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Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298
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30%, and block of current preceded the volume change by 1 min. Gd3+-induced cell shrinkage was proportional to ICir,swell when ICir,swell was varied by graded swelling or Gd3+ concentration and was voltage dependent, reflecting the voltage dependence of ICir,swell. Integrating the blocked ion flux and calculating the resulting change in osmolarity suggested that ICir,swell was sufficient to explain the majority of the volume change at –80 mV. In addition, swelling activated an outwardly rectifying Cl– current, ICl,swell. This current was absent after Cl– replacement, reversed at ECl, and was blocked by 1 mM 9-anthracene carboxylic acid. Block of ICl,swell provoked a 28% increase in swelling in hypotonic media. Thus, both cation and anion swelling-activated currents modulated the volume of ventricular myocytes. Besides its effects on cell volume, ICir,swell is expected to cause diastolic depolarization. Activation of ICir,swell also is likely to affect contraction and other physiological processes in myocytes.
Key Words: osmoregulation • stretch-activated channels • mechano-electrical feedback • arrhythmias
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; Morris, 1990; Sackin, 1995; Sukharev et al., 1996; Vandenberg et al., 1996). Members of this class of channels exhibit varying selectivity, conductance, kinetics, and requirements for activation. In cardiac myocytes, for example, osmotic or hydrostatic pressure-induced cell swelling activates Cl– channels (Tseng, 1992; Sorota, 1992; Hagiwara et al., 1992; Coulombe and Coraboeuf, 1992; Zhang et al., 1993), and mechanical deformation or swelling activate both poorly selective cation (Craelius, 1988; Bustamante et al., 1991; Sigurdson et al., 1992; Sadoshima et al., 1992; Kim, 1993; Kim and Fu, 1993; Ruknudin et al., 1993) and K+-selective channels (Brezden et al., 1986; Kim, 1992; Sasaki et al., 1992; Van Wagoner, 1993; Ruknudin et al., 1993).
The broad distribution of SACs suggests their sensitivity to mechanical stretch or swelling must subserve fundamental cellular functions or reflect fundamental properties of ion channels. One appealing physiological role is in cell volume regulation. After rapid swelling on exposure to hypotonic media, activation of transport pathways allows many cell lines to normalize their volume in a process called a regulatory volume decrease (RVD). SACs have been implicated in the RVD observed in Ehrlich ascites tumor cells (Lambert and Hoffman, 1994), renal cortical duct cells (Schwiebert et al., 1994), and embryonic chick cardiac myocytes (Rasmusson et al., 1993; Zhang et al., 1993). In contrast, rabbit myocytes can swell significantly without an RVD (Drewnowska and Baumgarten, 1991; Suleymanian and Baumgarten, 1996).
To investigate whether SACs regulate cell volume in mammalian cardiac myocytes, Suleymanian et al. (1995) used Gd3+ and 9-anthracene carboxylic acid (9-AC) as markers of channel activity. Gd3+ blocks stretch-activated, poorly selective cation channels (Yang and Sachs, 1989; Sadoshima et al., 1992; Hamill and McBride, 1996), and 9-AC blocks swelling-activated Cl– channels (Tseng, 1992; Sorota, 1992, 1994; Hagiwara, 1992; Vandenberg et al., 1994). Under physiological osmotic conditions, when SACs presumably are silent, neither blocker alters the volume of isolated rabbit ventricular cells. In hypotonic solutions, however, Gd3+ significantly decreases and 9-AC significantly increases rabbit ventricular myocyte volume (Suleymanian et al., 1995). These data on intact, unclamped cells were explained by suggesting that osmotic swelling activates Gd3+ and 9-AC–sensitive ion channels that mediate sustained cation and anion fluxes, respectively, and in turn modulate cell volume.
Volume regulation in cultured chick myocytes appears to be different than in freshly isolated rabbit myocytes. Chick heart cells exhibit a strong RVD in response to osmotic swelling that is attenuated by removing Cl– (Rasmusson et al., 1993). Osmotic and hydrostatic swelling is sustained and activates a Gd3+-sensitive Cl– current during ruptured patch whole-cell recordings (Zhang et al., 1993; Zhang and Lieberman, 1996). These workers concluded, however, that the swelling-induced Cl– current does not contribute to regulation of chick heart cell volume because a complete RVD occurred and a Cl– current was not generated under perforated patch conditions (Hall et al., 1995). This emphasizes that ruptured patch techniques may distort cell volume regulation.
The present work describes a swelling-induced, Gd3+-sensitive current in rabbit ventricular myocytes and examines its role in cell volume regulation. The perforated patch voltage-clamp technique and video microscopy were used to simultaneously determine whole-cell ionic currents and the relative cell volume in the absence and presence of osmotic swelling. The experiments demonstrated that: (a) osmotic swelling elicited a graded, inwardly rectifying, Gd3+-sensitive cation current, termed ICir,swell, that could be separated from an outwardly rectifying, 9-AC–sensitive anion current, ICl,swell; (b) ICir,swell poorly distinguishes between K+ and Na+; (c) block of ICir,swell by Gd3+ reduces the volume of osmotically swollen cells in a swelling- and voltage-dependent manner; and (d) the magnitude of the Gd3+-sensitive current can largely account for the Gd3+-induced cell shrinkage. A preliminary report appeared previously (Clemo and Baumgarten, 1995).
