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Monovalent cation and L-type Ca²⁺ channels participate in calcium paradox-like phenomenon in rabbit aortic smooth muscle cells

Sergey I. Zakharov, Dmitry A. Mongayt, Richard A. Cohen and Victoria M. Bolotina

Received 27 April 1998; accepted after revision 22 September 1998.

	ABSTRACT

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1. The effects of removal of extracellular divalent cations (experimental calcium paradox conditions) were studied on the whole-cell current in freshly isolated smooth muscle cells (SMCs), and on contraction in rabbit aortic rings.

2. Aortic rings treated for 30-60 min with extracellular Ca²⁺- and Mg²⁺-free solution contracted following readmission of extracellular Ca²⁺, even in the presence of nifedipine.

3. In isolated SMCs, the removal of extracellular Ca²⁺ and Mg²⁺ induced a non-inactivating whole-cell inward current and membrane depolarization. This current was a monovalent cation (MC) current which reversed at around 0 mV and conducted K⁺ Cs⁺ > Na⁺ > Li⁺. Extracellular divalent cations (Ca²⁺, Mg²⁺, Ba²⁺, Mn²⁺ and Ni²⁺) inhibited MC current.

4. Using noise analysis of the whole-cell MC current, the single MC channel conductance was estimated to be < 450 fS.

5. MC current was insensitive to nifedipine, TEA, 4-aminopyridine, SK&F 96365 and S-nitroso-N-acetyl-penicillamine (SNAP), but was decreased by amiloride and low pH.

6. When EGTA was present in Ca²⁺- and Mg²⁺-free solution, a significant nifedipine-sensitive Na⁺ current through L-type Ca²⁺ channels developed in addition to MC current.

7. It is concluded that upon the removal of extracellular Ca²⁺ and Mg²⁺ from resting SMCs, an inward MC current develops allowing Na⁺ influx and causing SMC depolarization which could be the important steps leading to vessel contraction upon Ca²⁺ readmission. Addition of EGTA to Ca²⁺- and Mg²⁺-free solution greatly potentiates Na⁺ influx and vessel contraction by allowing additional Na⁺ influx through L-type Ca²⁺ channels which are activated presumably by MC current-induced depolarization.

	INTRODUCTION

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Removal of extracellular Ca²⁺ followed by Ca²⁺ readmission produces pathological changes in a variety of mammalian cells and organs. This phenomenon was first described in the heart and called the 'calcium paradox' (Zimmerman & Hulsmann, 1966). It is thought (for review see Chapman & Tunstall, 1987) that during exposure of cardiomyocytes to Ca²⁺-free media, Na⁺ enters the cell significantly increasing the intracellular Na⁺ concentration. As a result, following extracellular Ca²⁺ readmission, there is a massive Ca²⁺ influx into the cell carried by the Na⁺-Ca²⁺ exchanger working in the reverse mode.

In spite of many studies, the role and mechanism for Na⁺ influx remain controversial (Van Echteld et al. 1998). Na⁺ current through L-type Ca²⁺ channels is thought to be the major pathway; however, inhibition of these channels is known to decrease but not eliminate calcium paradox. Recently, Mubagwa et al. (1997) showed that a monovalent cation (MC) current develops in cardiomyocytes in the absence of extracellular divalent cations which conduct Na⁺ and produce depolarization which can activate voltage-gated Ca²⁺ channels. They proposed that the MC current plays an important role in triggering calcium paradox in the heart. A similar MC current sensitive to extracellular divalent cations has also been found in amphibian epithelial cells (Van Driessche & Zeiske, 1985), chick embryo ectoderm (Li et al. 1994; Sabovik et al. 1996), and in Xenopus oocytes (Arellano et al. 1995). The physiological significance of MC current remains uncertain, but it can be one of the major mechanisms underlying the pathological calcium paradox in a variety of cells.

A calcium paradox-like phenomenon (similar to the one described in cardiomyocytes and the heart) has also been observed in smooth muscle cells (SMCs) and blood vessels (Kobayashi et al. 1985; Kutsky & Hester, 1986; Lindner & Heinle, 1987), but the mechanisms of its development are not understood. Contraction of blood vessels under conditions which induce calcium paradox in the heart is of particular interest because of the importance of coronary blood flow to cardiac function. In this respect, it is important to determine the distinct pathways which in the absence of extracellular divalent cations are responsible for pathological Na⁺ influx in SMCs leading to vessel contraction upon Ca²⁺ readmission.

