Nonselective Cation Currents Regulate Membrane Potential of Rabbit Coronary Arterial Cell
Modulation by Lysophosphatidylcholine
Background— The effects of lysophosphatidylcholine (LPC) on electrophysiological activities and intracellular Ca2+ concentration ([Ca2+]i) were investigated in coronary arterial smooth muscle cells (CASMCs).
Methods and Results— The patch clamp techniques and Ca2+ measurements were applied to cultured rabbit CASMCs. The membrane potential was −46.0±5.0 mV, and LPC depolarized it. Replacement of extracellular Na+ with NMDG+ hyperpolarized the membrane and antagonized the depolarizing effects of LPC. In Na+-, K+-, or Cs+-containing solution, the voltage-independent background current with reversal potential (Er) of approximately +0 mV was observed. Removal of Cl− failed to affect it. When extracellular cations were replaced by NMDG+, Er was shifted to negative potentials. La3+ and Gd3+ abolished the background current, but nicardipine and verapamil did not inhibit it. In Na+-containing solution, LPC induced a voltage-independent current with Er of approximately +0 mV concentration-dependently. Similar current was recorded in K+- and Cs+-containing solution. La3+ and Gd3+ inhibited LPC-induced current, but nicardipine and verapamil did not inhibit it. In cell-attached configurations, single-channel activities with single-channel conductance of ≈32pS were observed when patch pipettes were filled with LPC. LPC increased [Ca2+]i as the result of Ca2+ influx, and La3+ completely antagonized it.
Conclusions— These results suggest that (1) nonselective cation current (INSC) contributes to form membrane potentials of CASMCs and (2) LPC activates INSC, resulting in an increase of [Ca2+]i. Thus, LPC may affect CASMC tone under various pathophysiological conditions such as ischemia.
Received June 24, 2002; revision received August 29, 2002; accepted September 2, 2002.
Lysophosphatidylcholine (LPC), a major lysophospholipid in mammalian tissues, is formed from phosphatidylcholine by phospholipase A2. It accumulates in myocardial tissue during ischemia1,2⇓ and has toxic effects on myocardium, which include electrophysiological disturbances such as a decrease in membrane potential, followed by the occurrence of arrhythmia during ischemia.3 LPC is also known as a vasoactive phospholipid that has biological effects on arterial walls, including coronary artery.4 The prominent mechanism for vascular effects of LPC is to impair endothelium-dependent relaxing factor–mediated vasodilation, which inhibits nitric oxide production.5 In addition, LPC is reported to increase intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMCs) and increase vascular tone.4,6⇓ However, the underlying mechanisms for LPC effects on coronary arterial smooth muscle cells (CASMCs) have not been investigated.
Smooth muscle tone of arteries is an important determinant of coronary circulation and vascular resistance. Coronary arterial tone is regulated by various physiological and pathological changes such as O2 tension and an increase of H+, resulting in autoregulation of local blood flow. Membrane potential plays additional crucial roles in regulating arterial tone and hence arterial diameter; depolarization activates voltage-gated Ca2+ channels, subsequently increasing Ca2+ entry, which regulates muscle contractility and leads to vasoconstriction. The voltage-dependent K+ channel (KV) plays essential roles in forming membrane potential in VSMCs.7 ATP-sensitive K+ channels also regulate coronary tone under ischemia.8 However, the membrane potential of VSMCs including CASMCs is much less than K+ equilibrium potential,9,10⇓ proposing that additional membrane channels such as Cl− may contribute to form it.11 Alternatively, the background nonselective cation current (INSC), which forms membrane potential, is identified in cardiac myocytes12–14⇓⇓ and pulmonary smooth muscle cells,15 but it has not been reported in CASMCs.
Therefore, we have investigated the effects of LPC on electrophysiological activities and [Ca2+]i mobilization in rabbit CASMCs. We found that the background INSC contributes to form membrane potential of CASMCs, and LPC further induces INSC, followed by an increase in [Ca2+]i.
