a sine plus DC protocol using the Lock-in amplifier of the EPC-9 Pulse program. An 800-Hz sinusoidal command voltage was superimposed on the holding potential of 80 mV. The membrane capacitance (Cm), as well as membrane conductance (Gm) and series conductance (Gs), was continuously recorded before and after 30 s exposure to the drugs during the whole-cell recording configuration. Statistic analyses Data were analyzed using PulseFit software (HEKA Electronics, Lambrecht, Germany), IGOR Pro (WaveMetrics, Lake Oswego, OR, USA) and Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and reported as means SEM. Statistical significance was assessed by two-way ANOVA followed by Dunnett’s or Student’s ttest. A value of p < 0.05 was considered significant. Results and discussion Effects of statins on Kv1.3-channel currents in murine thymocytes Stepwise changes in the membrane potential, from the holding potential of 80 mV to the various depolarizing potential levels,
evoked membrane currents in the thymocytes, showing voltagedependent activation and inactivation patterns characteristic to Kv1.3 (Figs. 1A and B, 2A and 3A) [8,12]. In our previous study, since the application of margatoxin, a relatively selective Kv1.3-channel inhibitor, almost totally abolished the currents [12], we identified them with the Kv1.3-channel currents. To investigate the effects of statins on the channel currents, we applied external solution, containing either 1 mM pravastatin, 10 mM lovastatin or 10 mM simvastatin, to the thymocytes and examined the changes in the whole-cell currents (Figs. 1–3). Fig. 1A shows an example of a control recording with the application of the external solution alone, which confirmed that our procedure of reagent application, a puff application by constant hydrostatic pressure with a nearby pipette, was without significant effects on the Kv1.3-channel currents. In previous in vitro studies, single cells required less than 10 mM of lovastatin and simvastatin to effectively elicit their pharmacological effects on ion-channel currents, such as Ca2+ channel currents in vascular smooth muscle cells [13] and K+ channel currents in cardiomyocytes [14]. In those studies, the drugs were previously dissolved in whole culture media, or continuously infused into the bathing solution. In the present study, however, since we delivered the drugs to single, isolated cells by a puff application method, there was the possibility of partial and insufficient exposure of the drugs to the cells. Therefore, we applied significantly higher concentrations of lovastatin and simvastatin (10 mM) than those used in the previous studies (Figs. 2 and 3). In humans, the serum concentrations of lovastatin or simvastatin after oral administration reach around 0.1 mM [13,15], which is much lower than the concentrations used in our study. However, in patients with hypercholesterolemia, since statins are usually used for a long period of several years or more, their effects might be evoked at lower concentrations under chronic administration. Additionally, in the previous studies, pravastatin required much higher doses than lovastatin or simvastatin to exert its pharmacological effects [13,15]. In the present study, however, 100 mM pravastatin had little effect on Kv1.3-channel currents in thymocytes (data not shown) and considered to be still insufficient. Therefore, we applied a higher dose at 1 mM (Fig. 1B–D). Pravastatin suppressed the Kv1.3-channel currents in thymocytes (Fig. 1B), although this drug did not significantly lower the peak currents (Fig. 1C). However, it significantly suppressed the pulse-end currents expressed as percentages of the peak currents (I/Ipeak) (from 52.1 2.4 to 33.4 4.2%, n = 5, p < 0.05, Fig. 1D) and the effect continued after the drug withdrawal. As previously demonstrated with L-type Ca2+-channel currents in vascular smooth muscle cells [13], lovastatin also suppressed the Kv1.3-channel currents in thymocytes (Fig. 2A). However, in contrast to pravastatin, lovastatin both lowered the peak (from 301 12 to 237 5.0 pA/pF, n = 5, p < 0.05, Fig. 2B) and the pulse-end currents (from 55.1 0.4 to 26.8 0.7%, n = 5, p < 0.05, Fig. 2C). Then, shortly after the drug withdrawal, the currents mostly recovered (Fig. 2A) with significant increases in both the peak current density and the I/Ipeak (Fig. 2B and C), indicating that the effect of lovastatin was reversible. Similar to lovastatin, simvastatin suppressed the Kv1.3-channel currents (Fig. 3A) by lowering both the peak (from 307 3.7 to 225 28 pA/pF, n = 5, p < 0.05, Fig. 3B) and the pulse-end currents (from 47.3 3.9 to 12.1 2.2%, n = 5, p < 0.05, Fig. 3C). However, compared to lovastatin, its effect on the pulse-end currents was more marked (Fig. 3C). Additionally, in contrast to lovastatin, the suppressed currents failed to recover after the drug withdrawal (Fig. 3). The results indicated that the suppressive effect of simvastatin on the channel currents was irreversible during the observation period. In several in vitro studies, statins suppressed the production of pro-inflammatory cytokines from human leukocytes and thus
Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
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A
External solution
Before
3
Washout 500 pA 50 ms
+80 mV -80 mV
B
1 mM Pravastatin
Before
Washout 500 pA 50 ms
Peak current density (pA/pF)
C 300 200 100 0 Before
D
1 mM Pravastatin
Washout
Pulse-end current / peak current (I / Ipeak) (%)
60 # #
30
0
Before
1 mM Pravastatin
Washout
Fig. 1. Effects of pravastatin on Kv1.3 channel currents in murine thymocytes. The effects of 1 mM pravastatin. (A and B) Typical whole-cell current traces at different voltagesteps recorded before and after the application of external solution alone (A) or pravastatin (B), and after the washout. The currents were elicited by voltage-steps from the holding potential of 80 mV to 40, 0, 40 and 80 mV, as depicted in the voltage protocol. Each pulse was applied for a 200-ms duration between 10-s intervals. (C) Peak current densities (peak currents normalized by the membrane capacitance) obtained from the records in a at the voltage-step of 80 mV. (D) Percentages of the pulse-end currents relative to the peak currents (100*I/Ipeak) obtained from the records in A at the voltage-step of 80 mV. #p < 0.05 vs. before the drug application. Values are means SEM (n = 5). Differences were analyzed by ANOVA followed by Dunnett’s or Student’s t test.
exerted immunomodulatory effects [4,5]. In the present study, we showed for the first time that statins, such as pravastatin, lovastatin and simvastatin, inhibit the Kv1.3-channel currents in thymocytes (Figs. 1–3). Since the channels trigger Ca2+ influx, which is necessary for cytokine synthesis [7], and since channel blockade by highly selective inhibitors, including margatoxin and ShK-Dap22, actually repressed the immune response in lymphocytes [16,17], such effects of statins on the channel currents were thought to contribute to their immunomodulatory properties, which have also been demonstrated with NSAIDs, dihydropyridine Ca2+ channel blockers and macrolide antibiotics in our recent studies [8–10]. Among the drugs used in the present study, pravastatin did not affect the peak amplitude of the Kv1.3-channel currents (Fig. 1C), while lovastatin and simvastatin significantly inhibited it (Figs. 2B and 3B). Although kinetic studies are required to address the issue, our previous study demonstrated that the
amplitude of peak currents was deeply associated with the ‘‘activation’’ of the channel currents [12]. Therefore, our results indicated that, compared to pravastatin, lovastatin and simvastatin are more likely to induce membrane depolarization of lymphocytes, which counteracts the Ca2+ influx into the cells, leading to the decreased immune activity. This may explain the higher immunosuppressive potency of lovastatin and simvastatin than pravastatin, as previously demonstrated in in vitro studies [4]. Moreover, as we have recently shown with benidipine [9], the persistent effect of simvastatin on the decreased channel currents may predict its longer duration of action in thymocytes. However, to actually demonstrate such immunosuppressive properties of statins, functional analyses are required to determine their effects on lymphocyte activation kinetics. The possible approaches to address this issue in the future would include the measurement of cytokine production, the leukocyte migration assay [16] and the
Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
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A
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Peak current density (pA/pF)
B 300
#
*
200 100 0 Before
10 µM Lovastatin
Washout
C Pulse-end current / peak current (I / Ipeak) (%)
60
* #
30
0
Before
10 µM Lovastatin
Washout
Fig. 2. Effects of lovastatin on Kv1.3 channel currents in murine thymocytes. The effects of 10 mM lovastatin. (A) Typical whole-cell current traces at different voltage-steps recorded before and after lovastatin application, and after the washout. The currents were elicited by voltage-steps from the holding potential of 80 mV to 40, 0, 40 and 80 mV, as depicted in the voltage protocol. Each pulse was applied for a 200-ms duration between 10-s intervals. (B) Peak current densities (peak currents normalized by the membrane capacitance) obtained from the records in a at the voltage-step of 80 mV. (C) Percentages of the pulse-end currents relative to the peak currents (100*I/Ipeak) obtained from the records in A at the voltage-step of 80 mV; p < 0.05 vs. before the drug application. *p < 0.05 vs. after the drug application. Values are means SEM (n = 5). Differences were analyzed by ANOVA followed by Dunnett’s or Student’s t test.