The present study is the first attempt to determine the ion channel-mediated mechanisms of Na⁺ influx in experimental calcium paradox conditions in individual SMCs, and to determine their effects on the behaviour of the whole blood vessel. Using the perforated patch-clamp technique in isolated SMCs from rabbit aorta, we describe a nifedipine-insensitive monovalent cation (MC) current which develops in Ca²⁺,Mg²⁺-free solution. When all the residual extracellular Ca²⁺ was chelated by EGTA, in addition to MC current, a nifedipine-sensitive Na⁺ current through L-type Ca²⁺ channels develops in SMCs (Mironneau et al. 1982; for review also see Tsien et al. 1987). The results in rabbit aortic rings indicated that under calcium paradox conditions, nifedipine-insensitive MC current alone can produce Na⁺ influx and depolarization of SMCs sufficient for contraction of the vessel upon Ca²⁺ readmission. Activation of Na⁺ influx through L-type Ca²⁺ channels can be triggered by MC-induced depolarization and occurred only in the presence of EGTA which greatly potentiated vessel contraction. Thus, Na⁺ influx through both MC and L-type Ca²⁺ channels could precede the development of SMC contraction under experimental calcium paradox conditions. A preliminary report of this work has appeared as an abstract (Zakharov et al. 1998).

	METHODS

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Single-cell isolation procedure

New Zealand rabbits (2-2·5 kg) were killed by exsanguination after an I.V. dose of sodium pentobarbitone (40 mg kg^-1). All anaesthetic and surgical procedures used fall within the guidelines of the local Animal Ethics Committee. The thoracic aorta was excised and adhering fat and connective tissue were removed. Aortic smooth muscle cells (SMCs) were enzymatically isolated by the method of Clapp & Gurney (1991) with some modifications. Briefly, the aorta was opened longitudinally, endothelium was removed by carefully scraping the inside surface of the vessel, and 1 mm wide strips of the medial smooth muscle layer were separated from adventitia and transferred into a low CaCl₂ (150 µM)-containing dissociation medium (DM) of the following composition (mM): NaCl, 110; NaHCO₃, 10; NaH₂PO₄, 0·5; KCl, 5; KH₂PO₄, 0·5; CaCl₂, 0·16; MgCl₂, 2; glucose, 10; bovine serum albumin, 0·02 %; Hepes, 10; pH 7·0. The preparation was then stored at 5°C for 1 h. After that, the strips were transferred to the DM containing dl-dithiothreitol (2 mM) and papain (4 mg ml^-1), and were placed into an incubator at 37°C with gentle stirring for 30 min. Next, the digested strips were washed two times in the enzyme-free DM. Individual SMCs were obtained by tituration in fresh DM and stored at 5°C until used during the next 6 h. Only relaxed SMCs were used in our studies.

Electrophysiological recordings and data analysis

An aliquot of SMCs was added to the recording chamber (1 ml) which was mounted on the stage of an inverted microscope (Olympus IX70-S8F). Control solution was perfused through the chamber by gravity at a rate of 1 ml min^-1. Experimental solutions were applied to the individual SMCs through a special tube (3 mm i.d.) containing six smaller tubes which allowed rapid (< 3 s) changes of experimental solutions. Experiments were done at room temperature (22-24°C).

The patch-clamp technique (Hamill et al. 1981) was used for ion current and membrane potential (V_m) recording. Data were recorded with an Axopatch 200A amplifier (Axon Instruments), filtered at 2 kHz, sampled at 10 kHz and stored on a hard drive using an IBM computer with DigiData 1200 interface and pCLAMP 6 software (Axon Instruments). Pipettes were pulled from borosilicate glass capillaries (WPI) on a horizontal puller (model P-87, Sutter Instrument Company) and fire polished with a microforge (model MF-9, Narishige). Pipettes had the resistance of 1-3 M when filled with pipette solution.

Whole-cell currents and membrane potential were recorded using the perforated-patch technique (Horn & Marty, 1988) which allows the exchange of monovalent cations and anions, but not divalent cations, between the pipette and the cell. Electrical access to the cytoplasm was obtained by adding amphotericin B to the pipette solution, as described previously (Rae et al. 1991). Series resistance of 28 ± 4 M (n = 42) was achieved approximately 15 min after the cell-attached configuration was obtained. Before the recording of whole-cell current and membrane potential, series resistance and SMC capacitance were compensated using corresponding controls of the amplifier. The input resistance of SMCs was measured from the current response to a voltage step from -80 to -100 mV. Only resting SMCs with input resistance of more than 5 G were used for experiments. Leakage current was not subtracted.

Whole-cell currents were recorded using voltage ramps (1 s), or voltage steps (600 ms) applied from the holding potential of -80 mV. The time course of the changes in whole-cell current were monitored by voltage ramps applied from the holding potential every 5 s. Membrane potential was measured in the current-clamp mode of the patch-clamp amplifier. Data were corrected for a junction potential which was determined at the end of each experiment.