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; Clemo et al., 1992). Cells were stored in a modified Kraft-Brühe solution containing (mM): 132 KOH, 120 glutamic acid, 2.5 KCl, 10 KH2PO4, 1.8 MgSO4, 0.5 K2EGTA, 11 glucose, 10 taurine, 10 HEPES (pH 7.2, 295 mosm/liter). Typical yields were 50–70% Ca2+-tolerant, rod-shaped cells. Myocytes were used within 6 h of harvesting, and only quiescent cells with no evidence of membrane blebbing were selected for study.
Experimental Solutions
Cells were placed in a glass-bottomed chamber (0.3 ml) and superfused with room temperature (21–22oC) bathing solution at 3 ml/min. Solution changes were complete within 10 s, as estimated from the liquid junction potential of a microelectrode. The standard bathing solution contained (mM): 65 NaCl, 5 KCl, 2.5 CaSO4, 0.5 MgSO4, 5 HEPES, 10 glucose, and 17–283 mannitol (pH 7.4). The reduced, fixed NaCl concentration permitted adjustment of osmolarity with mannitol at a constant ionic strength. An osmolarity of 296 mosm/liter was taken as isotonicity (1T). Osmolarity ranged from 178 to 266 mosm/liter in hypotonic solutions (0.6T–0.9T) and was 444 mosm/liter in hypertonic solution (1.5T). Osmolarity routinely was verified with a freezing-point depression osmometer (Osmette S; Precision Systems Inc., Natick, MA).
To evaluate the roles of specific ions, nominally Ca2+-free, Na+-, K+-, and Ca2+-free, and Cl–- and Ca2+-free solutions were prepared. MgSO4 replaced CaSO4, N-methyl-d-glucamine (NMDG) Cl replaced NaCl and KCl, or Na and K methanesulfonate replaced NaCl and KCl on equimolar bases.
Voltage Clamp
Electrodes were pulled from 7740 glass capillary tubing (1.5 mm o.d., 1.12 mm i.d., filament; Glass Co. of America, Bargaintown, NJ) to give a final tip diameter of 3–4 µm and a resistance of 0.5–1 M
. The standard electrode filling solution contained (mM): 120 K aspartate, 10 KCl, 10 NaCl, 3 MgSO4, 10 HEPES, pH 7.1. In addition, a Na+- and K+-free pipette solution was made by replacing Na+ and K+ salts with Cs+ salts, and a low Cl– (5 mM) pipette solution was made by replacing NaCl and KCl with the corresponding aspartate salts.
Whole-cell currents were measured using an Axoclamp 200A amplifier (Axon Instruments, Inc., Foster City, CA). Pulse and ramp protocols, voltage-clamp data acquisition, and off-line data analysis were controlled with custom programs written in asyst (Keithley, Taunton, MA). Both step and ramp voltage-clamp protocols were applied (see Fig. 1, D and G), and holding potential (Eh) was either –80, –40, or 0 mV. In step protocols, the test voltage step was 500 ms in duration. Current during the last 50 ms of the step was averaged and called steady state current. In ramp protocols, the voltage was stepped from Eh to +40 mV for 20 ms, ramped to –100 mV over 5 s, and, after 10 ms, ramped back to +40 mV over 5 s. To cancel the capacitive current, the depolarizing and hyperpolarizing arms of the ramp were averaged. A very slow voltage ramp (28 mV/s) was used so as to approach steady state, and ramp current–voltage (I-V) relationships were in good agreement with those obtained by voltage steps. Nevertheless, the contribution of slowly activating currents such as the slow component of the delayed rectifier, IKs, may be underestimated. Both step and ramp currents were digitized at 1 kHz and low-pass filtered at 200 Hz. The reported Em was corrected for the liquid junction potential of the patch electrode as measured from the change in potential upon switching the bath between the superfusing and electrode-filling solutions (Neher, 1992), and the bath was grounded with a 3 M KCl agar bridge.
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) and other cell lines (Worrell et al., 1989; Doroshenko and Neher, 1992; Kinard and Satin, 1995), isosmotic pipette and bath solutions sometimes precipitated unpredictable cell swelling and changes in whole-cell currents. To prevent this problem, the amphotericin perforated patch technique (Horn and Marty, 1988; Rae et al., 1991) was employed. Both cell volume and whole-cell currents were stable under perforated patch recording conditions (Sorota, 1992; Hall et al., 1995).
Amphotericin-B (Sigma Chemical Co., St. Louis, MO) was freshly dissolved in dimethylsulfoxide, and then diluted in electrode filling solution to give final amphotericin and DMSO concentrations of 100 µg/ml and 0.2% (vol/vol), respectively. The electrode tip was dipped into amphotericin-free filling solution for 2 s, and then the barrel was backfilled with amphotericin-containing solution. Filled pipettes were quickly attached to a Ag/AgCl half-cell, placed in the bath solution, and zeroed. Gigaseals were formed without application of negative pressure, typically 30–45 s after backfilling the pipette. To follow the formation of amphotericin pores, access resistance and cell capacitance were monitored with 10-mV hyperpolarizing pulses from Eh. After 20 min, access resistance fell to 7–10 M, and cell capacitance was 70–200 pF.
