© 1995 American Heart Association, Inc.
Articles |
-Adrenergic Control of Volume-Regulated Cl- Currents in Rabbit Atrial Myocytes
Characterization of a Novel Ionic Regulatory Mechanism
From the Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada; the Department of Medicine, University of Montreal; and the Department of Medicine and the Research Center, Montreal Heart Institute.
Correspondence to Stanley Nattel, Research Center, Montreal Heart Institute, Montreal, Quebec, Canada H1T 1C8.
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Abstract
-Adrenergic stimulation is known to play a role
in cardiac arrhythmogenesis and to modulate a variety of cardiac
K+ currents. The effects of -adrenergic stimulation on
Cl- currents are largely unknown. Many cardiac cell types
show a volume-sensitive Cl- current induced by cell
swelling (ICl.swell). The present experiments were
designed to assess the potential -adrenergic modulation of
ICl.swell in rabbit atrial myocytes. ICl.swell
was induced with the use of a hypotonic superfusate, under conditions
designed to prevent currents carried by K+,
Na+, and Ca2+ ions. A basal
Cl- current (ICl.b) was observed under
isotonic conditions in 128 of 150 cells (85%), had the same dependency
on [Cl-]o as ICl.swell,
and was reduced by cell shrinkage induced by hypertonic superfusion,
suggesting that ICl.b is carried by the same
volume-sensitive Cl- conductance as ICl.swell.
Phenylephrine produced a concentration-dependent and
near-complete inhibition of ICl.b and
ICl.swell, with EC50 values of 86±5 and
72±7 (mean±SEM) µmol/L, respectively, at +20 mV.
Norepinephrine (administered in the presence of 1 µmol/L
propranolol) also inhibited ICl.b and
ICl.swell, with EC50 values of 2.6±0.1
and 2.8±0.4 µmol/L, respectively. The concentration-response curve
for phenylephrine was shifted significantly
(P<.001) to the right by the 1-adrenoceptor
antagonist prazosin and by the 1A-receptor
antagonists (+)-niguldipine and 5-methylurapidil but was
unaltered by the 1B-receptor antagonist
chloroethylclonidine (100 µmol/L). Inhibition of protein
kinase C (PKC) with staurosporine, H-7, or 18-hour
preincubation with the phorbol ester 4ß-phorbol 12-myristate
13-acetate (PMA, 500 nmol/L) blocked the effects of
phenylephrine on ICl.swell, and the
highly selective PKC inhibitor bisindolylmaleimide blocked
the effects of norepinephrine on ICl.swell and
ICl.b. Both PMA and 1-oleoyl-2-acetylglycerol inhibited
ICl.swell in a concentration-dependent fashion. In blinded
studies, the phorbol ester phorbol 12,13-didecanoate (PDD) reduced
ICl.swell by 91±3%; its inactive analogue 4-PDD had no
effect (mean change, 3±1%). Preincubation with pertussis toxin (PTX)
prevented the actions of phenylephrine on
ICl.swell, indicating a role for a PTX-sensitive
guanine nucleotide–binding (G) protein. We conclude that
-adrenergic agonists inhibit volume-sensitive Cl-
currents in rabbit atrial cells by interacting with an
1A-adrenoceptor mechanism that is coupled to PKC via a
PTX-sensitive G protein. These results suggest a potentially novel
mechanism of -adrenergic control of cardiac electrical activity, the
inhibition of volume-sensitive Cl- currents, and indicate
that PKC, well known to elicit
phosphorylation-dependent Cl- currents in
cat and guinea pig ventricular myocytes, is also capable of
potently inhibiting other forms of cardiac Cl- current.
Key Words: action potential • Cl- currents • ion channels • autonomic nervous system • phenylephrine
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Introduction |
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In most mammalian species, the stimulation of cardiac
-adrenoceptors increases action potential
duration and the force of cardiac contraction.1 2 A
variety of arrhythmia mechanisms may be enhanced by
-adrenergic stimulation,3 and -adrenoceptor blockade
can reduce the severity of arrhythmias induced by myocardial
ischemia and reperfusion.1 A number of actions of
-adrenergic receptor activation on ionic currents have been
described. Consistent with its ability to prolong action
potential duration, 1-adrenoceptor activation inhibits a
variety of K+ currents, including Ito,
in rat ventricular4 5 6 and rabbit
atrial7 8 9 myocytes, IK1 in rabbit atrial and
ventricular myocytes,10 11 and
IKACh in rabbit atrial and ventricular
cells.10 11 In guinea pig myocytes, -adrenergic
stimulation shortens action potential duration,12
apparently by enhancing
IK.13 Since the discovery in 1989 of ICl.cAMP in guinea pig and rabbit ventricular myocytes,14 15 16 the properties of several types of cardiac Cl- currents have been described. These include ICl.cAMP,14 15 16 17 18 ICl.Ca,19 20 ICl.b,21 22 ICl.swell,23 24 25 26 ICl.purinergic,27 and ICl.PKC.28 29 30 31 At physiological Cl- concentrations, the reversal potential for Cl- currents has been estimated at values ranging from -65 to -30 mV.26 32 33 Thus, Cl- current can play a role both in the repolarization of the cell from plateau potentials and in phase 4 depolarization underlying spontaneous activity.