measurement of [3H] thymidine incorporation into the lymphocyte DNA [17]. Recently, we have demonstrated in an animal study that the overexpression of Kv1.3-channels in lymphocytes was strongly associated with their in situ proliferation in kidneys and the progression of chronic renal failure [18]. In this study, margatoxin, a selective Kv1.3-channel inhibitor, actually decreased the number of infiltrating lymphocytes and slowed the progression of renal fibrosis. Our results demonstrated that statins that effectively suppress the Kv1.3-channel currents in thymocytes (Figs. 1–3) may also potentially be useful as ‘‘anti-fibrotic’’ agents in patients with advanced renal failure. Effects of statins on whole-cell membrane capacitance in murine thymocytes Our results demonstrated that pravastatin induced the current inactivation on a faster time scale than that before the drug
application (Fig. 1B). This represents an ‘‘N-type inactivation’’ pattern in kinetic studies [19], suggesting that this drug plugs into the open-pores of the channel to inhibit the currents. On the other hand, both lovastatin and simvastatin induced the current inactivation on a much slower time scale than pravastatin did (Figs. 2A and 3A), representing ‘‘C-type inactivation’’ patterns [19] and suggesting that these drugs induced conformational collapses of the selectivity filters (inactivation gates) within pore-forming domains of the K+-channels [12]. Concerning such properties, it was conceivable that lovastatin and simvastatin would generate structural changes in the thymocyte membranes. In thymocytes, microscopic changes in the cell membrane surface area were best monitored by measurement of the whole-cell membrane capacitance (Cm) [8–10]. Therefore, in the present study, we employed this electrophysiological approach to detect the structural changes in the thymocyte plasma membranes induced by the statins. Numerical changes in the parameter are summarized in Table 1.
Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
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10 µM Simvastatin
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Peak current density (pA/pF)
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#
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100 0 Before
10 µ M Washout Simvastatin
C
Pulse-end current / peak current (I / Ipeak) (%)
60
30
0
Before
#
#
10 µM Simvastatin
Washout
Fig. 3. Effects of simvastatin on Kv1.3 channel currents in murine thymocytes. The effects of 10 mM simvastatin. (A) Typical whole-cell current traces at different voltagesteps recorded before and after simvastatin application, and after the washout. The currents were elicited by voltage-steps from the holding potential of 80 mV to 40, 0, 40 and 80 mV, as depicted in the voltage protocol. Each pulse was applied for a 200-ms duration between 10-s intervals. (B) Peak current densities (peak currents normalized by the membrane capacitance) obtained from the records in a at the voltage-step of 80 mV. (C) Percentages of the pulse-end currents relative to the peak currents (100*I/Ipeak) obtained from the records in A at the voltage-step of 80 mV. #p < 0.05 vs. before the drug application. Values are means SEM (n = 5). Differences were analyzed by ANOVA followed by Dunnett’s or Student’s t test.
Because of the difference in the size of thymocytes used in each experimental group, the initial values of Cm before drug application were different among the groups. By simply applying the external solution alone to thymocytes, we confirmed that our procedure of reagent application, a puff application by constant hydrostatic pressure with a nearby pipette, was without significant effects on the Cm (Table 1), as well as the
other parameters including membrane conductance (Gm) and series conductance (Gs) (data not shown). Our results demonstrated that the inclusion in the pipette of 1 mM pravastatin did not change the Cm (Table 1). However, inclusion in the pipette of either 10 mM lovastatin or simvastatin induced a significant decrease in the Cm immediately after the application (Table 1), with minimal changes in Gm and Gs (data not shown). These results indicated that
Table 1 Summary of changes in membrane capacitance after application of statins. Agents
N
Cm before drug application (pF)
Cm after drug application (pF)
Cm after drug washout (pF)
External solution (control) 1 mM pravastatin 10 mM lovastatin 10 mM simvastatin
5 5 5 5
2.76 0.11 2.59 0.10 5.08 0.26 3.33 0.19
2.85 0.16 2.43 0.07 4.18 0.29# 2.89 0.18#
2.84 0.17 2.54 0.07 4.95 0.40* 2.90 0.15#
Values are means SEM. Cm, membrane capacitance. # p < 0.05 vs. Cm before drug application. * p < 0.05 vs. Cm after drug application.
Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
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6
both drugs actually induced structural changes in the thymocyte membranes. After withdrawal of the drugs, the washout of lovastatin almost recovered the decreased Cm within 2 min (Table 1). In contrast, however, the washout of simvastatin failed to recover the decreased Cm during the observation period (Table 1). These results suggested that simvastatin induced longer-lasting structural changes in the thymocyte membranes than lovastatin. The whole-cell Cm is mathematically calculated from a parallelplate capacitor formula: Cm = eA/d, where e is the dielectric modulus of the plasma membrane; A, the membrane surface area and d, the membrane thickness [20]. Assuming that e and A are relatively constant before and after the drug application, the increase in d is primarily considered to be responsible for the decrease in Cm [9,10]. Since lovastatin and simvastatin are much more lipophilic than pravastatin [11], they would disperse more easily into the lipid bilayers of the plasma membrane. Therefore, the lovastatin- or simvastatin-induced decrease in the Cm was thought to represent increased membrane thickness (d) as a result of the accumulation of the drugs in the plasma membrane. The accumulated drugs may directly perturb the composite domains of the channels from inside the membranes. This would include the constriction or the conformational collapse of the selectivity filters within the pore forming domains [19]. Thus, lovastatin and simvastatin were thought to induce the ‘‘C-type inactivation’’ patterns of the channel currents (Figs. 2A and 3A). Additionally, since simvastatin has a higher partition coefficient than lovastatin does [11], simvastatin was thought to accumulate in the membranes for a longer period of time than lovastatin. This may be responsible for the persistent inhibition of the channel currents by simvastatin (Fig. 3) and the long-lasting decrease in the Cm (Table 1). In summary, this study demonstrated for the first time that statins such as pravastatin, lovastatin and simvastatin, suppress thymocyte Kv1.3-channel currents. The slow inactivation patterns induced by lovastatin and simvastatin were thought to be associated with their accumulation in the plasma membranes, as detected by the decreases in the membrane capacitance. Conflicts of interest The authors declare no conflicts of interest. Funding source This work is supported by Grant-in-Aids for Young Scientists (B) (Grant number: 25860155) to I. Kazama from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) in Japan.
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Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
PHAREP-61; No. of Pages 6 Pharmacological Reports xxx (2014) xxx–xxx
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Short communication
HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes Itsuro Kazama *, Asuka Baba, Yoshio Maruyama Department of Physiology I, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan
A R T I C L E I N F O
Article history: Received 18 October 2013 Received in revised form 3 March 2014 Accepted 3 March 2014 Available online xxx Keywords: HMG-CoA reductase inhibitors (statins) Immunosuppressive effects Lymphocytes Kv1.3-channel Membrane capacitance (Cm)
A B S T R A C T
Background: Since lymphocytes predominantly express delayed rectifier K+-channels (Kv1.3) that trigger lymphocyte activation, statins, which exert immunosuppressive effects, would affect the channel currents. Methods: Employing the patch-clamp technique in murine thymocytes, we examined the effects of statins on Kv1.3-channel currents and the membrane capacitance (Cm). Results: Pravastatin significantly suppressed the pulse-end currents of the channels. Lovastatin and simvastatin also suppressed the peak currents, significantly decreasing the Cm. Conclusions: This study demonstrated for the first time that statins inhibit thymocyte Kv1.3-channels. The slow inactivation patterns induced by lovastatin and simvastatin may be associated with their accumulation in the plasma membranes. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (or statins), which potently inhibit cholesterol biosynthesis, are widely used in the treatment of hypercholesterolemia and atherosclerosis [1]. In concert with such lipid-lowering effects, statins have also been demonstrated to exert antiinflammatory or immunomodulatory properties that reduce the risk of cardiovascular events [2]. Regarding the mechanisms by which they exert immunosuppressive effects, Kwak et al. originally found the involvement of major histocompatibility complex class II (MHC-II) expressed on T-lymphocytes [3]. Then, later in vitro studies also revealed that statins, such as pravastatin, lovastatin and simvastatin, suppress the production of pro-inflammatory cytokines from leukocytes [4,5]. These cells, including macrophages and lymphocytes, predominantly express delayed rectifier K+-channels (Kv1.3) in their plasma membranes [6]. Since these channels play crucial roles in the initiation of the immune response [7], statins would affect the channel currents when they modulate the immune response. In our recent studies, we demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs), dihydropyridine Ca2+ channel blockers and macrolide antibiotics suppress the
* Corresponding author. E-mail address: [email protected] (I. Kazama).