Rabbit CASMCs were grown from explants of adult male Japanese White rabbit (2.5 to 3.0 kg, n=15) coronary arteries by explants methods. They exhibited typical “hill and valley” growth patterns and exhibited positive fluorescence with antibodies against α-smooth muscle actin but no fluorescence with antibodies against factor VIII antigen. They were grown in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma), 100U/mL penicillin, 100 μg/mL gentamicin, and 0.25 μg/mL amphotericin B (GIBCO BRL) in a humidified atmosphere of 5% CO2 and 95% air at 37°C. When cells became confluent, they were subcultured in the same medium with 0.5% trypsin in 0.02% EDTA. Confluent cells at passages 3 to 6 were used for the experiments.
Solutions and Drugs
The control Tyrode solution contained (in mmol/L) NaCl 136.5, KCl 5.4, CaCl2 1.8, MgCl2 0.53, glucose 5.5, and N-2-hydroxyethylpiperazine-N′-ethane sulfonic acid (HEPES)-NaOH buffer 5.5(pH 7.4). The Ca2+-free Tyrode solution contained EGTA (0.5 mmol/L). The high K+ bathing solution contained KCl 140, CaCl2 1.8, MgCl2 0.53, glucose 5.5, and HEPES-NaOH buffer 5.5 (pH 7.4). In NMDG+ solutions, Na+ was replaced with equimolar N-methyl-d-glucamine+ (NMDG+). In low Cl− solutions, Cl− was replaced with aspartate, and external concentration of Cl−([Cl−]o) was reduced to 10 mmol/L. The K+ internal solution in the patch pipette contained KCl 140, EGTA 5, MgCl2 2, Na2ATP 3, GTP 0.1, and HEPES-KOH buffer 5 (pH 7.2). The Cs+ internal solution contained CsCl 140, EGTA 5, MgCl2 2, Na2ATP 3, GTP 0.1, and HEPES-CsOH buffer 5 (pH 7.2). The K+, Cs+, or Na+ extracellular solution was as follows: KCl, CsCl, or NaCl 140, glucose 5.5, HEPES 5.5(pH 7.4). In cell-attached experiments, the Cs+-pipette solution contained CsCl 140, CaCl2 1.8, MgCl2 0.53, HEPES 5 (pH 7.4).
Palmitoyl-l-α-lysophosphatidylcholine (LPC), lanthanum (La3+), gadrinium (Gd3+), verapamil, nicardipine, 4-aminopyridine (4-AP), and tetraethylammonium (TEA) were purchased from Sigma. Fura-2 acetoxymethyl ester (fura-2/AM, molecular probes) was obtained from Dojin Chemicals.
Measurements of [Ca2+]i
Primarily cultured CASMCs on a glass culture dish were used. At confluence, CASMCs were loaded with 4 μmol/L fura-2/AM in DMEM for 60 minutes at 37°C. After loading, cells were washed 3 times, and the medium containing fura-2/AM was removed. The glass culture dish was mounted on an inverted microscope (Diaphot TMD, Nikon). Measurement of [Ca2+]i was performed with a SPEX dual-wavelength fluorolog spectrometer (SPEX Industries, Inc). Excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm were used. In assessment of [Ca2+]i, fluorescence intensity ratio of F340/F380 was used as an indicator of [Ca2+]i.16
Recording Technique and Data Analysis
Membrane potential and currents were recorded by using whole-cell clamp techniques.17,18⇓ The patch electrode had the tip resistance of 3 to 5 mol/LΩ, and series resistance was compensated. The data were reproduced, low-pass filtered at 1 kHz (−3dB) with a Bessel filter (FV-625, NF,48dB/octave slope attenuation), and sampled at 5 kHz. All data were acquired and analyzed on a Power Macintosh 7100/80 by using the PULSE+PULSEFIT software (HEKA Electronic) and Igor PRO (Wave Metrics). Single-channel currents were replayed from videotape either to a chart recorder (2400S, Gould Inc), or to the A/D broad for digitization.
Statistical data are expressed as mean±SD; n represents the number of cells tested. A Student’s t test was used for statistical analysis, and a value of P<0.05 was considered significant.