Noise analysis of the whole-cell current

Non-stationary noise analysis was done using the standard methods (Sigworth, 1977, 1980; Heinemann & Conti, 1992). Data were filtered at 2 kHz and sampled at 10 kHz. Whole-cell current during application of Ca²⁺,Mg²⁺-free extracellular solution was recorded and analysed in different SMCs at constant holding potentials (-100, -80 and -40 mV). Mean current (I) and current variance (²) were determined during 25-100 ms intervals. During the sampling intervals, the mean current changes by < 10 %. Analysis of the dependence of variance ² on mean current I was performed using ORIGIN (Microsoft, Redmond, WA, USA). The single channel current (i) was estimated for each experiment by fitting the equation ²(I) = Ii - I²/N + B, where ²(I) is the variance, I is the mean current, N is the number of functional channels, and B is a free parameter reflecting the background noise. Single channel conductance () was estimated from the slope of the single channel current-voltage relationship.

Tension measurement

Isometric tension was measured in rabbit aortic rings as previously described (Weisbrod et al. 1997). Briefly, the rabbit thoracic aorta was cleaned of fat and connective tissue, and endothelium was removed by gentle rubbing of the inner surface with forceps. The vessel was then cut into 6 mm wide rings which were mounted in organ chambers (25 ml) which contained oxygenated physiological solution. The rings were equilibrated under tension (6 g) for 60 min. Isometric tension recording was done with a Grass recorder (model 7D Polygraph). Experiments were done at 37°C.

Solutions and drugs

Tension experiments were done in physiological solution of the following composition (mM): NaCl, 118; KCl, 4·7; CaCl₂, 2; MgSO₄, 1·2; NaHCO₃, 25; KH₂PO₄, 1·2; glucose, 10 (with bubbling 95 % O₂-5 % CO₂ to give a pH of 7·35). All the drugs were added directly to the organ chambers. For patch-clamp experiments the control bath solution contained (mM): NaCl, 140; KCl, 2·8; CaCl₂, 2; MgCl₂, 1; Hepes, 10; glucose, 11 (pH 7·4 with NaOH). Ca²⁺,Mg²⁺-free solution was made by omitting these ions from the standard bath solution. The concentration of residual free Ca²⁺ in Ca²⁺-free solution (in the absence of EGTA) was 1-3 µM as measured using a Ca²⁺-sensitive electrode. In some experiments NaCl in the bath solution was replaced by equimolar CsCl, LiCl, KCl or N-methyl-D-glucamine chloride (NMDG⁺). In a few experiments intracellular Ca²⁺ was buffered by pretreatment with 20 µM BAPTA-AM (acetoxymethyl ester of 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid) for 20 min. For whole-cell current and V_m recording, pipettes were filled with a solution containing (mM): potassium aspartate, 100; KCl, 40; NaCl, 4·5; EGTA, 0·1; MgCl₂, 3; and Hepes, 10 (pH 7·2 adjusted with KOH). Nifedipine was diluted in the bath solution just before the experiment using stock solution in DMSO (final concentration of DMSO < 0·1 %). Amiloride, tetraethylammonium (TEA), 4-aminopyridine (4-AP), 1--[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl-1H-imidazol hydrochloride (SK&F 96365; Biomol, Plymouth Meeting, PA, USA) and S-nitroso-N-acetylpenicillamine (SNAP) were freshly prepared as aqueous solutions prior to use. Unless otherwise indicated, all salts and drugs used in this study were obtained from Sigma.

Statistical analysis

Results were expressed as means ± standard error of the mean; n represents the number of cells tested. Statistical significance was evaluated using Student's t test for paired observations. Differences were considered significant at P < 0·05.

	RESULTS

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Calcium paradox-like phenomenon in rabbit aortic rings

Removal of extracellular divalent cations (application of Ca²⁺,Mg²⁺-free solution) did not change the resting tone of rabbit aortic rings, but readmission of Ca²⁺ produced a sustained contraction (Fig. 1A) which increased with the time of vessel pre-exposure to Ca²⁺,Mg²⁺-free solution. After 30 and 60 min treatment of aortic rings with Ca²⁺,Mg²⁺-free solution in the continuous presence of nifedipine (10 µM), readdition of extracellular Ca²⁺ produced contraction of 0·6 ± 0·2 g (n = 7) and 1·5 ± 0·4 g (n = 5), respectively. There was no significant contraction upon Ca²⁺ readmission when Ca²⁺, but not Mg²⁺ was removed from the bath for 60 min (n = 5). Addition of EGTA (1 mM) to the Ca²⁺,Mg²⁺-free solution (to chelate the residual Ca²⁺) potentiated the contraction (Fig. 1B) upon Ca²⁺ readmission. Nifedipine (10 µM), when present throughout the whole experiment, eliminated the additional contraction induced by EGTA (Fig. 1C, n = 8), while the remaining nifedipine-insensitive part of contraction was similar to that observed in the absence of EGTA (see Fig. 1A). Nifedipine added shortly before Ca²⁺ readmission reduced the initial transient, but not the sustained part of the contraction (Fig. 2A, n = 5). The nifedipine-insensitive sustained contraction was also insensitive to the inhibitor of non-selective cation channels, SK&F 96365 (10 µM, n = 5), but was inhibited by nickel (5 mM, n = 4) which along with L-type Ca²⁺ and non-selective cation channels is also known to inhibit the Na⁺-Ca²⁺ exchanger. It is important to mention that sustained vessel contraction on Ca²⁺ readmission was also dependent on the time of vessel exposure to Ca²⁺,Mg²⁺-free solution containing EGTA (Fig. 2B). These results show that vessel contraction upon Ca²⁺ readmission depends on two distinct time-dependent processes which are activated before readmission of Ca²⁺ following vessel exposure to Ca²⁺,Mg²⁺-free conditions: (1) a nifedipine-insensitive process, and (2) a nifedipine-sensitive process which is present only when EGTA is added to Ca²⁺,Mg²⁺-free solution.