Although the actions of -adrenoceptor stimulation on K+
currents have been widely studied, -adrenergic effects on
Cl- currents have not been extensively investigated.
Preliminary data showing that -adrenoceptor stimulation activates a
Cl- current by stimulating PKC in cat
ventricular myocytes have been presented
previously.30 Cell swelling and cell stretch, both of
which activate ICl.swell,23 24 25 26 may
occur during a variety of clinical conditions, including acute
myocardial ischemia34 and congestive heart
failure. Increased catecholamine concentrations are a
feature of both conditions,35 36 so that the interactions
of adrenergic agonists with ICl.swell may be of
physiological and clinical importance.
Isoproterenol has been shown to enhance ICl.swell in canine
atrial myocytes, presumably by stimulating
ß-adrenoceptors.23 The ability of -adrenoceptor
stimulation to modify ICl.swell is presently unknown.
The present experiments were designed to determine (1) the effects
of -adrenoceptor stimulation on ICl.swell in rabbit
atrial myocytes, (2) the ability of subtype-selective -adrenoceptor
antagonists to block any response seen, and (3) the signal
transduction mechanisms linking -adrenergic stimulation to changes
in Cl- current. The results to be presented show
that in contrast to the effects of -adrenergic and PKC-mediated
enhancement of Cl- currents in cat and guinea pig
ventricular myocytes, -adrenoceptor stimulation and the
resulting activation of PKC cause concentration-dependent inhibition of
ICl.swell.
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Materials and Methods |
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Preparation of Single Cells
Single atrial cells were obtained from rabbit hearts by using a previously described dissociation technique.21 37 Briefly, rabbits (1.5 to 2.0 kg) were killed by a blow on the neck, and the hearts were quickly removed and perfused in the Langendorff mode, first with a modified HEPES-buffered Tyrode's solution at 37°C, then with a nominally Ca2+-free Tyrode's solution until the heart ceased to beat, and finally with the same solution containing 0.04% collagenase (CLS II, Worthington Biochemical) and 1.0% bovine serum albumin (Sigma Chemical Co) for 10 minutes. The left atrium was removed and further dissected into small pieces, and cell dissociation was achieved by gentle mechanical agitation. All cells studied were rod-shaped, had clear cross-striations, and lacked any visible blebs on their surfaces. Cell dimensions were determined with a calibrated graticule in the microscope, and cell volumes were estimated with assumed right cylindrical geometry according to the following equation:
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Electrophysiological
Recording
The tight-seal whole-cell voltage-clamp configuration of the
patch-clamp technique was used. Recordings were performed with an
Axopatch 1D or 200 amplifier (Axon Instruments). Voltage-clamp pulses
were generated by a 12-bit digital-to-analog (D/A) convertor. Membrane
current data were acquired by an analog-to-digital (A/D) conversion
board (Medical Systems) with a maximum sampling rate of 100 kHz and
simultaneously digitized (model TM 125, Scientific
Solutions) and stored on the hard disk of an IBM PC–compatible
computer under the control of PCLAMP software (Axon
Instruments). Recording pipettes were prepared from borosilicate
glass electrodes (outer diameter, 1.5 mm) with tip resistances of 2 to
5 M
(3.4±0.2 M, mean±SEM, n=118) when filled. Junction
potentials were corrected before achieving the membrane seal. After a
tight seal between the cell membrane and the pipette tip (seal
resistance >10 G) had been formed, the bath solution was changed
from Tyrode's solution to the standard isotonic experimental solution.
After seal formation, the membrane patch was ruptured with brief
additional suction.
The capacitive transients elicited by symmetrical 5-mV steps from -40
mV were recorded at 100 kHz for subsequent calculation of
capacitance and access resistance. The mean cellular capacitance was
75±2 pF, and the input resistance averaged 557±33 M, a value of
the same order as that obtained by Giles and van
Ginneken38 in rabbit atrial cells from the crista
terminalis. Series resistance was then compensated to minimize the
duration of the capacitive surge on the current record during 5-mV
hyperpolarizations from -40 mV, and over 70%
compensation was usually obtained. The time constant of the capacitive
transient averaged 205±7 microseconds (n=118), and series resistance
averaged 2.9±0.1 M (n=118) after compensation. In most experiments,
the maximum outward current was in the range of 1.5 nA, but in
occasional experiments, the current at very positive voltages (eg, +80
mV) was larger, exceeding 2 nA. All analyses of drug action
were therefore based on currents measured at +20 mV, a voltage in the
physiologically-relevant range at which currents
were always <1 nA.