Membrane Potentials and Effects of LPC
With K+ internal solution, the membrane potential was −46.0±5.0 mV (n=30). 4-AP (Figure 1Aa,10 mmol/L) depolarized the membrane potential from −51.6±4 mV to −34±6 mV (Figure 1Aa, n=4, P<0.05). TEA (10 mmol/L, n=4, Figure 1Ab) also depolarized it. Figure 1B illustrates the effects of replacement of extracellular Na+ with membrane-impermeable NMDG+ on membrane potentials. It hyperpolarized the membrane potential from −43±3 mV to −69±6 mV (Figure 1Ba, 1Bb, n=5, P<0.05) reversibly. However, the reduction of [Cl−]o failed to hyperpolarize it (Figure 1Bb, n=4).
The effects of LPC (10 μmol/L) on membrane potential were investigated in Figure 1C. LPC gradually depolarized it from −45±4 mV to −10±6 mV (n=8, P<0.05). Replacement of Na+ with NMDG+ antagonized the depolarizing effects. It hyperpolarized the membrane from −8±5 mV in control to −26±6 mV in NMDG+ solution (n=4, P<0.05). Reduction of [Cl−]o failed to hyperpolarize it. The depolarizing effects of LPC were similarly observed at any cells of the passage numbers examined.
Background Current in Rabbit CASMCs
Figure 2 shows the effects of replacement of extracellular Na+ by NMDG+ on membrane currents. The patch pipette contained Cs+ internal solution with 5 mmol/L EGTA and 3 mmol/L ATP to block K+ currents and Ca2+-dependent currents. The cell was held at −40 mV and the voltage pulses were applied from −90 to +40 mV. The background current without any time-dependent activation and inactivation was observed. The current-voltage relation measured at steady state is illustrated in Figure 2B. It is almost linear and crosses zero at −10 mV in Na+-containing solution. Replacement of Na+ by NMDG+ reduced the inward current (Figure 2A) and shifted the reversal potential (Er) toward more negative potentials. Er was shifted from −10±5 to −38±5 mV (n=4, P<0.05). The amplitude of the background inward current measured at hyperpolarizing pulses of −90 mV was −120±40 pA (n=36).
Figure 3 shows the effects of La3+, nicardipine, and verapamil on the background current. The cells were held at +0 mV and the command voltage pulses were applied from −90 to +60 mV. The current-voltage relations measured at steady state in the control and in the presence of La3+ are illustrated in Figure 3B. La3+ (1 mmol/L) abolished the background current reversibly. Gd2+ (1 mmol/L) also abolished it. However, nicardipine (10 μmol/L, Figure 3C) and verapamil (50 μmol/L, Figure 3D) reduced it at −90 mV only by −14±7% (n=4) and −6±5% (n=4), respectively.
Effects of LPC on Membrane Currents
Figure 4 illustrates the effects of LPC on membrane currents. The patch pipette contained 140 mmol/L Cs+ internal solution. The cell was held at −60 mV and ramp pulses from −60 to +60 mV (200 ms duration) were applied. In Figure 4A, LPC (10 μmol/L) gradually increased the inward current at a holding potential within 1 to 2 minutes and La3+ (1 mmol/L) completely inhibited it. Figure 4Ab illustrates the current-voltage relations in control, in the presence of LPC, and LPC plus La3+. The current-voltage relation of the LPC-induced current was obtained by subtracting control current from the current in the presence of LPC. It was linear with Er of approximately +0 mV. The effects of LPC (10 μmol/L) were partly reversible as shown in Figure 4B. Similar effects of LPC were observed at any cells of the passage number examined.
Figure 4, C and D show the effects of LPC (1 to 50 μmol/L) on current-voltage relations obtained by ramp pulses and dose-dependent effects of LPC. The current amplitude induced by 50 μmol/L LPC at −60 mV was defined as 1.0, and the relative current amplitude induced by LPC is plotted against each concentration. LPC concentration-dependently induced the current.