The cation/anion permeability ratio of a perforated patch depends on characteristics of both the ionophore and endogenous ion channels. Because the permeability ratio affects the transport numbers for ion fluxes between the pipette and cytoplasm, it may affect the cell volume attained under voltage clamp (see discussion). The PK/PCl ratio of amphotericin-treated cardiac sarcolemma was evaluated by exposing the entire sarcolemma to ionophore and determining the zero current potential under current clamp. For these measurements, the ruptured patch procedure was exploited. Seals were made while myocytes were incubated in a solution containing (mM): 140 KCl, 3 MgSO4, 5 HEPES, 10 glucose, pH 7.4; this represented the pipette filling solution in perforated patch experiments. The ruptured patch electrodes contained (mM): 40 KCl, 85 K aspartate, 3 MgCl2, 5 K2EGTA, 3 K2ATP, 10 HEPES, pH 7.1. After exposure to amphotericin (160 µg/ml) in the bath solution, the bath was switched to a series of solutions with varying concentrations of K+ (1–140 mM, K+ replaced with NMDG) or Cl– (1–140 mM, Cl– replaced with aspartate). The known intracellular and extracellular K+ and Cl– concentrations and measured Em data were fit to the Goldman-Hodgkin-Katz equation (TableCurve 2D; SPSS, Chicago, IL). PK/PCl was 0.9 ± 0.07 when extracellular Cl– was varied (n = 5) and was 0.8 ± 0.03 when extracellular K+ was varied (n = 5). Because experiments treating the entire sarcolemma with ampho-tericin could not exactly mimic the perforated patch experiments, these permeability ratios should be regarded as approximate.
Determination of Relative Cell Volume
Methods for determining relative cell volume have been described previously (Clemo and Baumgarten, 1991; Clemo et al., 1992; Suleymanian and Baumgarten, 1996). Myocytes were visualized with an inverted microscope (Diaphot; Nikon Inc., Garden City, NY) equipped with Hoffman modulation optics (40x; 0.55 NA) and a high resolution TV camera (CCD72; Dage-MTI; Michigan City, IN) coupled to a video frame-grabber (Targa-M8; Truevision, Santa Clara, CA). Images were captured on-line each time a ramp or step voltage-clamp protocol was performed by a program written in C and assembler and linked to the asyst voltage-clamp software. A combination of commercial (mocha; SPSS) and custom (asyst) programs were used to determine cell width, length, and the area of the image.
Changes in cell width and thickness on exposure to anisosmotic solutions are proportional (Drewnowska and Baumgarten, 1991). Using each cell as its own control, relative cell volume was calculated as:
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where t and c refer to test (e.g., 0.6T) and control (1T) solutions, respectively. These methods provide estimates of relative cell volume that are reproducible to within 1% (Clemo and Baumgarten, 1991; Clemo et al., 1992, Suleymanian and Baumgarten, 1996).
Statistics
Data are reported as mean ± SEM; n represents the number of cells. Except for Fig. 1, which depicts voltage clamp data from a typical experiment, all I-V relationships are averages, and mean current densities are expressed in pA/pF to account for differences in cell membrane area. When multiple comparisons were made, data were subjected to analysis of variance. Bonferroni's method for group comparisons was performed when appropriate. For simple comparisons, the Student's t test was used. All statistical analyses were conducted in SigmaStat (SPSS).
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, 1994; Rees et al., 1995; Wang et al., 1996). After recovery in 1T solution, the same cell was studied using the ramp protocol (28 mV/s) to define the steady state I-V relationship. As before, the currents in 0.6T were larger than those in 1T (Fig. 1 E). The I-V relationship for the osmotic stretch-induced difference current (Fig. 1 F, solid line) exhibited strong inward rectification at negative potentials and weaker outward rectification at positive potentials. Fig. 1 F also compares the results of the two voltage-clamp protocols. Steady state difference currents measured using the step protocol (•, data from Fig. 1 C) are superimposed on the difference current obtained with the ramp protocol. The good agreement verifies the adequacy of ramp protocols for determining swelling-induced steady state currents. Ramp clamps were used in subsequent experiments that were designed to study maintained currents that are likely to contribute to cell volume regulation.