To obtain whole-cell I-V relations, 300-millisecond hyperpolarizing and depolarizing pulses were imposed at 0.1 Hz in +10-mV increments between -100 and +80 mV from a holding potential of -40 mV. Current amplitudes were measured relative to the 0 current level. Leak-subtraction procedures were not used, but cells with evidence of a significant leak were rejected from study. All experiments were performed at 30±1°C. Na+ current was inactivated before the voltage steps by holding the cell at -40 mV. BaCl2 (500 µmol/L), CdCl2 (100 µmol/L), ouabain (10 µmol/L), and propranolol (1 µmol/L) were added to all superfusion solutions to block IK1, ICa, Na+,K+-ATPase, and ß-adrenoceptors, respectively.
Solutions and Drugs
The modified Tyrode's solution for cell isolation contained
(mmol/L) NaCl 126, KCl 5.4, CaCl2 2.0, MgCl2
1.0, NaH2PO4 0.33, glucose 10, and HEPES 10,
with pH adjusted to 7.4 with NaOH. The high-K+ storage
solution contained (mmol/L) KCl 20, KH2PO4 10,
glucose 10, potassium glutamate 70, ß-hydroxybutyric acid 10, taurine
10, and EGTA 10, along with 1% bovine serum albumin, pH 7.4
(KOH). K+-free pipette solutions were used to avoid
contamination by outward K+ currents. The pipette
(internal) solution contained (mmol/L) NMDG chloride 100, HEPES 10,
EGTA 5.0, and Mg2+-ATP 5.0, with pH adjusted to 7.4
with NMDG hydroxide and osmolarity adjusted to 270 to 290 mOsm/kg
H2O by adding mannitol (mean final osmolarity, 284±2
mOsm/kg H2O). Solution osmolarities were measured by
freezing-point depression (Osmomette A, Precision Systems Inc). In
experiments analyzing the response of currents to extracellular
Cl- replacement, the pipette solution contained (mmol/L)
NMDG chloride 24, NMDG aspartate 100, HEPES 10, EGTA 5, and
Mg2+-ATP 5, with pH adjusted to 7.4 with NMDG
hydroxide. [Cl-]o was modified by equimolar
replacement with aspartate. The standard isotonic bath solution
contained (mmol/L) NaCl 126, CsCl 5.4, MgCl2 0.8,
CaCl2 1.0, NaH2PO4 0.33, HEPES 10,
and glucose 5.5, with pH adjusted to 7.4 with NaOH (mean osmolarity,
294±3 mOsm/kg H2O). In some experiments, a hypertonic bath
solution was used, which was prepared by adding 75 mmol/L mannitol to
the standard isotonic solution to create a final osmolarity of 361±3
mOsm/kg H2O. The standard hypotonic bath solution contained
(mmol/L) NaCl 100, MgCl2 0.8, CaCl2 1.0,
NaH2PO4 0.33, HEPES 10, and glucose 5.5, pH
adjusted to 7.4 with NaOH (217±2 mOsm/kg H2O). In some
experiments, cell swelling was induced by using a hypertonic pipette
solution that contained (mmol/L) CsCl 160, CsOH 40, MgCl2
1.0, HEPES 10, EGTA 5.0, and Mg2+-ATP 5.0 (pH 7.4
with HCl, 400 to 420 mOsm/kg H2O) while cells were perfused
with standard bath solution (270 to 285 mOsm/kg H2O) as
described above. Similar results were obtained with either method of
inducing cell swelling, and the experiments presented were
performed with hypotonic superfusate-induced swelling.
Phenylephrine, norepinephrine,
propranolol, prazosin, ouabain, and PTX were purchased from
Sigma. H-7, staurosporine, PMA, and OAG were obtained from
ICN Biochemicals. The highly selective PKC inhibitor
bisindolylmaleimide, the phorbol ester PDD, and its inactive analogue
4-PDD were purchased from Calbiochem/Novobiochem. CEC, 5MU, and
S(+)-niguldipine were bought from Research Biochemicals Inc.
The disulfonic stilbene Cl- transport blockers DIDS and
SITS were purchased from Sigma and made up as fresh solutions on the
day of each experiment. Staurosporine, PMA, 4-PDD, and
PDD were prepared as 1 or 2 mmol/L stock solutions in dimethyl
sulfoxide. OAG was first dissolved in chloroform, then dispersed in
standard bath solution by sonication after evaporation of chloroform
with N2 gas, and finally diluted in the standard bath
solution to obtain the desired concentration. Stock solutions of the
other drugs were prepared in distilled water and added to known volumes
of superfusion solution to produce the desired concentrations. In all
experiments with staurosporine or H-7,
staurosporine (0.1 µmol/L) or H-7 (20 µmol/L) was
present in the pipette solution and was added to the superfusate at
the times indicated in "Results."