To determine the nature of the nifedipine-sensitive and insensitive events which occur in SMCs in the absence of extracellular divalent cations (in the absence or presence of EGTA), and which lead to vessel contraction upon Ca²⁺ readmission, ion currents were measured in individual SMCs under similar conditions.

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Figure 1. The effect of extracellular Ca2+ and Mg2+ removal and subsequent Ca2+ readmission on the tension of rabbit aortic rings A, B and C, representative tracings of tension recorded in the rings of rabbit aorta during their exposure to Ca2+,Mg2+-free solution followed by readmission of extracellular Ca2+. EGTA (1 mM) was added to Ca2+,Mg2+-free solution in B and C. Nifedipine (10 µM) was present during the whole experiment in C. Note that 3 instead of 2 mM Ca2+ was added to the rings in B and C to overcome the presence of EGTA.

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Figure 2. Transient and sustained contraction of rabbit aorta rings upon Ca2+ readmission: dependence on L-type Ca2+ channels and the time of vessel exposure to Ca2+, Mg2+-free solution A, representative tracings of simultaneous contraction of paired rings of rabbit aorta to phenylephrine (PE, 0·5 µM), and to Ca2+ (3 mM) readmission after 30 min of the ring treatment in Ca2+,Mg2+-free solution with EGTA (1 mM). Nifedipine (Nif; 10 µM) added before Ca2+ readmission (lower trace) reduces the initial transient, but not sustained part of the contraction. B, dependence of the sustained contraction (on Ca2+ readmission) on the time of exposure to Ca2+,Mg2+-free solution with EGTA. The contraction in each ring was normalized to the contraction produced by phenylephrine (0·5 µM) in the same ring. Bars represent means plus standard errors of the mean from 26 rings (from aortas of 6 rabbits). The value above each bar represents the number of experiments.

Monovalent cation (MC) current in single SMCs

A typical example of the effect of removal of divalent cations (Ca²⁺ and Mg²⁺) from the extracellular solution on macroscopic whole-cell current and membrane potential in SMCs (in the presence of the outward K⁺ currents) is shown in Fig. 3. In Ca²⁺,Mg²⁺-free bath solution (with no EGTA present), a pronounced inward current was activated at negative membrane potentials (Fig. 3A). Figure 3B shows the time course of the development of inward current measured at -80 mV. The effect of the removal of divalent cations was reversible, and either Ca²⁺ (2 mM) or Mg²⁺ (1 mM) were able to inhibit most but not all of the inward current when added separately (Fig. 3B). Removal of Ca²⁺ and Mg²⁺ caused significant and reversible depolarization of SMCs from a control level of -41 ± 1 mV to -28 ± 2 mV (n = 12, Fig. 3C). This depolarization was not prevented by nifedipine (10 µM, n = 5). Buffering of intracellular Ca²⁺ with BAPTA (pretreatment of SMCs with 20 µM BAPTA-AM for 20 min) did not change the inward current developed in Ca²⁺,Mg²⁺-free solution (Fig. 3A, n = 3).

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Figure 3. Effect of extracellular Ca2+ and Mg2+ removal on the whole-cell current and membrane potential in SMCs (when K+ currents were not inhibited) A, current-voltage relationships obtained by voltage ramps from -100 mV to +25 mV (holding potential -80 mV) in (1) control solution, (2) Ca2+,Mg2+-free solution and (3) Ca2+,Mg2+-free solution after treatment with BAPTA-AM (20 µM for 20 min). B, the time course of the effect of Ca2+ and Mg2+ removal and readmission on inward current recorded at -80 mV every 5 s. C, the effect of extracellular Ca2+ and Mg2+ removal on membrane potential in the same SMC as in B.

To separate the divalent cation-sensitive current, the outward K⁺ current was inhibited by substitution of K⁺ with Cs⁺ in the pipette and addition of TEA (5 mM) to the extracellular solution. Under such conditions removal of Ca²⁺ and Mg²⁺ evoked a non-inactivating 'leakage-like' current (Fig. 4A) which reversed at -3 ± 2 mV and showed some inward rectification (Fig. 4B and C, n = 13). This current was activated in all SMCs tested (n = 58), but its density varied from cell to cell. When measured at -80 mV, in a few SMCs it was as small as 0·1-0·2 pA pF^-1, or as large as 5-6 pA pF^-1, but in the majority of SMCs it was about 1 pA pF^-1 (0·9 ± 0·2 pA pF^-1, n = 52).