Kv1.3-channel currents in thymocytes and thus exert immunomodulatory effects [8–10]. Among them, lipophilic drugs induced microscopic structural changes in the membrane surface, as detected by the increase in the membrane capacitance [9,10]. Since statins, such as lovastatin and simvastatin, are highly lipophilic [11], they would directly disturb the thymocyte plasma membranes and thus affect the Kv1.3-channel currents. To test this, employing the standard patch-clamp whole-cell recording technique in murine thymocytes, we examined the effects of statins on the channel currents and the membrane capacitance. Here, we show for the first time that statins, such as pravastatin, lovastatin and simvastatin, suppress thymocyte Kv1.3-channel currents. We also show that the current suppression patterns of lovastatin and simvastatin were associated with their accumulation in the plasma membranes, as detected by decreases in the membrane capacitance. Materials and methods Cell sources and preparation Male ddy mice (4–5 weeks old), supplied by Japan SLC Inc. (Shizuoka, Japan), were deeply anaesthetized with isoflurane and then sacrificed by cervical dislocation. The protocol for animal use was approved by the Animal Care and Use Committee of the Tohoku University Graduate School of Medicine. Single thymocytes, isolated
http://dx.doi.org/10.1016/j.pharep.2014.03.002 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
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from murine thymus as described previously [8,12], were resuspended in standard external (bathing) solution containing (in mM): NaCl, 145; KCl, 4.0; CaCl2, 1.0; MgCl2, 2.0; Hepes, 5.0; bovine serum albumin, 0.01% (pH 7.2 adjusted with NaOH). They were maintained at room temperature (22–24 8C) for use within 4 h. Electrical setup and patch-clamp recordings We conducted standard whole-cell patch-clamp recordings using an EPC-9 patch-clamp amplifier system (HEKA Electronics, Lambrecht, Germany) as described previously [8,12]. The patch pipette resistance was 4–6 MV when filled with internal (patch pipette) solution containing (in mM): KCl, 145; MgCl2, 1.0; EGTA, 10; Hepes, 5.0 (pH 7.2 adjusted with KOH). After the giga-seal formation, we applied suction briefly to the pipette to rupture the patch membrane. The series resistance of the whole-cell recordings was maintained below 10 MV during the experiments. Peak currents were normalized by the membrane capacitance and expressed as the current densities (pA/pF). All experiments were carried out at room temperature. Drug delivery Pravastatin and simvastatin, purchased from Sigma Chemical Co. (St. Louis, MO, USA) and lovastatin, from Cayman Chemical (Ann Arbor, MI, USA), were separately dissolved in the external solution at final concentrations of 1 mM, 10 mM and 10 mM, respectively. We delivered one of the drugs to the cells by the standing hydrostatic pressure of 3 cm H2O from a nearby pipette as described previously [8,12]. Briefly, using a three-dimensional hydraulic manipulator (M-103, Narishige, Tokyo, Japan), the application pipette initially positioned outside the bathing solution was brought to within 10 mm of the cell surface. Then, to wash out the reagents, the pipette was brought up outside the bathing solution. Whole-cell membrane currents were recorded before and after 30 s exposure to the drug and after a 2 min washout. To rule out the possibility that the observed effect resulted from the procedure of the reagent application, we simply applied the external solution to the cells and confirmed the absence of any significant changes in the channel currents. Membrane capacitance measurements To measure the membrane capacitance of the thymocytes, we employed a sine plus DC protocol using the Lock-in amplifier of the EPC-9 Pulse program. An 800-Hz sinusoidal command voltage was superimposed on the holding potential of 80 mV. The membrane capacitance (Cm), as well as membrane conductance (Gm) and series conductance (Gs), was continuously recorded before and after 30 s exposure to the drugs during the whole-cell recording configuration. Statistic analyses Data were analyzed using PulseFit software (HEKA Electronics, Lambrecht, Germany), IGOR Pro (WaveMetrics, Lake Oswego, OR, USA) and Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and reported as means SEM. Statistical significance was assessed by two-way ANOVA followed by Dunnett’s or Student’s ttest. A value of p < 0.05 was considered significant. Results and discussion Effects of statins on Kv1.3-channel currents in murine thymocytes Stepwise changes in the membrane potential, from the holding potential of 80 mV to the various depolarizing potential levels,