Figure 5 illustrates the effects of La3+, Gd3+, and nicardipine on LPC-induced current. The voltage pulses were applied from −90 to +40 mV. The cells were held at +0 mV (Figure 5, A and B) and at −40 mV(Figure 5C). The current-voltage relations measured at steady state are shown in Figure 5, Ab through Cb. La3+ (1 mmol/L,Figure 5A) and Gd3+ (1 mmol/L,Figure 5B) abolished the LPC-induced current, whereas nicardipine (10 μmol/L, Figure 5C) and verapamil (50 μmol/L) only partly inhibited it at −90 mV by −20±5% and −15±5% (n=4), respectively.
Comparative Properties of the Background and LPC-Induced Current
Figure 6 compared ionic properties of the background current with those of LPC-induced current by using ramp pulses. The background current was obtained by subtracting the current in the presence of La3+ from the control current. The LPC-induced current was obtained by subtracting the control current from the current in the presence of LPC (10 μmol/L). Reduction of [Cl−]o caused no significant change of Er on the background and LPC-induced current. Er of background current was −6±5 and −5±5 mV (n=4, P>0.05) in control and low Cl− solutions, respectively (Figure 6Aa). Er of LPC-induced current was −2±3 and −1±3 mV (n=4, P>0.05) in control and low Cl− solution, respectively (Figure 6Ab). The replacement of Na+ by NMDG+ shifted Er of the background current from −6±5 to −31±10 mV (Figure 6Ba, n=4, P<0.05) and shifted Er of the LPC-induced current from −3±3 to −16±5 mV (Figure 6Bb, n=4, P<0.05).
To determine cationic selectivity of the background and LPC-induced current, Na+ was replaced by K+ and Cs+ (Figure 6C). The background current showed linear current-voltage relations (Figure 6Ca). Er was +5±3 mV (n=4) in 140 mmol/L K+ solution, +1±5 mV (n=4) in 140 mmol/L Cs+ solution, and −8±6 mV (n=4) in 140 mmol/L Na+ solution. The values of the slope conductance were 3.3±0.4 nS (n=4), 2.9±0.5 nS (n=4), and 2.2±0.5 nS (n=4) for K+, Cs+, and Na+, respectively. The permeability ratios were calculated according to the Goldman-Hodgkin-Katz equation Er=RT/ZF ln PX[X+]o/PCs[Cs+]i, where X+ is Na+, K+, or NMDG+ and F, R, T, Z have their usual meanings. [X+]o, a concentration of extracellular X+ ion, is 140 mmol/L and [Cs+]i, a concentration of internal Cs+ ion, is 140 mmol/L. The value of PK/PCs, PNa/PCs, and PNMDG/PCs, was 1.21, 0.73, and 0.30, respectively. The LPC-induced current also showed linear current-voltage relations (Figure 6Cb). Er was +1±4 mV (n=4), +0±3 mV (n=4), and −1±4 mV (n=4) for K+, Cs+, and Na+ bathing solution, respectively. The values of the slope conductance were 40±8 nS (n=4), 35±4 nS (n=4), and 33±6 nS (n=4) for K+, Cs+, and Na+, respectively. The values of PK/PCs, PNa/PCs, and PNMDG/PCs were 1.04, 0.96, and 0.54, respectively.
LPC-Induced Channels in Rabbit CASMCs
Figure 7 shows the results of single-channel recordings, using cell-attached methods. The patch pipette was filled with 140 mmol/L CsCl-pipette solution. The bath was perfused with high K+ bathing solution to settle membrane potential to approximately +0 mV. Under the conditions with LPC (10 μmol/L) in the patch pipette, marked channel activities were observed at a holding potential of −100 mV (lower trace), as compared with the control (upper trace). Figure 7B shows the amplitude histogram of LPC-induced channel, which was fitted by a sum of gaussian distribution using the least-squares method. The single channel amplitude measured at −100 mV was −3.4 pA. The current-voltage relations of LPC-induced channel are illustrated in Figure 7, C and D. The relations show linearity and slope conductance of 34 pS in this cell, and Er was approximately +0 mV. The mean slope conductance was 32±5 pS (n=4).
