From the * Department of Internal Medicine (Cardiology), and Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298
The role of swelling-activated currents in cell volume regulation is unclear. Currents elicited by swelling rabbit ventricular myocytes in solutions with 0.6-0.9× normal osmolarity were studied using amphotericin
perforated patch clamp techniques, and cell volume was examined concurrently by digital video microscopy. Graded swelling caused graded activation of an inwardly rectifying, time-independent cation current (ICir,swell) that
was reversibly blocked by Gd3+, but ICir,swell was not detected in isotonic or hypertonic media. This current was not
related to IK1 because it was insensitive to Ba2+. The PK/PNa ratio for ICir,swell was 5.9 ± 0.3, implying that inward current is largely Na+ under physiological conditions. Increasing bath K+ increased gCir,swell but decreased rectification. Gd3+ block was fitted with a K0.5 of 1.7 ± 0.3 µM and Hill coefficient, n, of 1.7 ± 0.4. Exposure to Gd3+ also
reduced hypotonic swelling by up to ~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.
Ion channels activated by mechanical stretch or cell
swelling (SAC)1 are present in cells ranging in complexity from primitive unicellular organisms to highly
differentiated neurons and myocytes (Sachs, 1988;
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).
Cell Isolation
Ventricular myocytes were freshly isolated from New Zealand
white rabbits (2.5-3 kg) using a collagenase-pronase dispersion method as previously described (Clemo and Baumgarten, 1991;
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.
Perforated Patch Technique
Volume measurements initially were attempted during ruptured-patch voltage clamp, but this approach proved unsuitable. As previously described for both cardiac myocytes (Sorota, 1992) 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 (40×; 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:
![]() |
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).
Characterization of Stretch-activated Currents
Membrane currents elicited by hyposmotic cell swelling
initially were studied by applying step and ramp voltage-clamp protocols to the same cell. Results for a typical cell in standard bath solution are shown in Fig. 1.
For the step protocol, Eh was 40 mV, and depolarizations to test potentials between
100 and +40 mV were
applied under control conditions (1T, Fig. 1 A) and
again 5 min after swelling in 0.6T hypotonic solution
(Fig. 1 B). The currents obtained after osmotic stretch
were much larger than those obtained in 1T solution.
The effects are depicted more clearly in Fig. 1 C in
which swelling-induced difference currents, measured as the current in 0.6T minus that in 1T, are plotted.
The difference currents were time independent over
most of the voltage range explored. Between 0 and
+40 mV, however, osmotic stretch provoked an increasing outward current that approached steady state
during the 500-ms pulse. This time-dependent current
resembles the delayed rectifier, IK, which is augmented
by osmotic and hydrostatic cell swelling of guinea pig
myocytes (Sasaki et al., 1992
, 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
).
Although intracellular K+ is controlled by dialysis
across the perforated patch, osmotic swelling initially
dilutes intracellular K+ and the resulting shift in IK1
might be mistaken for a novel swelling-activated cation
current. To test whether IK1 contributes to the Gd3+-sensitive current, Ba2+, a potent blocker of IK1 in rabbit
ventricular myocytes (Giles and Imaizumi, 1988; 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.
Kinetics of Current Activation
The kinetics of current activation also suggested that
separate processes contribute to osmotic swelling-
induced currents and positive and negative potentials.
To examine activation kinetics, I-V relationships were
determined every 30 s during swelling in 0.6T and recovery in 1T solutions in the absence and presence of
Gd3+. The currents at +40 and 100 mV are plotted in
Fig. 4 A. As expected from Fig. 2, the current at +40
mV was resistant to Gd3+, whereas the current at
100
mV consisted of nearly equal Gd3+-sensitive and -insensitive components. The kinetics of current activation
are shown more clearly in Fig. 4, B and C, in which the
amplitude of swelling-activated current is expressed as
a percentage of the maximum swelling-activated current. Upon exposure to 0.6T solution, the inwardly rectifying current at
100 mV activated significantly more
rapidly than the outwardly rectifying current at +40
mV (Fig. 4 B); the t1/2s were 18.7 ± 1.8 and 55.2 ± 3.4 s,
respectively (P < 0.05). This difference in t1/2 was eliminated when the 0.6T osmotic challenge was repeated
in the presence of 10 µM Gd3+. Gd3+ slowed the t1/2 for
current activation at -100 mV to 46.3 ± 3.1 s without
significantly affecting activation at +40 mV, 53.8 ± 3.5 s;
as a result, the t1/2s for inward and outward current activation no longer were significantly different (P > 0.21).
Thus, Gd3+ preferentially affected an inwardly rectifying component of osmotic stretch-induced current that
activated more rapidly than the rest of the current. Similar results were obtained when the kinetics of current
inactivation upon returning the cells to isotonic (1T) solution were considered. Half-times of activation and
inactivation are summarized in Table I. Slow activation
of swelling-induced outward current was noted previously (Sorota, 1995
).