Data Analysis
All analyses are based on comparisons of currents in the
presence of a drug with those recorded before drug superfusion in
the same cell. Dose-response experiments were performed by superfusing
the same cell with control solutions and then with each concentration
of the drug to be tested. All drug effects were assessed after a
superfusion interval long enough to achieve steady state effects, which
was 10 to 15 minutes unless otherwise indicated.
EC50 was calculated from the concentration-response relation with an Emax model,39 in which the measured effect (E) of an agent at a known concentration C was fitted to the relation E=EmaxC/(C+EC50), where the variables determined by curve fitting are Emax (maximum effect) and EC50 (concentration for 50% of maximum effect). All results are expressed as mean±SEM. Statistical comparisons were performed either by ANOVA with Scheffé contrasts for group data or by Student's t test when only two groups were compared. A two-tailed probability of <5% was taken to indicate statistical significance.
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Results |
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Properties of ICl.swell and ICl.b
Fig 1A
shows the properties of swelling-induced
current in a myocyte lacking any significant conductance under basal
conditions (Fig 1A, a). After exposure to hypotonic conditions, a
substantial current is seen (Fig 1A, b). The current shows outward
rectification and has a reversal potential (-36 mV) that is close to
the calculated Cl- equilibrium potential (-38.5 mV). Fig 1A, c and d, shows the effects of extracellular Cl-
replacement with aspartate on swelling-induced currents in the same
cell. Reduction of [Cl-]o shifted the
reversal potential to more positive values and strongly reduced the
amplitude of outward currents. Fig 1B shows the I-V relations for
swelling-induced currents in three cells that lacked any significant
basal Cl- conductance. Results (mean±SEM) before
hypotonic cell swelling are shown by open symbols; the results after
cell swelling are shown by filled symbols. Under basal conditions (open
circles), the conductance is extremely small and reverses at 0 mV. Cell
swelling induces a large outwardly rectifying conductance whose
reversal potential shifts strongly with changes in
[Cl-]o. Fig 1C shows the relation between
mean reversal potential in these cells and the logarithm of
[Cl-]o. The relation was highly linear
(r2=.999) and had a slope of -57 mV per decade.
Experimentally determined values were close to the relation predicted
for a pure Cl--specific conductance, as calculated from
the Nernst equation and shown by the line in the figure.
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) and in the presence of cell swelling at
three values of [Cl]o (filled symbols). Results are
mean±SEM in three cells. C, Relation between reversal potential
(Erev) of ICl.swell and
[Cl]o in cells lacking ICl.b. Results
are mean±SEM, but error bar falls within symbol for mean. The square
of the correlation coefficient for the relation between
Erev and [Cl]o is .999, and the slope is -57
mV per decade. The line shown is the relation predicted for a pure
Cl- conductance.
A minority of cells had the basal conductance characteristics shown in
Fig 1
. Of a total of 150 cells studied, 128 (85.3%) showed a
significant basal conductance as illustrated in Fig 2A.
ICl.b was present immediately after membrane rupture
and remained constant under isotonic conditions for observation periods
of up to 40 minutes. ICl.bs were outwardly rectifying and
reversed at a potential (-46 mV) very close to the calculated
Cl- equilibrium potential (-45.4 mV) in the presence of
136 mmol/L [Cl-]o and 24 mmol/L
[Cl-]i. Reductions in
[Cl-]o to 25 (Fig 2B) and 5 (Fig 2C) mmol/L
reduced the outward current amplitude and shifted the reversal
potential, changes that were reversible upon returning to a
[Cl-]o of 136 mmol/L (Fig 2D). Exposure to
hypotonic conditions caused cell swelling and greatly increased the
conductance (Fig 2E). Overall, cell size increased upon hypotonic
swelling from 114±2x10.6±0.2 µm to 107±2x16.4±0.5 µm,
corresponding to a calculated volume increase of 140±15%. Reducing
[Cl-]o reduced the magnitude of
swelling-induced currents and shifted their reversal potential in the
positive direction, as illustrated in Figs 2F and 2G.