Previous work on mammalian cardiac myocytes has described both a Gd3+-sensitive cation channel in cell-attached and excised patches that is activated by pipette suction (Bustamante et al., 1991) or mechanical stretch (Sadoshima et al., 1992) and a Gd3+-sensitive osmotic swelling of intact, unclamped cells (Suleymanian et al., 1995). Fig. 2 shows that Gd3+ also blocked a component of whole-cell current elicited by osmotic swelling and decreased cell volume under perforated patch conditions. Em was held at –40 mV except during voltage ramps. During the initial 20 min control period in 1T, the I-V relationship was stable (Fig. 2 A, curve a), and no change in cell volume occurred (Fig. 2 D). Exposure of myocytes to 0.6T solution for 5 min increased cell volume by 37 ± 2%, a swelling similar to that previously observed in intact, unclamped rabbit ventricular myocytes (e.g., 34 ± 2%, Drewnowska and Baumgarten, 1991). Both inward current at negative and outward current at positive potentials simultaneously increased in amplitude, at –100 mV from –1.68 ± 0.19 to –7.55 ± 0.21 pA/pF and at +40 mV from +0.74 ± 0.05 to +3.82 ± 0.14 pA/pF (Fig. 2 A). After recovery of cell volume and current in 1T solution, the osmotic challenge was repeated in the presence of 10 µM Gd3+ (Fig. 2 B; 1T, curve c; 0.6T, curve d). Osmotic swelling still affected membrane currents after Gd3+ treatment, but the inward shift of the I-V relationship at negative potentials in 0.6T solution was less pronounced (e.g., from –1.63 ± 0.19 to –3.54 ± 0.18 pA/pF at –100 mV; Fig. 2 B). Furthermore, Gd3+ decreased the amount of cell swelling significantly; relative cell volume in 0.6T was 1.37 ± 0.02 in the absence of Gd3+ and 1.33 ± 0.01 in its presence (P < 0.05). The Gd3+-sensitive difference currents in 0.6T and 1T solution are plotted in Fig. 2 C. During osmotic stretch in 0.6T solution, Gd3+ blocked (Fig. 2 C, curves b – d) an inwardly rectifying current that reversed near –57 mV. Inward rectification also is exhibited by a Gd3+-sensitive cation SAC studied at the single channel level in chick heart (Ruknudin et al., 1993), but other SACs in chick and rat heart have a linear I-V relationship under nearly symmetrical and asymmetrical conditions (Craelius et al., 1988; Ruknudin et al., 1993). In contrast, Gd3+ had no effect on currents in 1T (Fig 2 C, curves a – c). This argues that Gd3+ blocked only a swelling-induced component of steady state current. Gd3+ did not block all of the swelling-induced current, however. The outwardly rectifying component at positive potentials was unaffected (compare Fig. 2, A and B). The Gd3+-resistant current is likely to be the osmotic swelling-induced anion current, ICl,swell, previously described in atrial, ventricular, and SA nodal myocytes from dog (Tseng, 1992; Sorota, 1992), rabbit (Hagiwara et al., 1992; Duan et al., 1995), rat (Coulombe and Coraboeuf, 1992), guinea pig (Vandenberg et al., 1994; Shuba et al., 1996), and man (Oz and Sorota, 1995; Sakai et al., 1995).
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; Shimoni et al., 1992), was employed. Under isosmotic conditions, 0.2 mM Ba2+ markedly attenuated the inward rectification of the whole-cell current at negative potentials attributable to IK1 (Fig. 3 A).2 Nevertheless, swelling in 0.6T + Ba2+ still turned on an inwardly rectifying current (Fig. 3 B, curve c) that was blocked by 10 µM Gd3+ (Fig. 3 B, curve d). The magnitude of the swelling-activated, Gd3+-sensitive current in the presence of Ba2+, –4.29 ± 0.30 pA/pF at –100 mV (Fig. 3 C, curve c – d) was similar to that in the absence of Ba2+, –4.01 ± 0.23 pA/pF (Fig. 2 C, curve b – d). These data argue that IK1 does not significantly contribute to the swelling-activated Gd3+-sensitive current.
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; Hamill and McBride, 1996), Robson and Hunter (1994) describe a direct effect of Gd3+ on swelling-activated Cl– currents in Rana temporaria proximal tubule cells, and Zhang et al. (1994) and Zhang and Lieberman (1996) describe a Ca2+-dependent indirect effect on swelling-activated Cl– currents in chick myocytes. The experiments depicted in Fig. 4 were conducted in Ca2+-free bathing media. Nevertheless, the same Gd3+-sensitive and -resistant current components were observed as in the presence of bath Ca2+ (see Fig. 2). This argues that swelling-activated currents in mammalian myocytes are not Ca2+ dependent (Tseng, 1992; Sorota, 1992; Hagiwara et al., 1992), and we previously showed that the amount of swelling in hypotonic solution is not Ca2+ dependent (Suleymanian et al., 1995). To exclude the possibility that the Gd3+-sensitive current was carried by Cl–, cells were studied using Cl–-free (methanesulfonate) bath and low Cl– (aspartate; Cl– = 5 mM) pipette solutions. The I-V relationships depicted in Fig. 5 A were obtained in Cl–-free 1T (Fig. 5 A, curve a) and 0.6T (Fig. 5 A, curve b) solutions without Gd3+. Osmotic swelling still increased the current amplitude at negative potentials, but removal of Cl– sharply attenuated the outwardly rectifying current at positive potentials (see Fig. 2 A). The remaining swelling-activated current was blocked almost completely by 10 µM Gd3+ (Fig. 5 B; Cl–-free 1T, curve c; Cl–-free 0.6T, curve d). On the other hand, removal of Cl– had no effect on the Gd3+-sensitive, inwardly rectifying difference current in 0.6T (Fig. 5 C, curve b – d), or on the reduction of cell swelling caused by adding Gd3+ to 0.6T solution (Fig. 5 D). As before, Gd3+ was efficacious only after cell swelling; Gd3+ failed to alter either cell volume or the I-V relationship in Cl–-free 1T solution, and the Gd3+-sensitive difference current was negligible (Fig. 5 C, curve a – c). These data suggest that Cl– contributed to the outwardly rectifying current evoked by hypotonic solution, but was not a significant component of the Gd3+-sensitive, inwardly rectifying current.