Table I. Kinetics of Swelling-induced Current Activation and Inactivation |
Ionic Nature of Gd3+-sensitive Current
Whereas Gd3+ generally is held to be a blocker of cation SACs (Yang and Sachs, 1989; 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.
If physiologic cations are the major charge carriers of
the Gd3+-sensitive, osmotic stretch-activated inward rectifier, removal of Na+, K+, and Ca2+ from the bath and
electrode solutions should markedly attenuate both the
inwardly rectifying current in hypotonic solution and the Gd3+ sensitivity of the remaining current. These
predictions were tested by replacing Na+ and K+ in the
bath with NMDG and in the pipette with Cs+ and replacing bath Ca2+ with Mg2+. Fig. 6 A depicts the I-V relationships in 1T, 0.6T, and 0.6T plus Gd3+ (Fig. 6 A,
curves a-c, respectively) NMDG solutions. Hypotonic NMDG solution still provoked outwardly rectifying current at positive potentials. As predicted, however, the
inward rectifier previously observed at negative potentials was abolished (see Figs. 1 A and 5 A); instead, the
inward current was linear. Moreover, 10 µM Gd3+ did
not significantly alter the I-V relationship in NMDG-0.6T solution; the negligible Gd3+-sensitive difference
current is shown in Fig. 6 B, curve b c. Cation replacement also affected the cell volume response. Gd3+ no
longer reduced cell volume in 0.6T NMDG solution
(Fig. 6 C; compare Figs. 2 D and 5 D). These data
strongly argue that the inwardly rectifying current induced by osmotic swelling was carried by cations.
Therefore, the Gd3+-sensitive current was designated
ICir,swell (cation inward rectifier, swelling activated).
The suggestion that the Gd3+-resistant current elicited by 0.6T solution is ICl,swell also was supported by experiments in NMDG solutions. Under these conditions,
the ICl,swell blocker 9-AC (1 mM) did not affect the I-V
relationship in 1T solution when SACs should be
closed (data not shown). On the other hand, 9-AC totally prevented activation of the remaining swelling-
induced current in 0.6T NMDG solution (Fig. 6 A, curve
d; compare 1T, curve a). The 9-AC-sensitive current (Fig.
6 B, curve c d) exhibited outward rectification at positive potentials and a reversal potential (Erev) of
35.6 ± 1.8 mV. Assuming equilibration across the perforated patch, ECl calculated from the bath and pipette solutions was
32.9 mV after accounting for the effect of
ionic strength on activity coefficients (
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
).
Table II. Electrophysiological Characteristics of ICir,swell |
Replacing Na+ with K+ not only altered the difference current, it also caused cell swelling in 0.6T solution to significantly increase from 1.279 ± 0.021 to 1.343 ± 0.018, and 1.428 ±0.020 in 5, 35, and 65 mM [K+]o bathing media, respectively. The relationship between the current induced by swelling and cell volume in 0.6T solution is illustrated in Fig. 7 C. The data were well described by a straight line with a slope of 0.048 and a y-intercept of 1.22 (r = 0.99).
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).
Dose-response curves for current blocked by Gd3+ at
80 mV and the reduction of cell volume caused by
Gd3+ in 0.6T solution are presented in Fig. 9 A. The
data from Fig. 8, B and C were fitted to the Hill equation, and the responses are plotted with the fitted maximum taken as 100%. The K0.5 and Hill coefficient, n,
were: 1.7 ± 0.3 and 1.7 ± 0.4 µM (r = 0.95) for block
of current (
) 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.
The excellent correlation between the effects of Gd3+ on current and volume are emphasized in Fig. 9 B, in which responses were expressed as a percentage of the maximum. The relationship was well described by a straight line with a slope 0.87 and a y-intercept of 8.6% (r = 0.98). The slope and y-intercept were not significantly different than 1 and 0, respectively.
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.
Because activation of ICir,swell should depend on cell
volume rather than osmolarity per se, Gd3+-sensitive
current at 80 mV and Gd3+-induced cell shrinkage
are plotted as a function of relative cell volume before
adding Gd3+ in Fig. 11, A and B. Both effects of Gd3+
were apparent in 0.9T solution at a relative volume of
1.075 ± 0.025, the smallest amount of swelling examined, and were fully activated in 0.7T solution at a relative volume of 1.305 ± 0.033. Decreasing bath osmolarity to 0.6T did not alter the responses to Gd3+ despite
causing additional cell swelling. Furthermore, the Gd3+-sensitive current and shrinkage, expressed as a percentage of the maximum Gd3+-induced change, were proportional over a wide range of osmolarities (0.6-1.5T).
This linear relationship (Fig. 11 C) suggests a tight coupling between the processes.