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Fig 3 shows a quantitative analysis of the
effects of changes in [Cl-]o on currents
recorded under both hypotonic and isotonic conditions in six cells
exposed to various [Cl-] values before and after cell
swelling. For a given value of
[Cl-]o, the I-V relation has the same
form and a similar reversal potential under both isotonic and hypotonic
superfusate conditions (Fig 3A). The relations between reversal
potentials and [Cl-]o under isotonic and
hypotonic conditions are shown in Fig 3B. The relations were highly
linear (r2=.997 and .999 for isotonic and
hypotonic conditions, respectively), with a slope of 56 mV per decade
under each condition. Since currents were recorded with both
hypotonic and isotonic superfusate at corresponding values of
[Cl-]o in each cell, we were able to
determine the response of the swelling-induced component to changes in
[Cl-]o by subtracting ICl.b
(under isotonic conditions) from the current recorded in the
presence of hypotonic cell swelling. The resulting values have the I-V
relations shown by the open squares in Fig 3A. The reversal potential
of the swelling-induced component follows the Cl-
equilibrium potential (Fig 3B) and has a linear relation to log
([Cl-]o), with an
r2 of .999 and a slope of 57 mV per decade.
Values of the reversal potential of swelling-induced current in the
absence of ICl.b, as illustrated in Fig 1C, are
reproduced as open triangles in Fig 3B. Note that the reversal
potentials of ICl.b (open circles), the total current in
the presence of swelling in cells with ICl.b (open
triangles), the swelling-induced component in cells with
ICl.b (open squares), and the swelling-induced current in
cells without ICl.b (open diamonds) virtually superimpose
on each other at each [Cl-]o (Fig 3B).
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)
hypotonic-induced cell swelling at [Cl-]o
values of 105 (a), 25 (b), and 5 (c) mmol/L in six cells with
ICl.b exposed to various [Cl-] values under
both control and swelling conditions. 
indicates the current
induced by swelling, obtained by subtracting ICl.b from
total current in the presence of swelling. B, Relation between reversal
potential (Erev) and [Cl]o for currents shown
in panel A (graphs a through c). Values are also shown for
swelling-induced currents in cells lacking ICl.b (
,
same data as in Fig 1C
The results shown in Fig 3 suggest that ICl.b may be
carried by the same ionic current mechanism as the swelling-induced
current. Therefore, we sought to determine whether ICl.b is
volume sensitive and can be suppressed by reducing cell volume with
hypertonic superfusates. These experiments (and all others shown
subsequently) were performed with pipettes containing 100 mmol/L
[Cl-] in the pipette, bringing the Cl-
reversal potential toward 0 mV. Fig 4A, a, shows
ICl.b recorded immediately after rupturing the membrane
and compensating for capacitance and series resistance. After 30
minutes of continued superfusion with isotonic solution,
ICl.b was unchanged (Fig 4A, b). Subsequent exposure to
hypertonic superfusate caused a gradual reduction in current amplitude,
with results after 30 minutes shown in Fig 4A, c. Similar results were
obtained in a total of four cells studied in this fashion, for which
the mean I-V relations before and after 30 minutes of exposure to
hypertonic solution are shown in Fig 4B. Overall, exposure to
hypertonic solution for 30 minutes reduced ICl.b by 52±8%
(P<.001) at +20 mV. Calculated mean volume of these cells
averaged 9056±1514 µm3 (length, 123±13 µm; width,
9.5±0.4 µm) under isotonic conditions and 4163±884
µm3 (length, 121±12 µm; width, 6.5±0.4 µm) after
hypertonic superfusion (a mean reduction in cell volume of
53±6%).
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The data shown in Figs 1 through 3 indicate that ICl.b and
the current induced by swelling are anion currents that have a similar
and substantial selectivity for Cl- ions over aspartate
and a similar I-V relation. Fig 4 shows that ICl.b is
volume sensitive and can be decreased by hypertonic superfusate-induced
cell shrinkage. Therefore, it is quite likely that ICl.b
and the current induced by swelling are carried by the same underlying
volume-sensitive anion conductance. Since relatively few cells lack
ICl.b, making it difficult to study swelling-induced
current in isolation, we studied the -adrenergic regulation of the
total current in the presence of cell swelling, which we will call
ICl.swell in this article and which we believe to
represent the total magnitude of volume-regulated
Cl- current in the presence of cell swelling in each cell.
Several additional series of experiments were performed with both
ICl.b and ICl.swell to determine whether they
respond similarly to drug interventions, as would be expected if they
are carried by the same underlying volume-sensitive current
mechanism.