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Figure 4. The effect of extracellular Ca2+ and Mg2+ removal on whole-cell current when K+ currents were inhibited by 40 mM Cs+ in the pipette and 5 mM TEA in the bath A, current traces during voltage steps (shown in inset) in control and in Ca2+,Mg2+-free solution in a single SMC. The difference current activated by the removal of extracellular divalent cations was obtained by subtraction of the control current from the corresponding current in Ca2+,Mg2+-free solution. B, current-voltage relationships from the same cell obtained during voltage ramps (from -100 to +60 mV) in control and in Ca2+,Mg2+-free solution and when all extracellular Na+ was replaced by NMDG+. C, the current-voltage relationship of the difference current activated by the removal of extracellular divalent cations in the presence of extracellular Na+ (Ca2+,Mg2+ free) obtained from B by subtraction of the current in control solution from the corresponding current in Ca2+,Mg2+-free solution. D, the time course of inward current development upon extracellular Ca2+ and Mg2+ removal measured at -80 mV in extracellular solutions containing 140 mM Li+, Na+ or Cs+.

Figure 4B and C shows that, in the absence of extracellular divalent cations, replacement of extracellular Na⁺ with the impermeable cation NMDG⁺ eliminated the inward component of the current (with no change in outward current) causing a negative shift in reversal potential from -4 ± 2 mV to -60 ± 3 mV (n = 4). This is consistent with a very high cation selectivity of this current. Such high cation selectivity discriminates this current from a simple passive leak which can develop at the junction of the cell membrane with the glass patch-clamp pipette. Figure 4D shows that equimolar substitution of extracellular Na⁺ by other monovalent cations (Li⁺, Cs⁺) did not prevent the development of the inward current in Ca²⁺,Mg²⁺-free solution. The density of K⁺, Cs⁺ and Li⁺ currents was 137 ± 14 % (n = 4), 117 ± 16 % (n = 7) and 52 ± 5 % (n = 6) of the corresponding Na⁺ current. Thus, the ionic preference of this current for monovalent cations had the following order K⁺ Cs⁺ > Na⁺ > Li⁺. To determine if divalent cations can also permeate through the same channels, we recorded and compared inward whole-cell currents at low and high concentrations of extracellular divalent cations. An increase in the concentration of extracellular Ca²⁺ from 2 to 10 mM did not increase, but rather decreased, the inward current (see Fig. 5B). Addition of 10 mM Ba²⁺ did not affect the whole-cell current at -80 mV, but evoked an additional inward current which was a typical Ba²⁺ current through L-type Ca²⁺ channels in SMCs (Ohya et al. 1986; Bolton et al. 1988).

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Figure 5. Extracellular divalent cations block the MC current A, the time course of concentration-dependent inhibition by extracellular Ca2+ of MC current recorded at -80 mV. B, the dependence of the normalized MC current (at -80 mV) on the concentration of extracellular Ca2+. The basal (zero) level of the current (shown by the dashed line) corresponds to the current under control conditions (in the presence of 2 mM Ca2+ and 1 mM Mg2+), and the MC current activated by the removal of extracellular Ca2+ and Mg2+ was taken as 100 %. Ca2+ was added in the continuous absence of Mg2+. The data points were fitted by the Hill equation: I = 100(1 + KCa/[Ca2+]onH)-1, where KCa is an affinity constant, [Ca2+]o is the extracellular Ca2+ concentration and nH is the Hill coefficient. The best fit was generated with a KCa value of 250 µM and n = 0·8. The values above the standard error bars represent the number of experiments. C, activation of MC current by the removal of all extracellular divalent cations, and its inhibition by extracellular application of 1 mM Ba2+, Mn2+ or Ni2+. MC current was recorded at -80 mV every 5 s.

These data demonstrate that the current developing upon removal of extracellular divalent cations is a non-selective monovalent cation (MC) current. Because the electrogenic exchangers possess high cation selectivity (O'Donnell & Owen, 1994), the poor selectivity of the current to a variety of monovalent cations and its reversal at around 0 mV suggests that monovalent cation channels rather than cation exchangers are responsible for the MC current.

