HMG-CoA Reductase Inhibitors Suppress Intracellular Calcium Mobilization and Membrane Current Induced by Lysophosphatidylcholine in Endothelial Cells
Background— Lysophosphatidylcholine (LPC) is known to increase intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells. This study was conducted to investigate the effects of HMG-CoA reductase inhibitors (statins) on the increase in [Ca2+]i and membrane current induced by LPC.
Methods and Results— [Ca2+]i was determined in cultured human aortic endothelial cells by fura-2 assay, and membrane current was measured by whole-cell patch clamp. The [Ca2+]i increase induced by LPC was abolished by inhibitors of phospholipase C (PLC). Statins markedly decreased the [Ca2+]i increase caused by LPC. This suppressive effect was quickly reversed by geranylgeranylpyrophosphate (GGPP) and was mimicked by inhibitors of Rho and Rho kinase. LPC induced the translocation of the GTP-bound active form of RhoA into membranes within 1 minute as determined by a pull-down assay and reduced the levels of RhoA in the cytoplasm, indicating that LPC quickly increases the GTP/GDP ratio of RhoA and induces membrane translocation. Statins prevented the GTP/GDP exchange of RhoA and its membrane translocation from the cytoplasm caused by LPC, and these effects of statins were reversed by GGPP. The responses of RhoA activation to statins and GGPP concurred with their effects on Ca2+ mobilization. LPC also induced a nonselective cation current after a lag. Statins prolonged the lag and decreased the current amplitude, and GGPP abolished the inhibitory effect on the current.
Conclusions— LPC induced Ca2+ mobilization and membrane current via a Rho activation–dependent PLC pathway in endothelial cells, and statins blocked these effects by preventing the GGPP-dependent lipid modification of Rho. The present study implicates Rho in LPC stimulation of Ca2+ movement.
Received October 9, 2001; revision received December 17, 2001; accepted December 21, 2001.
The dysfunction of endothelial cells is one of the initial steps in atherosclerosis.1 Hyperlipidemia impairs endothelium-dependent relaxation of coronary arteries in humans.2,3⇓ Oxidized low-density lipoprotein (LDL) plays an important role in endothelial dysfunction, and lysophosphatidylcholine (LPC) is a phospholipid component of oxidized LDL. When LDL is oxidized, approximately 60% of the phosphatidylcholine seems to be converted to LPC, which induces atherogenic activities, including the induction of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) in endothelial cells.4,5⇓
LPC also increases intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells.6 [Ca2+]i increase consists of the release of Ca2+ from intracellular Ca2+ stores and influx of extracellular Ca2+ across the plasma membrane. However, the mechanisms by which LPC induces intracellular Ca2+ mobilization and the influx of extracellular Ca2+ are not clear.
HMG-CoA reductase inhibitors (statins) prevent the incidence of coronary events and improve endothelium-dependent relaxation in hyperlipidemic patients with coronary artery disease.2,3,7⇓⇓ In addition, statins have been shown in vitro and in vivo to improve endothelial nitric oxide synthesis by direct actions independent of cholesterol lowering.8,9⇓ However, it is not known whether statins alter the effects induced by LPC on [Ca2+]i and membrane current in endothelial cells.
In the present study, we examined the effects of LPC on [Ca2+]i and membrane current and their possible mechanisms and the effects of statin on the [Ca2+]i and membrane current induced by LPC in human aortic endothelial cells.
L-α-LPC (palmitoyl, Sigma Chemical Co) was dissolved in chloroform and methanol (3:1) evaporated to dryness and dissolved in Tyrode solution. DL-mevalonic acid lactone, geranylgeranylpyrophosphate (GGPP), and farnesylpyrophosphate (FPP) were obtained from Sigma. Fura-2/acetoxymethyl ester, neomycin sulfate, and U-73122 were from Wako Pure Chemicals. Fluvastatin was kindly provided by Novartis Pharm Inc (Basel, Switzerland), cerivastatin was from Bayer, pravastatin from Sankyo Pharmaceutical, and Y-27632, a Rho-kinase inhibitor, from Welfide. C3 exoenzyme, a Rho inhibitor, was purchased from Upstate Biotechnology.
Preparation of Endothelial Cells
Passage 4 endothelial cells from human aortic artery were purchased from Clonetics and cultured according to the supplier’s instructions and were used for experiments after 5 to 10 passages.