Effects of -Adrenoceptor Stimulation on ICl.b
and ICl.swell
Fig 5 shows the effects of varying concentrations
of phenylephrine on ICl.b. ICl.b
was present as soon as the first voltage-clamp steps could be made
after membrane rupture, capacitance compensation, and measurement of
series and input resistance (Fig 5A) and did not change during 20
minutes of observation (Fig 5B). Subsequent superfusion of the cells
with the -adrenoceptor agonist phenylephrine in the
presence of the ß-adrenoceptor antagonist
propranolol (1 µmol/L) caused a concentration-dependent
inhibition of ICl.b (Fig 5C through 5E). At +20 mV in four
experiments in which all drug concentrations could be tested in each
cell, 5 µmol/L phenylephrine caused a 9.8±1.5%
reduction (P<.05), and 100 µmol/L
phenylephrine reduced the current by 48±1%
(P<.001). At a greater concentration (800 µmol/L),
phenylephrine suppressed ICl.b by 82±4%, an
effect that disappeared in the example shown after 45 minutes of
washout (Fig 5F). The concentration-response curve for
phenylephrine inhibition of outward current at +20 mV is
shown in Fig 5G. The EC50 for phenylephrine
inhibition of ICl.b averaged 86±5 µmol/L in four cells
in which all drug concentrations could be studied.
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Fig 6 shows the response of ICl.swell to
phenylephrine. Under isotonic conditions, ICl.b
is present (Fig 6A). Membrane conductance started to increase 3 to
5 minutes after exposure to the hypotonic solution and approached
steady state values within 20 minutes (Fig 6B). Subsequent exposure to
phenylephrine caused a concentration-dependent inhibition
of ICl.swell, which was partially reversible upon
washout of the largest concentration (Fig 6F). At +20 mV, the
EC50 for phenylephrine action on
ICl.swell averaged 72±7 µmol/L (n=9), not significantly
different from the EC50 for phenylephrine
inhibition of ICl.b. Although phenylephrine was
somewhat more potent at inhibiting ICl.swell at more
positive potentials (EC50 of 90±13, 71±10, 72±7, 77±8,
64±4, and 64±5 µmol/L at test potentials of -100, -80, +20, +40,
+60, and +80 mV, respectively; n=9 for each), these differences were
not statistically significant (ANOVA).
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To determine whether the changes observed with
phenylephrine are also produced by the
endogenous neurotransmitter norepinephrine, we
studied the concentration-dependent effects of
norepinephrine (in the presence of 1 µmol/L
propranolol) on ICl.b and
ICl.swell, as shown in Fig 7.
Norepinephrine produced a concentration-dependent and
voltage-independent inhibition of ICl.b (Fig 7A) and
ICl.swell (Fig 7B), with EC50 values of
2.6±0.1 µmol/L (n=4) and 2.8±0.4 µmol/L (n=4), respectively. The
results shown in Figs 5 through 7 indicate that -adrenergic
stimulation is highly effective in inhibiting the volume-sensitive
Cl- currents (ICl.b and ICl.swell)
in rabbit atrial myocytes. The similar sensitivity of ICl.b
and ICl.swell to both phenylephrine and
norepinephrine supports the contention that both currents
are carried by the same underlying mechanism.
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Effects of Selective -Receptor Antagonists on
Phenylephrine Action
Fig 8 illustrates the antagonism of the
effects of phenylephrine on ICl.swell by the
1-adrenoceptor antagonist prazosin. Fig 8A,
a and b, shows currents before and after the induction of cell swelling
in a representative myocyte. Exposure to prazosin (2
µmol/L) in the absence of phenylephrine did not alter
ICl.swell (Fig 8A, c). Exposure to 800 µmol/L
phenylephrine in the continued presence of prazosin
inhibited ICl.swell (Fig 8A, d) but to a much smaller
extent than the same concentration of phenylephrine in
cells not exposed to prazosin (eg, compare with Figs 5E and 6E).
Subsequent exposure to a superfusate containing the same concentration
of phenylephrine in the absence of prazosin resulted in
much stronger inhibition of ICl.swell (Fig 8A, e), which
was partially reversed by the reintroduction of prazosin (Fig 8A,
f).
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), in the presence of PZ (
), and in the presence of PE alone
(
) in four cells studied under all conditions. C,
Concentration-response curve for PE inhibition of ICl.swell
at +20 mV in the absence (
Fig 8B shows mean I-V relations for ICl.swell under control
conditions, in the presence of 2 µmol/L prazosin alone, in the
presence of 800 µmol/L phenylephrine and 2 µmol/L
prazosin, and in the presence of 800 µmol/L phenylephrine
alone in four cells exposed to all conditions. Prazosin alone did not
alter the I-V curve, and phenylephrine strongly inhibited
the current at all voltages. The effect of phenylephrine
was greatly attenuated by coadministration with prazosin. Prazosin (2
µmol/L) shifted the phenylephrine concentration-response
curve in a parallel fashion to the right (Fig 8C), with the
phenylephrine EC50 at +20 mV increased by
prazosin from 72±7 to 737±99 µmol/L (n=6, P<.001).