Sensitivity of the monovalent cation (MC) current to Ca²⁺ and other divalent cations

Addition of extracellular Ca²⁺ or Mg²⁺ to the bath solution inhibited the MC current (Fig. 3B). Figure 5A and B shows that the MC current was blocked by extracellular Ca²⁺ in a dose-dependent manner with a dissociation constant (K_Ca) of 250 µM and a Hill coefficient of 0·8. It is important to mention that 1 or 2 mM extracellular Ca²⁺ (when added alone without Mg²⁺) did not inhibit the MC current completely, while 10 mM Ca²⁺ reduced the current even below the basal level (which was observed in the presence of 2 mM Ca²⁺ and 1 mM Mg²⁺). Extracellular application of 1 mM Mg²⁺, Ba²⁺, Mn²⁺, Ni²⁺ (Figs 3B and 5C) or Co²⁺ (not shown) also reversibly inhibited the current (Ba²⁺ being the least potent).

Noise analysis of MC current

To characterize the single channels which underlie the whole-cell MC current, noise analysis of the whole-cell current was made. Figure 6A shows an example of the whole-cell current recorded in a SMC at -100 mV in control and in Ca²⁺,Mg²⁺-free solution. Figure 6B shows an example of the distribution of current variance at -40 and -100 mV in the same SMC fitted with the equation ²(I) = iI - I²/N + B, where I is a mean whole-cell current , and i is an estimated single channel current. Figure 6C summarizes the dependence of single channel current amplitudes on membrane potential in different SMCs (n = 4). Single MC channel conductance was estimated to be 450 fS, which at the present time is below the level of resolution of individual single channel currents in cell-attached or excised membrane patches.

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Figure 6. Noise analysis of MC current A, the traces of the whole-cell current are shown at -100 mV in external 140 mM NaCl solution containing 2 mM Ca2+ and 1 mM Mg2+ (control), and 1 min after the removal of extracellular Ca2+ and Mg2+ (Ca2+,Mg2+ = 0) when MC current reached plateau. B, representative plot of current variance versus mean current (I) at -40 and -100 mV in the same SMC. Each point represents the current variance during consecutive (50 ms long) intervals recorded during the development of MC current in Ca2+,Mg2+-free solution (with no EGTA present). C, summary of estimated single channel amplitude (i) versus membrane potential (Vm) in different SMCs (n = 4). Single channel conductance = 450 fS is obtained from the slope of i-V relation.

Effect of pharmacological inhibitors on divalent cation-sensitive MC current

Looking for possible inhibitors of MC current (other than divalent cations), several inhibitors of Ca²⁺, K⁺ and non-selective cation channels were tested. Figure 7 shows that the MC current was insensitive to nifedipine (10 µM), 4-AP (1 mM), SK&F 96365 (10 µM) and the nitric oxide donor, SNAP (200 µM). Neither NMDG⁺ (5 mM) nor TEA (5 mM) in the presence of Na⁺ inhibited MC current, demonstrating that complete inhibition of inward MC current by substitution of these non-permeable cations for extracellular Na⁺ (Figs 4 and 8) is due to complete removal of extracellular Na⁺. Amiloride, in high concentration (1 mM), was the only inhibitor tested which reversibly decreased MC current by approximately 50 %. We also found that the lowering of extracellular pH from 7·4 to 6·3 significantly reduced MC current.

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Figure 7. Effect of nifedipine (Nif; 10 µM), SK&F 96365 (10 µM), TEA (5 mM), NMDG+ (5 mM), 4-AP (1 mM), amiloride (Amil; 1 mM) and SNAP (100 µM) on the monovalent cation (MC) current recorded at -80 mV The 'basal leak' current was subtracted, and the MC current activated by the removal of extracellular Ca2+ and Mg2+ was taken as 100 % (Con; control). The value above each bar shows the number of tested cells. Data are means ± S.E.M. * P < 0·05 .

Divalent cation-sensitive MC current and Na⁺ current through L-type Ca²⁺ channels are two different currents

In SMCs from rabbit aorta, L-type Ca²⁺ current was not resolvable at physiological concentrations of extracellular Ca²⁺ (2 mM). In Ca²⁺,Mg²⁺-free solution in the majority of SMCs there was no inward current other than MC. However, addition of EGTA (1 mM) to the external Ca²⁺,Mg²⁺-free solution caused the development of nifedipine-sensitive Na⁺ current in all SMCs tested (n = 14). Figure 8 shows the presence of two different Na⁺-conducting currents which we could experimentally separate in SMCs by varying the amount of extracellular Ca²⁺. Figure 8A shows changes in the amplitude of the whole-cell current at -80 mV () and -20 mV () throughout the whole experiment. Corresponding I-V relationships obtained under different conditions are shown in Fig. 8B, C and D. Removal of extracellular Ca²⁺ and Mg²⁺ evoked a typical 'leakage-like' MC current (Fig. 8A and B). When EGTA (1 mM) was added to Ca²⁺,Mg²⁺-free bath solution, an additional inward current developed at membrane potentials from -40 to +30 mV (with a maximum near -20 mV) with no change in inward current at more negative membrane potentials (Fig. 8A and C). This 'L-type Ca²⁺ channel'-like component of inward current was effectively blocked by nifedipine (10 µM, n = 9), but nifedipine did not affect the residual MC current (n = 9). The amplitude of the current through L-type Ca²⁺ channels measured at -20 mV varied significantly from cell to cell and sometimes exceeded the MC current measured at -80 mV. Both components of inward current were reversibly eliminated by substitution of extracellular Na⁺ with NMDG⁺ (Fig. 8A and D, n = 6), or TEA⁺ (n = 4, not shown) consistent with Na⁺ being the main cation responsible for both currents.