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Cl,bath = 0.78; Cl,cell = 0.74). The small difference between Erev and the calculated ECl is likely to be due in part to imperfect selectivity of stretch-activated anion channels (Tseng, 1992; Sorota, 1992; Hagiwara et al., 1992; Zhang et al., 1993; Vandenberg et al., 1994) and difficulty in controlling intracellular Cl– with the perforated patch method. Furthermore, Fig. 6 C shows that 9-AC significantly increased swelling in 0.6T + Gd3+ by 28%, from 1.295 ± 0.011 (Fig. 6 C, curve c) to 1.377 ± 0.016 (Fig. 6 C, curve d). A similar effect was noted in intact, un-clamped myocytes; in 0.6T without Gd3+, 9-AC increased cell volume 32%, from 1.311 ± 0.019 to 1.413 ± 0.027 (Suleymanian et al., 1995). However, others using the conventional ruptured patch voltage clamp technique failed to detect an acute effect of 9-AC on the volume of swollen myocytes (Tseng, 1992; Sorota, 1992).
The selectivity of ICir,swell for K+ and Na+ was determined next. I-V relationships were recorded in 1T and 0.6T solutions in which the K+ concentration ([K+]o) was raised from 5 to 35 and 65 mM by equimolar replacement of Na+ and Cl– was replaced by methanesulfonate. The difference currents (0.6T – 1T) are depicted in Fig. 7 A ([K+]o: 5 mM, curve b – a; 35 mM, curve d – c ; 65 mM, curve f – e). The Erev and slope conductance at Erev for each condition are presented in Table II. A 13-fold increase of [K+]o shifted Erev by 40 mV to more positive voltages and increased the slope conductance 1.5-fold. Also included in Table II are the constant field PK/PNa ratios calculated from Erev and ion concentrations and the rectifier ratio. For the three combinations of K+ and Na+, the PK/PNa ratio was 5.9 ± 0.3, indicating only a modest selectivity for K+, and increasing K+ decreased the extent of rectification by approximately twofold. Because extracellular Na+ is much greater than K+ under physiologic conditions, inward current normally would be carried predominantly by Na+. PK/PNa ratios of 0.7–7.2 have been estimated for Gd3+-sensitive unitary cation SAC currents elicited in chick ventricle by pipette suction (Bustamante et al., 1991; Ruknudin et al., 1993). The selectivity of the present channel for Ca2+ was not examined, although Ca2+ is known to permeate cation SACs in a number of tissues, including the heart (Sigurdson et al., 1992).
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Dose Dependence of Gd3+
Yang and Sachs (1989) reported that 10 µM Gd3+ fully blocks stretch-activated cation channels in frog oocytes, but Bustamante et al. (1991) indicated a 10-fold higher dose is necessary in chick and guinea pig myocytes. To characterize the dose dependence of Gd3+s effects on current and volume, cells swollen in Cl–-free 0.6T bath solution were treated with successively higher concentrations of Gd3+ (1–30 µM). Fig. 8 A shows ICir,swell (curve b – a) and the effect of 30 µM Gd3+ (curve f – a). This dose of Gd3+ appears to have totally blocked the inward rectifier, leaving a small, nearly linear, swelling-induced current. The residual current is likely to reflect in part the permeation of anions other than Cl– through the swelling-activated anion channel. The Gd3+-sensitive current in 0.6T solution is depicted in Fig. 8 B. As the Gd3+ concentration was increased, the amount of current blocked by Gd3+ increased significantly (P < 0.01). The effect of Gd3+ on cell volume in 0.6T solution also was dose dependent (Fig. 8 C). As the Gd3+ concentration was increased, relative cell volume in 0.6T solution significantly decreased (P < 0.001).
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) and 1.8 ± 0.4 and 1.3 ± 0.2 µM (r = 0.96) for the Gd3+-induced cell shrinkage (
). This means that the 10-µM dose of Gd3+ used in other experiments should have blocked 97% of the Gd3+-sensitive current and caused 92% of the maximum volume change.