To gain insights into the 1-receptor subtype mediating
the effect of phenylephrine on
ICl.swell, we studied the effect of the
1A-receptor antagonists (+)-niguldipine and
5MU and the 1B-receptor antagonist CEC on
the phenylephrine concentration-response curve. As shown in
Fig 9, even large concentrations of CEC (100 µmol/L)
did not perceptibly alter the response to phenylephrine. On
the other hand, 0.1 µmol/L 5MU substantially inhibited the action of
phenylephrine, with relatively little current inhibition
occurring at a concentration of 800 µmol/L.
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The actions of several antagonists were assessed
quantitatively by studying their effects on the
phenylephrine concentration-response curve (Fig 10). While CEC (100 µmol/L) did not significantly
alter the phenylephrine EC50 (84±12 µmol/L
in presence of CEC, n=8, P=NS vs phenylephrine
alone), (+)-niguldipine, 5MU, and prazosin all significantly
(P<.001 for each) increased the phenylephrine
EC50 by shifting the concentration- response curve to
the right in a parallel fashion (Fig 10). The action of (+)-niguldipine
was concentration dependent [EC50 of 368±74 and 691±47
µmol/L for 0.1 and 1.0 µmol/L (+)-niguldipine, respectively; n=4
for each], and its potency was substantially less than that of 5MU.
5MU, at a concentration of 0.1 µmol/L, increased the
phenylephrine EC50 in five cells to 2418±53
mmol/L, a greater increase than produced by a 10 times higher
concentration of (+)-niguldipine. None of the antagonists
significantly altered the Emax for
phenylephrine, which averaged 93±4% under control
conditions and 94±3%, 93±3%, 91±5%, 96±2%, and 99±1% in the
presence of prazosin, CEC, niguldipine at 0.1 µmol/L, niguldipine at
1 µmol/L, and 5MU, respectively.
|
) and 1.0 (
). The best-fit curves
(shown) to the Emax equation provided in the text had
EC50 values of 61 (PE alone), 69 (PE+CEC), 367 (PE+NIG, 0.1
µmol/L), 840 (PE+NIG, 1 µmol/L), and 1869 µmol/L (PE+5MU) and
Emax values of 91%, 88%, 95%, 98%, and 97%,
respectively.
Effects of PKC Inhibition on the Response of ICl.swell
and ICl.b to -Adrenoceptor Agonists
To determine whether the -adrenoceptor–induced decrease in
ICl.swell is mediated by the activation of PKC, we applied
phenylephrine in the presence of the PKC
inhibitors staurosporine40 and
H-7.41 Fig 11A shows the effect of
staurosporine on the response of ICl.swell to
phenylephrine. In contrast to the reproducible inhibition
caused by phenylephrine in the absence of PKC
inhibitors (eg, Figs 5 and 6), 800 µmol/L
phenylephrine had minimal effect on Cl-
current in their presence. Fig 11B shows overall data for the
concentration-dependent inhibitory effects of
phenylephrine on ICl.swell under control
conditions (n=9) and in the presence of 100 nmol/L
staurosporine (n=6) and 20 µmol/L H-7 (n=5). In the
presence of PKC inhibitors, no statistically significant
effects of phenylephrine on ICl.swell could be
demonstrated at concentrations up to 1600 µmol/L.
|
Organic PKC inhibitors like H-7 and
staurosporine are not completely specific in their actions.
Therefore, additional experiments were performed to establish the
effects of PKC inhibition on phenylephrine action.
Prolonged stimulation (>6 hours) of PKC by phorbol esters such as PMA
leads to a loss of PKC enzymatic activity and high-affinity phorbol
ester binding.9 11 42 This allows for the role of PKC to
be tested in a fashion independent of the use of organic PMA
inhibitors. Therefore, we incubated cells in the
high-K+ storage solution (for contents, see "Materials
and Methods") overnight (>15 hours) at room temperature, with or
without the addition of 500 nmol/L PMA. Experiments were done in a
paired fashion, with the cells from each atrial isolate divided into
two lots, one to be incubated with and the other without PMA.
ICl.swell and its response to phenylephrine
were then assessed in cells from both groups in random order the next
day. Fig 11C shows the mean concentration-response curve for
phenylephrine inhibition of ICl.swell at +20 mV
in cells incubated with (n=5) or without (n=5) PMA, along with data
from nine cells studied without prior overnight incubation. In cells
incubated without PMA, phenylephrine caused a
concentration-dependent inhibition of ICl.swell with an
EC50 of 66±8 µmol/L (n=5), a value not significantly
different from that in cells studied without preincubation. In
contrast, exposure to phenylephrine at concentrations up to
2 mmol/L did not significantly alter ICl.swell in cells
incubated overnight with PMA before study.