Kinetics of Gd3+'s Effects on Current and Volume
The data in Figs. 9 B and 11 C establish that block of
current by Gd3+ and cell shrinkage are linearly related.
It is unclear, though, whether block of current causes
cell shrinkage or whether the primary effect of Gd3+ is
cell shrinkage, which in turn is responsible for the diminution of current. This question can be addressed by
examining the kinetics of the two effects. Fig. 12 depicts whole-cell current at 80 mV (
) 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.
Voltage Dependence of the Sensitivity of Cell Volume to Gd3+
Gd3+-sensitive ICir,swell was larger at physiologically relevant diastolic potentials than at plateau voltages. At
80 mV, for example, the Gd3+-sensitive current was
>2 pA/pF in 0.6T solution (Fig. 10 A). A sustained current of this magnitude represents a significant transmembrane ion flux and could cause significant alterations in intracellular osmolarity and, thus, cell volume.
Because of its inward-going rectification, however, the
Gd3+-sensitive current should have little effect on cell
volume at more positive potentials. To investigate this
idea, Eh was varied from
80 and
40 mV under isosmotic conditions (1T) and during hyposmotic cell
swelling (0.6T) in the absence and presence of 10 µM
Gd3+. The I-V relationships for the Gd3+-sensitive current holding at
40 mV (Fig. 13 A; 1T, curve a
e ;
0.6T, curve b
f ) and at
80 mV (Fig. 13 B; 1T, curve
d
h; 0.6T, curve c
g) are plotted. As expected, the
Gd3+-sensitive current in 0.6T was not affected by Eh.
Relative cell volume during the protocol is shown in
Fig. 13 C. If Gd3+ blocks an inwardly rectifying cation
current, cell volume in 0.6T plus Gd3+ should be less
than in 0.6T without Gd3+ at
80 mV (see Fig. 13 C, c
and g), but because depolarization limits the current,
Gd3+ should fail to alter cell volume at
40 mV (see Fig
13 C, b and f ). Both of these predictions were observed.
The Gd3+-sensitive current was not the only mechanism
regulating volume under these conditions, however. Hyperpolarization from
40 to
80 mV in 0.6T without
Gd3+ should lead to the activation of inward current
and cell swelling at
80 mV if this current acted alone.
Instead, a very small cell shrinkage was observed (see
Fig. 13 C, b and c). This suggests that the osmotic effect
of Gd3+-sensitive cation influx was opposed by a voltage-dependent efflux of osmolytes. When the cation influx was blocked by Gd3+, a significant shrinkage was
unmasked on hyperpolarization (see Fig. 13 C, f and g).
These portions of data can be explained by the contribution of ICl,swell to osmolyte fluxes.
Osmotic swelling of rabbit ventricular myocytes caused
the graded activation of ICir,swell, a Gd3+-sensitive cation
current with a PK/PNa ratio of ~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, ~25× more than
at
40 or 0 mV. This amounted to a 5.5% decrease in
osmolarity, whereas volume decreased ~7% at
80 mV.
Table III. Effect of Gd3+-sensitive Current on Intracellular Concentrations |
An additional issue complicates the quantitative relationship between ionic currents under voltage clamp
and cell volume changes. There is also a flux between
the pipette and cell, and the pipette transport numbers
for cations, t+, and anions, t, must be considered, as is
illustrated in Fig. 14. If the membrane and patch pipette are both perfectly selective for cations or anions,
the ion flux between the pipette and cell will exactly balance the transmembrane ion flux and no volume
change will occur. The other extreme assumes the pipette and membrane have opposite cation/anion selectivity. In such a case, total fluxes into or out of the cell
are exactly twice the transmembrane flux, and observed volume changes are exactly twice those expected
from transmembrane currents. The optimal situation is
for the pipette cation and anion transport numbers
both to equal 0.5. In that case, cation and anion fluxes
between the pipette and cell are equal in magnitude
but opposite in direction, and their effects on cell volume cancel. As a result, the observed volume change will exactly equal the change expected from the measured ionic current.
Unfortunately, the transport numbers of a perforated patch are difficult to predict. The fluxes are determined by the combined characteristics of the pore-former and native membrane channels. Amphotericin
pores are permeant to both monovalent cations and anions (Marty and Finkelstein, 1975; 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.
Address correspondence to Dr. C.M. Baumgarten, Department of Physiology, Box 980551, Medical College of Virginia, Richmond, VA 23298-0551. FAX: 804-828-7382; E-mail: baumgart{at}gems.vcu.edu
Received for publication 3 April 1997 and accepted in revised form 20 June 1997.
1 Abbreviations used in this paper: 9-AC, 9-anthracene carboxylic acid; Eh, holding potential; I-V, current-voltage; NMDG, N-methyl-d-glucamine; RVD, regulatory volume decrease; SAC, stretch- or swelling-activated channel.This work was supported by National Heart, Lung, and Blood Institute grants HL-46764 and HL-02798.
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