evoked membrane currents in the thymocytes, showing voltagedependent activation and inactivation patterns characteristic to Kv1.3 (Figs. 1A and B, 2A and 3A) [8,12]. In our previous study, since the application of margatoxin, a relatively selective Kv1.3-channel inhibitor, almost totally abolished the currents [12], we identified them with the Kv1.3-channel currents. To investigate the effects of statins on the channel currents, we applied external solution, containing either 1 mM pravastatin, 10 mM lovastatin or 10 mM simvastatin, to the thymocytes and examined the changes in the whole-cell currents (Figs. 1–3). Fig. 1A shows an example of a control recording with the application of the external solution alone, which confirmed that our procedure of reagent application, a puff application by constant hydrostatic pressure with a nearby pipette, was without significant effects on the Kv1.3-channel currents. In previous in vitro studies, single cells required less than 10 mM of lovastatin and simvastatin to effectively elicit their pharmacological effects on ion-channel currents, such as Ca2+ channel currents in vascular smooth muscle cells [13] and K+ channel currents in cardiomyocytes [14]. In those studies, the drugs were previously dissolved in whole culture media, or continuously infused into the bathing solution. In the present study, however, since we delivered the drugs to single, isolated cells by a puff application method, there was the possibility of partial and insufficient exposure of the drugs to the cells. Therefore, we applied significantly higher concentrations of lovastatin and simvastatin (10 mM) than those used in the previous studies (Figs. 2 and 3). In humans, the serum concentrations of lovastatin or simvastatin after oral administration reach around 0.1 mM [13,15], which is much lower than the concentrations used in our study. However, in patients with hypercholesterolemia, since statins are usually used for a long period of several years or more, their effects might be evoked at lower concentrations under chronic administration. Additionally, in the previous studies, pravastatin required much higher doses than lovastatin or simvastatin to exert its pharmacological effects [13,15]. In the present study, however, 100 mM pravastatin had little effect on Kv1.3-channel currents in thymocytes (data not shown) and considered to be still insufficient. Therefore, we applied a higher dose at 1 mM (Fig. 1B–D). Pravastatin suppressed the Kv1.3-channel currents in thymocytes (Fig. 1B), although this drug did not significantly lower the peak currents (Fig. 1C). However, it significantly suppressed the pulse-end currents expressed as percentages of the peak currents (I/Ipeak) (from 52.1 2.4 to 33.4 4.2%, n = 5, p < 0.05, Fig. 1D) and the effect continued after the drug withdrawal. As previously demonstrated with L-type Ca2+-channel currents in vascular smooth muscle cells [13], lovastatin also suppressed the Kv1.3-channel currents in thymocytes (Fig. 2A). However, in contrast to pravastatin, lovastatin both lowered the peak (from 301 12 to 237 5.0 pA/pF, n = 5, p < 0.05, Fig. 2B) and the pulse-end currents (from 55.1 0.4 to 26.8 0.7%, n = 5, p < 0.05, Fig. 2C). Then, shortly after the drug withdrawal, the currents mostly recovered (Fig. 2A) with significant increases in both the peak current density and the I/Ipeak (Fig. 2B and C), indicating that the effect of lovastatin was reversible. Similar to lovastatin, simvastatin suppressed the Kv1.3-channel currents (Fig. 3A) by lowering both the peak (from 307 3.7 to 225 28 pA/pF, n = 5, p < 0.05, Fig. 3B) and the pulse-end currents (from 47.3 3.9 to 12.1 2.2%, n = 5, p < 0.05, Fig. 3C). However, compared to lovastatin, its effect on the pulse-end currents was more marked (Fig. 3C). Additionally, in contrast to lovastatin, the suppressed currents failed to recover after the drug withdrawal (Fig. 3). The results indicated that the suppressive effect of simvastatin on the channel currents was irreversible during the observation period. In several in vitro studies, statins suppressed the production of pro-inflammatory cytokines from human leukocytes and thus
Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
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Fig. 1. Effects of pravastatin on Kv1.3 channel currents in murine thymocytes. The effects of 1 mM pravastatin. (A and B) Typical whole-cell current traces at different voltagesteps recorded before and after the application of external solution alone (A) or pravastatin (B), and after the washout. The currents were elicited by voltage-steps from the holding potential of 80 mV to 40, 0, 40 and 80 mV, as depicted in the voltage protocol. Each pulse was applied for a 200-ms duration between 10-s intervals. (C) Peak current densities (peak currents normalized by the membrane capacitance) obtained from the records in a at the voltage-step of 80 mV. (D) Percentages of the pulse-end currents relative to the peak currents (100*I/Ipeak) obtained from the records in A at the voltage-step of 80 mV. #p < 0.05 vs. before the drug application. Values are means SEM (n = 5). Differences were analyzed by ANOVA followed by Dunnett’s or Student’s t test.