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Graded Activation of Gd3+ Sensitivity
Gd3+-sensitive cation SAC activity in cell-attached patches is a graded function of the negative pressure applied to the pipette and the degree of membrane stretch (Guharay and Sachs, 1984; Sigurdson et al., 1987). For a range of stimuli, the open probability of the nonselective cation mechanoelectrical transduction channel in the bullfrog saccular hair cell has been postulated to be linearly related to membrane tension (Howard et al., 1988), whereas Guharay and Sachs (1984) postulated that gating of the cation SAC in tissue-cultured embryonic chick skeletal muscle cells varies with the square of membrane tension. One might expect the magnitude of ICir,swell and Gd3+-induced volume changes also would depend on the amount of swelling-induced membrane stretch. To test this idea, myocytes were placed in a series of hypotonic (0.6–0.9T), isotonic (1T), and hypertonic (1.5T) solutions, and the I-V relationship and relative cell volume were monitored. ICir,swell was measured as the difference between the I-V relationships ± 10 µM Gd3+ in each of the superfusates. Fig. 10 A shows that Gd3+ did not affect membrane current in isotonic solution or when myocytes were shrunken in 1.5T solution. On the other hand, as bath solution osmolarity was gradually stepped from 1 to 0.6T, ICir,swell increased in a graded fashion. At the same time, osmotic swelling in the presence of Gd3+ was attenuated (Fig. 10 B). These effects are summarized in Fig. 10 C where Gd3+-sensitive current and cell shrinkage are plotted versus bath osmolarity.
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) and cell volume () at 20-s intervals during osmotic swelling and exposure to 10 µM Gd3+. Gd3+ decreased the inward current with a t1/2 of 50.3 ± 2.5 s (Fig. 12, down arrow), whereas the Gd3+-induced cell shrinkage was significantly slower, with a t1/2 of 116.3 ± 3.4 s (up arrow). These data argue that the primary effect of Gd3+ is block of ICir,swell rather than cell shrinkage. They do not, however, rule out the possibility that cell shrinkage secondary to block of current leads to a further reduction of ICir,swell.
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6. ICir,swell was not detectable under isosmotic conditions, but was readily measured after a swelling of only 7.5%, the smallest perturbation explored. The I-V relationship for ICir,swell revealed a strong inward-going rectification and was insensitive to Ba2+, a blocker of IK1. This swelling-induced current appeared to be time independent at potentials negative to 0 mV. Several lines of evidence indicate that ICir,swell modulates cell volume at physiologically relevant potentials. (a) A linear relationship was found between the amount of ICir,swell blocked by Gd3+ during graded osmotic swelling and with varying concentrations of Gd3+ and the subsequent Gd3+-induced reduction of cell volume. In 0.6T, 10 µM Gd3+ reduced swelling by 30%, and Gd3+-induced block of ICir,swell preceded Gd3+-induced cell shrinkage by 1 min. (b) Elimination of the Gd3+-sensitive current in hypotonic solution containing NMDG instead of Na+ and K+ also eliminated the effect of Gd3+ on cell volume. (c) The amount of swelling in hypotonic solution was linearly related to ICir,swell when bath Na+ was partially replaced by K+. (d) Gd3+ also reduces cell volume in osmotically swollen, unclamped myocytes (Suleymanian et al., 1995). These data indicate that osmotic swelling elicited a cation influx via a poorly selective channel. Rather than provoking an RVD, the influx of cations contributed to cell swelling.
A number of studies in mammalian myocytes focused on a swelling-induced, outwardly rectifying anion current, ICl,swell (Tseng, 1992; Sorota, 1992, 1995; Hagiwara et al., 1992; Coulombe and Coraboeuf, 1992; Vandenberg et al., 1994; Duan et al., 1995; Shuba et al., 1996), and the characteristics of the 9-AC–sensitive ICl,swell observed here were consistent with these reports. However, insensitivity to Gd3+ and to omission of extracellular Ca2+ distinguishes ICl,swell in rabbit from that found in chick heart (Zhang et al., 1994). Although previous reports suggested that brief pharmacological blockade of ICl,swell does not affect cell volume (Tseng, 1992; Sorota, 1992), the present paradigm detected a 28% augmentation of cell swelling in hypotonic solution on blocking ICl,swell with 9-AC, and the effect of 9-AC on cell volume under voltage clamp was similar to that observed in unclamped myocytes (Suleymanian et al., 1995). A 9-AC–induced swelling was expected because inward current carried by ICl,swell at diastolic potentials represents the efflux of anions. It is uncertain why we observed increased swelling with 9-AC, whereas others did not. Species differences cannot be ruled out. For example, 9-AC (1 mM) blocks only 50–60% of ICl,swell in canine and guinea pig myocytes (Tseng, 1992; Sorota, 1994; Vandenberg et al., 1994), but it blocked all of ICl,swell in rabbit ventricular (Fig. 6 A, a and d) and atrial (Hagiwara et al., 1992) myocytes. In addition, the use of ruptured patch technique (Tseng, 1992; Sorota, 1992) may have blunted changes in intracellular osmolarity caused by 9-AC. Finally, the present digital video microscopy technique for estimating volume has a much greater resolution than measurements of cell width with an ocular reticle (Tseng, 1992; Sorota, 1992).