Measurement of [Ca2+]i
[Ca2+]i was measured as described previously.10 Briefly, endothelial cells were grown to confluence in 35-mm glass-bottom dishes (MatTek). The cells were incubated at 37°C for 10 minutes with 1 μmol/L fura-2/acetoxymethyl ester and then washed with the Tyrode solution. The dish was mounted on an inverted fluorescence microscope (IX-FLA, Olympus) and then placed in a flow-through chamber (0.2 mL volume). Fura-2–loaded cells were perfused continuously (4 mL/min) with prewarmed (37°C) Ca2+-containing or Ca2+-free Tyrode solution in the absence or presence of agents. The cells were exposed to alternating excitation wavelengths of 340 and 380 nm, and the fluorescence image was monitored by a cooled CCD camera (Ultra Pix, Olympus). Data were recorded and processed using a MERLIN ratio image system (Olympus), and the 340/380-nm fluorescence emission was calculated.
Western Blotting of RhoA
Western blotting was performed as previously described.11 Briefly, cells were lysed with a hypotonic buffer, and a part of lysate was sonicated and centrifuged at 15 000g for 10 minutes to prepare membrane and cytoplasmic fractions. Aliquots containing 20 μg of proteins were subjected to SDS/polyacrylamide gel electrophoresis (10% running gel, 5% stacking gel, PAGEL, ATTO Co) and were then transferred onto polyvinylidene difluoride membranes (MILLIPORE). After incubating with blocking solution at room temperature for 30 minutes, the membranes were incubated for 60 minutes at room temperature with a mouse monoclonal antibody to RhoA diluted 1:500 (Santa Cruz Biotechnology Inc, Santa Cruz, Calif) and then for 45 minutes with a horseradish peroxidase–conjugated goat anti-mouse IgG diluted 1:10000 (Santa Cruz). The signals from immunoreactive bands were visualized by an Amersham ECL System (Amersham Pharmacia Biotech UK Lit).
Determination of GTP-Rho
RhoA activity was determined by measuring GTP-Rho using the Rho-binding domain of rhotekin, as previously described.12 Briefly, samples of the membrane fractions of the cells were extracted with a lysis buffer. The extracts were incubated for 45 minutes at 4°C with glutathione-S-transferase (GST)-rhotekin fusion protein bound to glutathione-Sepharose 4B beads. The beads were washed 3 times with a washing buffer and boiled for 5 minutes in a loading buffer. Bound GTP-Rho proteins were quantitatively determined by Western blotting.
Membrane Current Recording
Whole-cell patch clamp was performed using the external and pipette solutions, as previously described.13 The external solution was prewarmed to ≈35 to 37°C by a water jacket. The resistance of the pipette filled with the pipette solution was 2 to 4 megaOHgr;. The patch-clamp amplifier was TM-1000 (Act ME). Ramp pulses were given every 10 seconds from the holding potential of −60 mV, initially depolarizing to +60 mV and then hyperpolarizing to −110 mV and depolarizing back to −60 mV, with a constant speed of 0.72 V/sec. The data were collected online and stored in a computer (PC-9801 RX, NEC).
Statistical analyses were performed using ANOVA with Scheffé’s post hoc test, as appropriate. A level of P<0.05 was considered significant. Data are expressed as mean±SEM.
Effects of LPC on [Ca2+]i
Figure 1 demonstrates the mean fluorescent values indicating [Ca2+]i of 100 endothelial cells each in the presence or absence of external Ca2+. LPC (10 μmol/L) induced a rapid and transient increase in [Ca2+]i in the absence of external Ca2+ (Figure 1A, top), whereas in the presence of external Ca2+, a sustained increase was observed (Figure 1B, top). When 1 μmol/L neomycin or U-73122, PLC inhibitors, perfused the cells starting 3 minutes before LPC treatment, each agent markedly suppressed the increase in [Ca2+]i induced by LPC in the presence or absence of external Ca2+ (Figures 1A and 1B, middle). These observations suggest that the increase in [Ca2+]i induced by LPC is mediated by a PLC-signaling pathway.
Effects of Statins and Rho/Rho-Kinase Inhibitors on [Ca2+]i Increase Induced by LPC
Fluvastatin markedly suppressed the [Ca2+]i increase induced by LPC in the presence or absence of external Ca2+ (Figure 1, bottom and Figures 2A and 2B). However, fluvastatin (FLV) did not reduce the elevated [Ca2+]i induced by LPC when FLV was perfused after LPC. Moreover, FLV had no effect on [Ca2+]i induced by ATP (100 μmol/L), a G-protein–coupled receptor agonist (data not shown). To examine whether the effect is mediated by a mevalonate pathway, 100 μmol/L mevalonate, 2.5 μmol/L GGPP, or 2.5 μmol/L FPP was added to the cells 3 minutes before FLV and LPC. The suppressive effect of FLV was abolished by mevalonate or GGPP but not by FPP (Figures 2A and 2B), suggesting an involvement of GGPP-dependent isoprenylation of signaling molecules, such as Rho, in LPC-induced effects. Therefore, we examined the effects of C3 exoenzyme and Y-27632 on the [Ca2+]i increase evoked by LPC. Treatment with C3 exoenzyme overnight (0.25 μg/mL) markedly inhibited the [Ca2+]i increase induced by LPC (Figures 2A and 2B). Perfusion with Y-27632 (10−7 mol/L) induced a suppressive effect similar to statins on [Ca2+]i increase induced by LPC (Figures 2A and 2B).