Effects of LPC on [Ca2+]i
Figure 8 shows the effects of LPC on [Ca2+]i. In Ca2+-free solution, LPC (50 μmol/L) did not affect [Ca2+]i significantly (Figure 8A). The addition of Ca2+ induced a sustained rise of [Ca2+]i, which was abolished by the removal of Ca2+ from the extracellular solution. The effects of LPC on [Ca2+]i were partly reversible (Figure 8B). La3+ (0.5 mmol/L, Figure 8C) blocked LPC-induced, sustained [Ca2+]i rise. However, verapamil (50 μmol/L, Figure 8D) and nicardipine (10 μmol/L, Figure 8E) partly inhibited it by −30±6% (n=3) and −19±7% (n=3), respectively. LPC (1 to 50 μmol/L) concentration-dependently increased [Ca2+]i (Figure 8F). Similar results were obtained from 4 different cells. To investigate whether LPC-induced [Ca2+]i rise is related to membrane potential, the bathing solution was changed from 5.4 to 140 mmol/L K+ solution to settle membrane potential to approximately +0 mV. LPC-induced [Ca2+]i rise was decreased (Figure 8F).
Voltage-dependent K+ current (KV) regulates vascular tone and forms membrane potential in VSMCs, including CASMCs.19 In our experimental conditions, 4-AP and TEA, K+ channel blockers that produce coronary spasm,20 inhibited KV and resulted in depolarizing the membrane. The membrane potential has been reported to be −56±2 mV and to 40.4±4.9 mV in canine and porcine CASMCs.9,10⇓ It was −46.0±5.0 mV in cultured CASMCs. These values were less than K+ equilibrium potential of approximately −80 mV, suggesting that additional membrane channels contribute to form membrane potential. The contribution of Cl− currents has been reported in VSMCs.11 In our study, however, reduction of [Cl−]o failed to affect the membrane potential, whereas replacement of Na+ by NMDG+ markedly hyperpolarized it. These results suggest that the contribution of Cl− current is minimal, and the background INSC contributes to form membrane potential in rabbit CASMCs.
The role of INSC has been investigated in pacemaking cells of hearts12,13⇓ and cardiac myocytes,14 where the background Na+ current is important to generate action potentials by raising resting membrane potential to the threshold for activation of Ca2+ current. The background INSC of rabbit CASMCs had the same tendencies of linear current-voltage relations and permeability sequences (K+>Cs+>Na+) as the properties of the background INSC described previously12–15⇓⇓⇓ and may play important roles in forming membrane potentials as reported in pulmonary VSMCs.15 Depolarization through INSC may open voltage-operated Ca2+ channels and subsequently increase [Ca2+]i, which regulates muscle contractility and leads to vasoconstriction.
The activation of the background INSC was not mediated by [Ca2+]i rise because it was still observed under the conditions with high EGTA in the patch pipette. Similar Ca2+-independent properties were observed in the background INSC of cardiac myocytes and pulmonary VSMCs,12–15⇓⇓⇓ though they were different from that reported in other cells.21
INSC has been reported to be activated by membrane stretch.22 Here, we provided evidence that LPC was a potent activator of INSC in rabbit CASMCs, which was consistent with previous studies of whole-cell clamp conditions showing that LPC induced INSC in guinea pig ventricular myocytes and dog renal VSMCs.23,24⇓ The effects of LPC on INSC were not dependent on the passage number, and similar effects were observed in cultured cells of any passage number and freshly isolated CASMCs (data not shown). In our study, the ionic permeability ratio of K+, Cs+, Na+, and NMDG+ of the LPC-induced current was 1.04, 1.00, 0.96, and 0.54, which showed the same tendencies as the previous study using renal VSMCs.24 However, it does not seem to be the same INSC reported in cardiac myocytes by Magishi et al,23 because NMDG+ passed the channel similarly as Na+ and K+, and Gd3+ did not inhibit it. Thus, it is likely that there are several types of LPC-induced INSC, depending on cell types investigated. Actually, the present study provided direct evidence showing that LPC activated a specific type of channels. However, further studies with single-channel analysis are needed to clarify the characteristic and molecular mechanisms underlying the activation of INSC by LPC.