Basis for the Gd3+-sensitive Swelling-activated Cation Current
This appears to be the first description at the whole-cell level of a Gd3+-sensitive, poorly selective cation current elicited by swelling cardiac myocytes. Single channel recordings from chick, guinea pig, and rat cardiac cells demonstrate that several different cation channels activated by pipette suction are blocked by Gd3+ (Bustamante et al., 1991; Sadoshima et al., 1992, Ruknudin et al., 1993). A 25-pS channel in chick heart exhibits strong inward-going rectification that is unaffected by switching Na+ and K+ on one side (Ruknudin et al., 1993). In contrast, the rectification found here was markedly diminished when bath Na+ was replaced by K+ (Fig. 7 A). Other Gd3+-sensitive cation and K+ channels in chick (Ruknudin et al., 1993) and rat (Sadoshima et al., 1992) have linear unitary I-V relationships in both symmetrical K+ solutions and with Na+ replacing K+ on one side, and the voltage dependence of their open probability would not generate inward rectification of whole-cell currents. Thus, none of the Gd3+-sensitive SACs described at the single channel level can fully account for the behavior of ICir,swell. Nevertheless, the apparent K0.5 for block of current and for Gd3+- induced cell shrinkage, 1.7 and 1.8 µM, and Hill coefficients of 1.7 and 1.3, were consistent with Gd3+ block of SACs in Xenopus oocytes (Yang and Sachs, 1989). Yang and Sachs (1989) suggested that low concentrations of Gd3+ screen negative charges near the vestibule of the channel and interact with an allosteric site outside the membrane field to induce a short-lived closed state, whereas higher concentrations cause a cooperative transition to a long-lived closed state.
At positive potentials, an increasing component of outward current also was observed during swelling (Fig. 1 C) and attributed to ICl,swell because of its sensitivity to 9-AC and Cl– replacement. Swelling-activated outward current may reflect in part a stimulation of the delayed rectifier, IK, that previously was noted during both osmotic and hydrostatic swelling of guinea pig ventricular myocytes (Sasaki et al., 1992; 1994; Rees et al., 1995; Wang et al., 1996). Both groups agree that swelling primarily increases the slowly activating component, IKs, but the rapidly activating component, IKr, is said to either decrease (Rees et al., 1995) or increase (Wang et al., 1996). It is possible that swelling-activated delayed rectifier contributes to the total current in 0.6T, although this current is much smaller in rabbit ventricle than in several other species (Giles and Imaizumi, 1988). The time-dependent current observed here was found at potentials more appropriate for IKs, although its approaching steady state within 500 ms was suggestive of IKr. To the extent IK contributes to swelling- induced current, it also might contribute to the Gd3+-sensitive current. IKr is blocked by 
1 µM of another lanthanide, La3+, and 10 µM La3+ shifts IKs activation in a positive direction (Sanguinetti and Jurkiewicz, 1990). On the other hand, no component of current attributable to block of IKr or IKs by Gd3+ was present at appropriate voltages.
Cell swelling or mechanical stretch also has been reported to activate Gd3+-insensitive, poorly selective, cation channels in neonatal rat atrial cells (Kim, 1993; Kim and Fu, 1993). Such channels did not appear to be present in adult rabbit ventricular cells. As judged by the effect of replacement of Na+ and K+ with NMDG and Ca2+ with Mg2+ in Cl–-free solution (Fig. 6, A and B), all of the swelling activated cation current was blocked by Gd3+. Furthermore, the strong inward rectification of ICir,swell argues against the participation of free fatty acid–activated (Kim, 1992) or ATP-sensitive (Van Wagoner, 1993) K+ channels in the response to cell swelling. Activation of either of these channels should have elicited a significant outward cation current in 5 mM [K+]o, but virtually none was observed.
Osmotic swelling may or may not be equivalent to mechanical stretch or localized membrane deformation as a stimulus for activating SACs (Vandenberg et al., 1996). Although both osmotic swelling and mechanical stimuli distort the membrane and cytoskeleton, osmotic swelling also dilutes intracellular ions and macromolecules. Dilution of the intracellular contents can modulate ion transport by multiple mechanisms (Baumgarten and Feher, 1998). In particular, reduction of the intracellular K+ concentration, [K+]i, (Fozzard and Lee, 1976) raises an important concern for the present study: is ICir,swell simply a manifestation of dilution of [K+]i and the resulting positive shift of EK on IK1? This is unlikely because Ba2+, a blocker of IK1 in rabbit ventricular myocytes (Giles and Imaizumi, 1988; Shimoni et al., 1992), did not inhibit ICir,swell (Fig. 3), whereas ICir,swell was blocked by Gd3+, which did not affect the background currents, including IK1 in 1T (Fig. 2). Moreover, osmotic dilution of [K+]i is transient under patch clamp conditions. By the time I-V curves were recorded, at least 5 min after the onset of an osmotic challenge, dialysis of the cytoplasm by the patch pipette should have substantially reduced changes in [K+]i. Sasaki et al. (1994) found that IK1 was hardly affected only 2 min after exposing dialyzed myocytes to 0.7T solution. Despite these compelling arguments that swelling-induced shifts in EK cannot fully explain the data, the possibility remains that cellular dialysis did not completely restore [K+]i after an osmotic challenge. Such incomplete dialysis, to the extent that it occurred, could have affected characterization of ICir,swell.