Cells were treated with 3 kinds of statins starting 3 minutes before LPC perfusion. Figure 3 shows that there was no significant difference among 3 agents (5 μmol/L FLV, 0.1 μmol/L cerivastatin, and 20 μmol/L pravastatin) in their suppressive effect on [Ca2+]i increase evoked by 10 μmol/L LPC.
Translocation of RhoA Into Membranes and GTP/GDP Exchange
To examine the correlation between Rho activation and Ca2+ mobilization, we determined the translocation of Rho into membranes and the activity of Rho. Western blotting revealed that LPC increased the levels of RhoA in membrane fractions within 1 minute and decreased its levels in the cytoplasm. A 3-minute treatment with FLV before the addition of LPC blocked the membrane translocation and the reduction of cytoplasmic RhoA induced by LPC (Figure 4A). Figure 4B shows that the translocated RhoA is in a GTP-bound active form, indicating that LPC markedly increases the GTP/GDP ratio of RhoA, which is followed by membrane translocation within 1 minute. A 3-minute incubation with FLV prevented the GTP/GDP exchange and membrane translocation of RhoA. GGPP fully restored the LPC-induced membrane translocation and reduction of cytoplasmic RhoA, which were inhibited by FLV, whereas FPP had no effect (Figure 4C). A 3-minute incubation with FLV alone did not significantly alter the distribution of RhoA in membranes and cytoplasm or the GTP/GDP ratio in the membranes (data not shown).
Membrane Current Induced by LPC
Because the sustained increase in [Ca2+]i in the presence of external Ca2+ is probably mediated by Ca2+ influx across the plasma membrane, we measured membrane currents. After a lag, LPC increased the membrane current. Figure 5A shows a representative time course of the membrane current induced by 10 μmol/L LPC (top), the current-voltage (I-V) curves of the control (a) and during the application of LPC (b) (middle), and the difference in the I-V curve before and at 130 seconds after addition of 10 μmol/L LPC (bottom). The reversal potential of the current induced by LPC was 0 mV, suggesting that LPC induces a nonselective cation current (Figure 5A, bottom).
A significant difference in the duration of the lag was observed between the cells treated with 0.2 μmol/L and those treated with >2 μmol/L (Figure 6A, P<0.001). LPC induced a significant membrane current at concentrations >2 μmol/L, and the current magnitude was concentration dependent (Figure 6B). The EC50 of LPC in the nonselective cation current was 2 μmol/L.
Effects of Statins on the Current Induced by LPC
Pretreatment with FLV (5 μmol/L) for 3 minutes prolonged the lag (Figures 5B and 6⇑C) and markedly suppressed the current density induced by 10 μmol/L LPC (Figures 5B and 6⇑D). Other statins, such as cerivastatin and pravastatin, had similar effects on the current induced by LPC (data not shown). Similar to the effect on [Ca2+]i, FLV could not inhibit the current induced by LPC in the presence of 100 μmol/L mevalonate (Figures 5C, 6C, and 6⇑D).
We added 2.5 μmol/L GGPP to the pipette solution and found that it completely abolished the inhibitory effect of FLV (Figure 7). This suggests that the effect of FLV is mediated through inhibition of geranylgeranylation. Furthermore, Y-27632 partially inhibited the current induced by LPC (data not shown).
The present study showed that in human aortic endothelial cells, LPC increased [Ca2+]i via PLC and that this was accompanied by translocation of the GTP-bound active form of RhoA into membranes, providing evidence for a novel signaling pathway in the RhoA-dependent PLC activation of Ca2+ regulation induced by LPC. Statins quickly prevented the [Ca2+]i increase and the activation of RhoA caused by LPC, and these effects of statins were reversed by GGPP.
In this study, PLC inhibitors markedly inhibited the stimulatory effect of LPC on [Ca2+]i in the absence or presence of external Ca2+. We showed that in the presence of external Ca2+, the LPC-induced sustained increase in [Ca2+]i is mediated by Ca2+ influx through the nonselective cation channel. Therefore, our findings suggest that in endothelial cells the intracellular Ca2+ mobilization and the membrane current induced by LPC are mediated by the PLC-signaling pathway.