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Figure 8. Monovalent cation (MC) current and Na+ current through L-type Ca2+ channels are two different currents A, the time course of changes in inward current monitored at -80 mV () and at -20 mV () in control, in Ca2+,Mg2+-free solution, after EGTA (1 mM) and nifedipine (10 µM) were added to the bath solution, and when external Na+ was replaced by NMDG+. B, C and D, corresponding current-voltage relationships at different times during the experiment (indicated in A). Voltage ramps (from -100 to +60 mV) were applied every 5 s from a holding potential of -80 mV.

	DISCUSSION

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The present study demonstrates for the first time that Na⁺ influx through two different pathways (MC channels and L-type Ca²⁺ channels conducting Na⁺) can occur in SMCs upon removal of extracellular divalent cations (calcium paradox-like conditions).

Monovalent cation (MC) current in SMCs

We found that removal of Ca²⁺ and Mg²⁺ from the extracellular solution induces a pronounced inward current in the physiological range of membrane potentials and causes SMC depolarization similar to that recently described in cardiomyocytes (Mubagwa et al. 1997). The current appeared to be selective for the monovalent cations K⁺, Cs⁺, Na⁺ and Li⁺, but was virtually impermeable to divalent cations (Ca²⁺ and Ba²⁺). High cation selectivity of the MC current clearly discriminates it from a leakage current. On the other hand, poor selectivity of this current to a variety of monovalent cations is consistent with ion channels, rather than any specific ion exchanger, being responsible for MC current. The prominent feature of MC current is its high sensitivity to a variety of extracellular divalent cations (Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Ba²⁺ and Co²) which all inhibited it when applied in millimolar concentrations. It is important to mention that extracellular Ca²⁺ (which inhibited MC current with K_Ca = 250 µM) did not inhibit MC current completely when added alone (without Mg²⁺) at physiological concentrations of 1-2 mM. All these important features allowed us to distinguish MC current from a variety of other non-selective cation currents described previously in SMCs (see Isenberg, 1993).

The single channel properties of the MC current were estimated by noise analysis of whole-cell current evoked by the removal of extracellular divalent cations under conditions in which other ion channels in SMCs were blocked. The unitary conductance of MC channels appeared to be extremely low (less than 450 fS) which does not allow us to record and study single MC channel currents in cell-attached or excised membrane patches.

In an attempt to inhibit the MC current, several different ion channel inhibitors were tested including nifedipine, TEA, 4-AP, amiloride, SK&F 96365 and SNAP. Only a high concentration of amiloride (1 mM) decreased, but did not completely inhibit, the MC current. Looking for other ways to inhibit MC current, we found that lowering extracellular pH decreased MC current in SMCs. Interestingly, amiloride and acidic pH have been reported to have a protective effect on Ca²⁺ paradox-induced cellular damage in rat kidney and heart (Duncan & Morton, 1996; Harding & Duncan, 1997).

Na⁺ current through L-type Ca²⁺ channels in SMCs

It is known that L-type Ca²⁺ channels become permeable to monovalent cations when all divalent cations are removed from the extracellular solution (Prosser et al. 1977; Almers & McCleskey, 1984; Hess et al. 1986; Matsuda, 1986; for review also see Tsien et al. 1987). In SMCs significant Na⁺ influx through Ca²⁺ channels was previously reported when extracellular Ca²⁺ was buffered to nanomolar concentrations by the addition of EGTA to Ca²⁺-free solution (Mironneau et al. 1982; Ohya et al. 1986; Jmari et al. 1987). We found that Na⁺ current through L-type Ca²⁺ channels developed in addition to MC current, but only when EGTA was added to Ca²⁺,Mg²⁺-free solution. This additional current, in contrast to the MC current, was completely blocked by 10 µM nifedipine. Thus, based on different sensitivity to extracellular Ca²⁺ and to nifedipine, MC and L-type Ca²⁺ channel-mediated pathways for Na⁺ influx in SMCs can be clearly distinguished. It is attractive to speculate that, at physiological resting potentials, removal of extracellular cations can first activate MC current which will cause membrane depolarization and promote the opening of L-type Ca²⁺ channels which can produce additional significant influx of Na⁺ into SMCs. Note that because of the differences in the current-voltage relationship of the MC and Ca²⁺ channels, cell depolarization will tend to decrease Na⁺ influx through MC channels and increase it through L-type Ca²⁺ channels.