Corr et al3 reported that LPC was increased during ischemia, and the concentration of LPC corresponded to 990 μmol/L. In addition, atherosclerotic arteries have been reported to be chronically exposed to high concentrations of LPC as compared with normal arteries.25,26⇓ Thus, LPC may accumulate under pathophysiological conditions such as ischemia and atherosclerosis. Our results indicate that LPC at concentrations of 1 to 50 μmol/L induced INSC, depolarized the membrane, and resulted in [Ca2+]i rise. Thus, the effects of LPC observed in this study may play significant roles in pathophysiological conditions such as ischemia.
LPC increased [Ca2+]i in rabbit CASMCs, which was consistent with the previous studies in ASMCs or cardiac myocytes.27,28⇓ LPC may increase [Ca2+]i through an increase of Ca2+ entry or release from Ca2+ storage sites. In the absence of extracellular Ca2+, it failed to increase [Ca2+]i, suggesting that LPC increased Ca2+ influx as reported in rat ASMCs.28,29⇓ Several mechanisms by which LPC might influence [Ca2+]i have been proposed. The [Ca2+]i increase induced by LPC may relate to detergent or toxic effects of this amphiphile.29 However, we used low concentrations of LPC (<50 μmol/L), and cell viability measured by trypan blue exclusion was not changed in between control and LPC-treated cells. Moreover, La3+ and Gd3+ completely abolished it, suggesting that detergent action of LPC is not likely. LPC depolarized the membrane, proposing that LPC increased [Ca2+]i indirectly through voltage-dependent L-type Ca2+ channels. Actually, nicardipine and verapamil partly inhibited LPC-induced [Ca2+]i rise. However, La3+ and Gd3+ abolished it, suggesting that LPC increased [Ca2+]i mainly by activating Ca2+ entry pathways other than L-type Ca2+ channels. In our study, including single-channel analysis, LPC activated INSC with a large amplitude of approximately −1 nA at around −50 mV near resting membrane potential. La3+ and Gd3+ inhibited both the activation of INSC by LPC and LPC-induced [Ca2+]i rise. In addition, when cells were bathed with high K+ solution to settle the membrane potential to approximately +0 mV, LPC-induced [Ca2+]i rise was decreased. These observations also support that LPC increased [Ca2+]i through activation of INSC.
LPC has been reported to increase cGMP-dependent verapamil-sensitive [Ca2+]i in ASMCs.28 However, verapamil (50 μmol/L) and nicardipine (10 μmol/L) only partly inhibited LPC-induced [Ca2+]i rise, proposing that verapamil-sensitive Ca2+ influx pathways do not largely contribute to [Ca2+]i rise induced by LPC. The discrepancy among these results may depend on different concentrations of LPC or different cell types. LPC enhances cell proliferation and migration of CASMCs.29,30⇓ Since [Ca2+]i is known to be related to cell proliferation and migration, activation of INSC by LPC may play essential roles in these events as well as CASMC tone.
In conclusion, INSC plays important roles in forming membrane potentials of CASMCs and LPC induces INSC and then depolarizes the membrane, resulting in an increase of [Ca2+]i. Thus, LPC may affect CASMCs tone under various pathophysiological conditions such as ischemia.
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- ↵Harder DR, Belardinelli L, Sperelakis N, et al. Differential effects of adenosine and nitroglycerin on the action potentials of large and small coronary arteries. Circ Res. 1979; 44: 176–182.
- ↵Hume JR, Giles W. Ionic currents in single isolated bullfrog atrial cells. J Gen Physiol. 1983; 81: 153–194.
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- ↵Nakajima T, Iwasawa K, Oonuma H, et al. Troglitazone inhibits voltage-dependent calcium currents in guinea pig cardiac myocytes. Circulation. 1999; 99: 2942–2950.
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