We found that within 1 minute, LPC increased the translocation of RhoA into membranes and that the translocated RhoA was in a GTP-bound active form. This was accompanied by a decrease in the levels of cytoplasmic RhoA. These findings suggest that LPC rapidly activates Rho metabolism, including the GTP/GDP exchange reaction and membrane translocation.
We also found that a 3-minute incubation with FLV prevented the GTP loading, membrane translocation, and reduction of cytoplasmic RhoA induced by LPC and that these were abolished by GGPP. This suggests that there is a pool of ungeranylgeranylated Rho in the cytoplasm and that the GTP/GDP exchange reaction and the levels of the pool of ungeranylgeranylated Rho are quickly altered by a GGPP-dependent process in LPC stimulation. To prove this, it will be necessary to measure directly the pool of ungeranylgeranylated Rho.
Taken together, our findings suggest that the geranylgeranylation of unprocessed GDP-Rho might limit the rate of GTP/GDP exchange and membrane translocation when stimulated with LPC, although it is generally accepted that the GTP/GDP exchange reaction is a rate-limiting step in Rho activation.14 In the present study, we describe a novel mechanism of Rho activation. It would be of interest to see if geranylgeranylation is also involved in the activation of Rho by receptor agonists such as sphingosine 1-phosphate and lysophosphatidic acid, which seem to exert biological activities through Rho activation.15
Statins have been shown to decrease the membrane translocation of Rho slowly, taking several hours.16 Although a similar phenomenon was observed in cells not stimulated with LPC (data not shown), we show that a GGPP-dependent lipid modification takes place quickly and is associated with Rho activation and Ca2+ movement when stimulated with LPC and that statins quickly modulate Rho metabolism and Ca2+ movement via inhibiting geranylgeranylation. Moreover, the [Ca2+]i increase evoked by ATP was not affected by statins, suggesting that LPC does not directly activate the heterotrimeric Gq family. Thus, the rapid responses of Rho metabolism and Ca2+ movement to statins and GGPP seem to be specific for LPC stimulation.
Rho inactivation by C3 exoenzyme inhibited the [Ca2+]i increase caused by LPC, suggesting that Rho activation is upstream of Ca2+ movement. It is of interest to determine which effector is associated with Rho activation–induced [Ca2+]i increase. A Rho-kinase inhibitor induced a similar inhibitory effect as the Rho inhibitor, indicating that Rho/Rho-kinase activation induces the Ca2+ mobilization caused by LPC stimulation. Moreover, the responses of Rho activation to statins and GGPP concurred with those of Ca2+ mobilization. Therefore, the present study strongly implicates Rho in the LPC stimulation of Ca2+ movement.
With respect to LPC-induced Ca2+ influx, we found that LPC activated a membrane current in endothelial cells. The current was similar to that reported in ventricular myocytes, in which the reversal potential was 0 mV and a lag was required for the induction of current.13 Thus, the current induced by LPC in endothelial cells was characterized as a nonselective cation current. Similar to the [Ca2+]i response, the LPC-induced current was inhibited by statins and the inhibitory effect was abolished by GGPP, suggesting that a GGPP-dependent process is also involved in the nonselective cation current induced by LPC.
Intracellular PLC/IP3 signaling for Ca2+ release was immediate in cells stimulated with LPC, whereas there was a lag in the current activation. Although [Ca2+]i was markedly suppressed by Y-27632, it only partially inhibited the LPC-induced current. These findings suggest that the Rho/Rho-kinase pathway may play some role in the current induced by LPC and that intracellular Ca2+ signaling and membrane current may be regulated independently. The detailed signaling pathway in current activation needs to be clarified.
In LPC stimulation, an association of adhesion molecule induction with Ca2+ signaling is unknown. A recent study showed that [Ca2+]i increase is required for activation and binding of nuclear factor-κB for induction of the ICAM-1 and VCAM-1 genes.17 Together with our findings, it seems that Rho-mediated [Ca2+]i increase may induce adhesion molecule expression in LPC-treated endothelial cells and statins may prevent endothelial dysfunction by suppressing Ca2+ signaling through Rho-dependent PLC signaling.
In conclusion, the present study shows that the inhibitory effects of statins on intracellular Ca2+ mobilization and nonselective cation current evoked by LPC prevent GGPP-dependent Rho activation in endothelial cells.
This work was supported in part by a Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (13670732) and a Research Grant for Cardiovascular Diseases from the Ministry of Health, Labor and Welfare of Japan. We thank Dr Yasuhide Watanabe for data analysis of EC50 and Sanae Sato for data preparation.
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