The role of MC and L-type Ca²⁺ channels in contraction of SMCs upon Ca²⁺ readmission

Previously it was shown that after perfusion with Ca²⁺,Mg²⁺-free (EGTA containing) solution, aortic smooth muscle contracts upon Ca²⁺ readdition, and that the contraction is only partially sensitive to the L-type Ca²⁺ channel inhibitor, methoxyverapamil (D600) (Kutsky & Hester, 1986). A significant intracellular Na⁺ rise was reported in rabbit aorta during the period of Ca²⁺,Mg²⁺-free perfusion with EGTA (Ahn et al. 1984); however, the pathways for Na⁺ influx into SMCs remained unclear. Intracellular Na⁺ rise can produce Ca²⁺ influx and SMC contraction via the Na⁺-Ca²⁺ exchanger functioning in reverse mode (Ashida & Blaustein, 1987; Bova et al. 1990; Matlib, 1991).

Comparison of our results in individual SMCs and in intact aortic rings suggests that both MC and L-type Ca²⁺ channels can mediate the influx of Na⁺ during Ca²⁺,Mg²⁺-free conditions. This could lead to time-dependent Na⁺ overload of SMCs followed by Na⁺-Ca²⁺ exchanger-dependent Ca²⁺ influx and vessel contraction on Ca²⁺ readmission. Indeed, in the absence of EGTA (or in the presence of EGTA and nifedipine) Na⁺ influx through MC channels could be the main reason and trigger for vessel contraction upon Ca²⁺ readmission. When EGTA is present in Ca²⁺,Mg²⁺-free solution, additional Na⁺ influx through L-type Ca²⁺ channels could increase Na⁺ overload leading to a time-dependent potentiation of contraction. MC channels could still be crucial for such contraction providing sustained depolarization of SMCs leading to activation of L-type Ca²⁺ channels. Inhibition of the initial transient phase of contraction by nifedipine applied just before Ca²⁺ readmission is consistent with Ca²⁺ channels being active at that moment (most probably due to MC current-induced SMC depolarization). The fact that in the presence of EGTA nifedipine (present throughout the experiment) considerably reduced, but did not eliminate, the sustained contraction is consistent with both nifedipine-insensitive MC channels and nifedipine-sensitive L-type Ca²⁺ channels accounting for Na⁺ influx and its time-dependent accumulation in SMCs. Na⁺ overload and depolarization of SMCs can activate the Na⁺-Ca²⁺ exchanger which seems to be responsible for sustained vessel contraction upon Ca²⁺ readmission. Indeed, this sustained contraction appeared to be insensitive to nifedipine and to SK&F 96365, which is known to inhibit a variety of non-selective cation channels in different cells including aortic SMCs (Merritt et al. 1990; Krautwurst et al. 1994; Minowa et al. 1997). On the other hand, this contraction was inhibited by nickel which, along with Ca²⁺ and non-selective cation channels, also is known to block the Na⁺-Ca²⁺ exchanger.

Summarizing these data we propose the following:

(1) Na⁺ influx via MC channels during experimental calcium paradox conditions can trigger blood vessel contraction upon Ca²⁺ readmission. The magnitude of this nifedipine-insensitive contraction increases with the time of vessel exposure to Ca²⁺,Mg²⁺-free solution.

(2) Activation of MC channels can depolarize SMCs, activate L-type Ca²⁺ channels and allow additional Na⁺ influx which can significantly increase vessel contraction upon Ca²⁺ readmission.

Therefore, there is a close relationship between activation of MC current and the Ca²⁺ paradox-like phenomenon in SMCs. The fact that the MC current was activated and studied under conditions which are not likely to exist physiologically, does not necessarily exclude the possibility of its activation under physiological conditions (i.e. in the presence of physiological levels of extracellular divalent cations). Indeed, a sizable component of the control inward current is carried by Na⁺ and is eliminated by substituting NMDG⁺ for Na⁺ (Fig. 4). It is attractive to speculate that MC channels can be spontaneously active under physiological conditions and can be involved in normal Na⁺ homeostasis of SMCs. It is also possible that some (still unknown) pathological changes in cell metabolism or environment can activate this current producing pathological Na⁺ influx and cell depolarization. Further studies of MC channels are necessary to determine the mechanisms and conditions of their activation, and to find effective inhibitors of these channels which could prevent the development of the calcium paradox-like phenomenon in a variety of cells.

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Acknowledgements

This study was supported by the NIH (grants HL54150-02, H31607-15, HL55993-02) and the American Heart Association (grant 9417730). We thank Dr Stephen Sims and Dr Marion Gericke for helpful discussion and critical comments on the manuscript, and especially thank Dr Igor Medina for help in noise analysis.

Corresponding author

V. M. Bolotina: Vascular Biology Unit, R408, Boston University Medical Center, 88 East Newton Street, Boston MA 02118-2393, USA.

Email: vbolotina{at}med-med1.bu.edu

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