exerted immunomodulatory effects [4,5]. In the present study, we showed for the first time that statins, such as pravastatin, lovastatin and simvastatin, inhibit the Kv1.3-channel currents in thymocytes (Figs. 1–3). Since the channels trigger Ca2+ influx, which is necessary for cytokine synthesis [7], and since channel blockade by highly selective inhibitors, including margatoxin and ShK-Dap22, actually repressed the immune response in lymphocytes [16,17], such effects of statins on the channel currents were thought to contribute to their immunomodulatory properties, which have also been demonstrated with NSAIDs, dihydropyridine Ca2+ channel blockers and macrolide antibiotics in our recent studies [8–10]. Among the drugs used in the present study, pravastatin did not affect the peak amplitude of the Kv1.3-channel currents (Fig. 1C), while lovastatin and simvastatin significantly inhibited it (Figs. 2B and 3B). Although kinetic studies are required to address the issue, our previous study demonstrated that the
amplitude of peak currents was deeply associated with the ‘‘activation’’ of the channel currents [12]. Therefore, our results indicated that, compared to pravastatin, lovastatin and simvastatin are more likely to induce membrane depolarization of lymphocytes, which counteracts the Ca2+ influx into the cells, leading to the decreased immune activity. This may explain the higher immunosuppressive potency of lovastatin and simvastatin than pravastatin, as previously demonstrated in in vitro studies [4]. Moreover, as we have recently shown with benidipine [9], the persistent effect of simvastatin on the decreased channel currents may predict its longer duration of action in thymocytes. However, to actually demonstrate such immunosuppressive properties of statins, functional analyses are required to determine their effects on lymphocyte activation kinetics. The possible approaches to address this issue in the future would include the measurement of cytokine production, the leukocyte migration assay [16] and the
Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
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Fig. 2. Effects of lovastatin on Kv1.3 channel currents in murine thymocytes. The effects of 10 mM lovastatin. (A) Typical whole-cell current traces at different voltage-steps recorded before and after lovastatin application, and after the washout. The currents were elicited by voltage-steps from the holding potential of 80 mV to 40, 0, 40 and 80 mV, as depicted in the voltage protocol. Each pulse was applied for a 200-ms duration between 10-s intervals. (B) Peak current densities (peak currents normalized by the membrane capacitance) obtained from the records in a at the voltage-step of 80 mV. (C) Percentages of the pulse-end currents relative to the peak currents (100*I/Ipeak) obtained from the records in A at the voltage-step of 80 mV; p < 0.05 vs. before the drug application. *p < 0.05 vs. after the drug application. Values are means SEM (n = 5). Differences were analyzed by ANOVA followed by Dunnett’s or Student’s t test.
measurement of [3H] thymidine incorporation into the lymphocyte DNA [17]. Recently, we have demonstrated in an animal study that the overexpression of Kv1.3-channels in lymphocytes was strongly associated with their in situ proliferation in kidneys and the progression of chronic renal failure [18]. In this study, margatoxin, a selective Kv1.3-channel inhibitor, actually decreased the number of infiltrating lymphocytes and slowed the progression of renal fibrosis. Our results demonstrated that statins that effectively suppress the Kv1.3-channel currents in thymocytes (Figs. 1–3) may also potentially be useful as ‘‘anti-fibrotic’’ agents in patients with advanced renal failure. Effects of statins on whole-cell membrane capacitance in murine thymocytes Our results demonstrated that pravastatin induced the current inactivation on a faster time scale than that before the drug
application (Fig. 1B). This represents an ‘‘N-type inactivation’’ pattern in kinetic studies [19], suggesting that this drug plugs into the open-pores of the channel to inhibit the currents. On the other hand, both lovastatin and simvastatin induced the current inactivation on a much slower time scale than pravastatin did (Figs. 2A and 3A), representing ‘‘C-type inactivation’’ patterns [19] and suggesting that these drugs induced conformational collapses of the selectivity filters (inactivation gates) within pore-forming domains of the K+-channels [12]. Concerning such properties, it was conceivable that lovastatin and simvastatin would generate structural changes in the thymocyte membranes. In thymocytes, microscopic changes in the cell membrane surface area were best monitored by measurement of the whole-cell membrane capacitance (Cm) [8–10]. Therefore, in the present study, we employed this electrophysiological approach to detect the structural changes in the thymocyte plasma membranes induced by the statins. Numerical changes in the parameter are summarized in Table 1.
Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
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Fig. 3. Effects of simvastatin on Kv1.3 channel currents in murine thymocytes. The effects of 10 mM simvastatin. (A) Typical whole-cell current traces at different voltagesteps recorded before and after simvastatin application, and after the washout. The currents were elicited by voltage-steps from the holding potential of 80 mV to 40, 0, 40 and 80 mV, as depicted in the voltage protocol. Each pulse was applied for a 200-ms duration between 10-s intervals. (B) Peak current densities (peak currents normalized by the membrane capacitance) obtained from the records in a at the voltage-step of 80 mV. (C) Percentages of the pulse-end currents relative to the peak currents (100*I/Ipeak) obtained from the records in A at the voltage-step of 80 mV. #p < 0.05 vs. before the drug application. Values are means SEM (n = 5). Differences were analyzed by ANOVA followed by Dunnett’s or Student’s t test.
Because of the difference in the size of thymocytes used in each experimental group, the initial values of Cm before drug application were different among the groups. By simply applying the external solution alone to thymocytes, we confirmed that our procedure of reagent application, a puff application by constant hydrostatic pressure with a nearby pipette, was without significant effects on the Cm (Table 1), as well as the
other parameters including membrane conductance (Gm) and series conductance (Gs) (data not shown). Our results demonstrated that the inclusion in the pipette of 1 mM pravastatin did not change the Cm (Table 1). However, inclusion in the pipette of either 10 mM lovastatin or simvastatin induced a significant decrease in the Cm immediately after the application (Table 1), with minimal changes in Gm and Gs (data not shown). These results indicated that
Table 1 Summary of changes in membrane capacitance after application of statins. Agents
N
Cm before drug application (pF)
Cm after drug application (pF)
Cm after drug washout (pF)
External solution (control) 1 mM pravastatin 10 mM lovastatin 10 mM simvastatin
5 5 5 5
2.76 0.11 2.59 0.10 5.08 0.26 3.33 0.19
2.85 0.16 2.43 0.07 4.18 0.29# 2.89 0.18#
2.84 0.17 2.54 0.07 4.95 0.40* 2.90 0.15#
Values are means SEM. Cm, membrane capacitance. # p < 0.05 vs. Cm before drug application. * p < 0.05 vs. Cm after drug application.
Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
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6
both drugs actually induced structural changes in the thymocyte membranes. After withdrawal of the drugs, the washout of lovastatin almost recovered the decreased Cm within 2 min (Table 1). In contrast, however, the washout of simvastatin failed to recover the decreased Cm during the observation period (Table 1). These results suggested that simvastatin induced longer-lasting structural changes in the thymocyte membranes than lovastatin. The whole-cell Cm is mathematically calculated from a parallelplate capacitor formula: Cm = eA/d, where e is the dielectric modulus of the plasma membrane; A, the membrane surface area and d, the membrane thickness [20]. Assuming that e and A are relatively constant before and after the drug application, the increase in d is primarily considered to be responsible for the decrease in Cm [9,10]. Since lovastatin and simvastatin are much more lipophilic than pravastatin [11], they would disperse more easily into the lipid bilayers of the plasma membrane. Therefore, the lovastatin- or simvastatin-induced decrease in the Cm was thought to represent increased membrane thickness (d) as a result of the accumulation of the drugs in the plasma membrane. The accumulated drugs may directly perturb the composite domains of the channels from inside the membranes. This would include the constriction or the conformational collapse of the selectivity filters within the pore forming domains [19]. Thus, lovastatin and simvastatin were thought to induce the ‘‘C-type inactivation’’ patterns of the channel currents (Figs. 2A and 3A). Additionally, since simvastatin has a higher partition coefficient than lovastatin does [11], simvastatin was thought to accumulate in the membranes for a longer period of time than lovastatin. This may be responsible for the persistent inhibition of the channel currents by simvastatin (Fig. 3) and the long-lasting decrease in the Cm (Table 1). In summary, this study demonstrated for the first time that statins such as pravastatin, lovastatin and simvastatin, suppress thymocyte Kv1.3-channel currents. The slow inactivation patterns induced by lovastatin and simvastatin were thought to be associated with their accumulation in the plasma membranes, as detected by the decreases in the membrane capacitance. Conflicts of interest The authors declare no conflicts of interest. Funding source This work is supported by Grant-in-Aids for Young Scientists (B) (Grant number: 25860155) to I. Kazama from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) in Japan.
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Please cite this article in press as: Kazama I, et al. HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.03.002