Gd3+-sensitive Current Alters Cell Volume
In response to graded osmotic swelling, equimolar partial replacement of bath Na+ with K+, and varying the concentration of Gd3+, the magnitude of the cation current blocked by Gd3+ at –80 mV was linearly related to the ensuing reduction of cell volume. Also, Gd3+'s effect on ICir,swell preceded its effect on cell volume. Although these data imply that ICir,swell modulates cardiac cell volume, a more quantitative comparison may be helpful. To estimate the expected volume change, the Gd3+-sensitive current was integrated over time and converted to changes in intracellular molarity. Table III shows the result of calculations based on the experiments depicted in Fig. 13 in which Eh was set at –80 and –40 mV and analogous studies switching Eh between –40 and 0 mV. A much greater Gd3+-sensitive cation influx occurred when myocytes were held at –80 mV than at –40 or 0 mV or during the voltage ramp. The integral of ICir,swell accounted for a 16.2 ± 1.2 mM change in concentration at –80 mV, 25x more than at –40 or 0 mV. This amounted to a 5.5% decrease in osmolarity, whereas volume decreased 7% at –80 mV.
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; Horn and Marty, 1988; Ebihara et al., 1995; Kyrozis and Reichling, 1995), and amphotericin may affect the properties of native channels (Hsu and Burnette, 1993). In preliminary studies, PK/PCl of amphotericin-treated myocytes was estimated as 0.8–0.9 (see methods). Using PK/PCl to approximate Pcation/Panion and taking the potential across the perforated patch as 0 mV, t+ was 0.44– 0.47. Although only an approximation, these data suggest that the cation and anion fluxes between pipette and cell were roughly comparable and, therefore, that cell volume changes measured under perforated patch conditions should approximate those expected from the measured ionic currents.
Physiological and Pathophysiological Implications
Activation of ICir,swell and ICl,swell may directly affect cardiac electrical activity, alter ionic gradients, and contribute to cell volume regulation. These currents are likely to be activated during ischemia and reperfusion (Reimer and Jennings, 1992) and surgical cardioplegia (Drewnowska et al., 1991; Handy et al., 1996) when myocyte swelling is pronounced. The present studies do not establish whether cell swelling is the required stimulus or if stretch also activates the same channels. However, several effects of stretch are blocked by Gd3+ and are consistent with activation of ICir,swell by stretch. For example, 10 µM Gd3+ blocks stretch-induced depolarizations and ventricular premature beats initiated by rapid inflation of a ventricular balloon in canine ventricle (Hansen et al., 1991; Stacy et al., 1992), 80 µM Gd3+ blocks stretch-induced delayed afterdepolarizations, premature beats, and poststretch augmentation of contractile force in rat atria (Tavi et al., 1996), and 5 µM Gd3+ decreases stretch-induced release of atrial natriuretic peptide from rat atrium (Laine et al., 1994). Furthermore, infusion of GdCl3 (76 µmol/kg) attenuates the upward shift of the left ventricular diastolic pressure– volume relationship caused by pacing-induced cardiac ischemia in dogs (Takano and Glantz, 1995). Another intriguing possibility is the involvement of stretch-activated channels in congestive heart failure. We found that ICir,swell was chronically activated in isosmotic solution in ventricular myocytes from dogs with pacing-induced congestive failure (Clemo et al., 1995). Thus, activation of ICir,swell by cell swelling or mechanical stretch may have important implications for myocyte volume regulation, and electrical and mechanical activity under both physiologic and pathophysiologic conditions.
2 IK1 current density in 1T, estimated as Ba2+-sensitive current, was less than that recorded by Giles and Imaizumi (1988) and Shimoni et al. (1992) in rabbit ventricular cells. This difference may arise from the methods. They used ruptured rather than perforated patch. Concentrations of inorganic and organic modulators of IK1, including Mg2+ and polyamines (Nichols and Lopatin, 1997), are likely to differ. Furthermore, greater cell capacitance, cell diameter, and animal weight suggest the present experiments used older animals or selected a different population of ventricular cells.
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ACKNOWLEDGMENTS |
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Submitted: 3 April 1997
Accepted: 20 June 1997
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TopABSTRACTintroductionmaterials and methodsresultsdiscussionreferences |
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-Adrenergic control of volume-regulated Cl–currents in rabbit atrial myocytes. Characterization of a novel ionic regulatory mechanism, Circ Res, 1995, 77, 379–393.
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); n = 4 cells. Time course of current activation in the absence (B) and presence (C) of Gd3+ are compared; current expressed as a percentage of the maximum swelling-induced current. Gd3+ significantly increased the t1/2 for activation of current at –100 mV (18.7 ± 1.8 vs. 46.3 ± 3.1 s), but did not affect the t1/2 at +40 mV. Eh, –40 mV; bath, Ca2+-free.
0.7T. In contrast, Gd3+ did not alter the I-V relationship or cell volume in isotonic (1T) or hypertonic (1.5T) solutions. (C) Gd3+-sensitive current and cell shrinkage are plotted as a function of bath-relative osmolarity. Eh, –80 mV; bath, 0 Cl–; pipette, low Cl–.