Finally, we used the recently developed and highly selective PKC
inhibitor bisindolylmaleimide,43 to determine
whether PKC inhibition alters the response to
norepinephrine (coadministered with 1 µmol/L
propranolol). Fig 12 shows the effect of 30
nmol/L bisindolylmaleimide on the response of ICl.b
(panel A) and ICl.swell (panel B) to
norepinephrine at concentrations up to 20 µmol/L. In
marked contrast to the strong inhibition of these currents caused by
norepinephrine in the absence of bisindolylmaleimide (Fig 7), PKC inhibition completely prevented -adrenergic actions on these
currents. Furthermore, the similar effects of PKC inhibition on the
response to norepinephrine of ICl.b and
ICl.swell suggest that these volume-sensitive currents are
both inhibited by -adrenergic agonists via the activation of
PKC.
|
Effects of PKC Activators on
ICl.swell
To further assess the ability of PKC activation to inhibit
ICl.swell, we examined the effects of addition to
the superfusate of PKC activators.44 Fig 13 shows that PMA (50 to 800 nmol/L) induced a
concentration-dependent decrease in ICl.swell. The
EC50 for inhibition of ICl.swell at +20 mV by
PMA was 210±23 nmol/L (n=5) (Fig 13E). Similar results were obtained
with OAG but at higher concentrations (5 to 200 µmol/L). The
EC50 for inhibition of ICl.swell by OAG was
47±14 µmol/L (n=5) at +20 mV (Fig 13F). To exclude a nonspecific
action of phorbol esters unrelated to PKC activation, we performed
blinded experiments in which coded stock solutions of either PDD or
4-PDD (structurally very similar to PDD but ineffective in
activating PKC) were used in a randomized fashion to study the changes
in ICl.swell caused by 1 µmol/L of each in the
superfusate. Fig 14 shows results in one cell exposed
to both compounds. 4-PDD had no effect on
ICl.swell, whereas PDD produced strong inhibition.
Overall, PDD reduced ICl.swell by 91±3% (n=4,
P<.001), whereas the mean change in ICl.swell
occurring in the presence of 1 µmol/L 4-PDD averaged 3±1% (n=6,
P=NS). These results indicate that activators of
PKC are capable of mimicking the effect of -adrenoceptor stimulation
on ICl.swell.
|
|
Effects of PTX on Phenylephrine-Induced Inhibition
of ICl.swell
Cardiac 1-adrenoceptors are functionally linked to
heterotrimeric GTP-binding regulatory proteins (G
proteins),45 which are thought to play an important role
in mediating 1-adrenoceptor–induced increases in
phospholipase C activity46 47 that result in PKC
activation. PTX inactivates G proteins (Go and
Gi) that are potentially coupled with
1-adrenoceptors,1 48 49 50 by catalyzing the
ADP ribosylation of the subunit at a C-terminal cysteine residue
and thus blocking the interaction between activated receptors and the
holo-G protein.51
To determine whether the 1-adrenergic inhibition of
ICl.swell is coupled via PTX-sensitive G proteins, we
incubated myocytes for over 18 hours at room temperature in storage
solution containing 0.5 µg/mL of PTX. This procedure has been
reported to cause the ADP ribosylation of up to 90% of the
available PTX-sensitive G proteins in rabbit atrial
cells.9 11 As shown in Fig 15,
pretreatment of cells with PTX abolished the effect of
phenylephrine (25 to 3200 µmol/L) on
ICl.swell (n=5). Preincubation of cells in the storage
solution alone did not alter the response to phenylephrine
(Fig 11C). These results indicate that PTX-sensitive G proteins are
essential for the inhibition of ICl.swell by
-adrenoceptor stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown that ICl.b and ICl.swell in rabbit atrial myocytes share properties of outward rectification, Cl- selectivity, volume regulation, and concentration-dependent inhibition by
-adrenergic agonists,
suggesting that they are carried by the same underlying
volume-sensitive anion conductance. The inhibitory actions
of phenylephrine on ICl.swell are antagonized
by the 1-receptor antagonist prazosin, not
altered by the 1B-receptor antagonist CEC,
and prevented by the 1A-selective receptor
antagonists (+)-niguldipine and 5MU, indicating mediation
by an 1A-receptor subtype. The actions of -adrenergic
stimulation on volume-regulated Cl- currents are mimicked
by the PKC-stimulating phorbol esters PDD, PMA, and OAG and are
prevented when PKC is inhibited by staurosporine, H-7,
bisindolylmaleimide, or downregulation by prolonged exposure to PMA.
Exposure to PTX also blocks -adrenergic inhibition of
ICl.swell, indicating the participation of a
PTX-sensitive G protein in the signal transduction pathway leading to
PKC activation by 1A-receptor stimulation.
Properties of Cl- Currents Studied
The properties of ICl.b in the present experiments
resemble those we have previously reported in the same
preparation.21 22 Although ICl.swell has not
previously been reported to exist in rabbit atrium, the properties of
ICl.swell described in the present article resemble
those described in other cardiac preparations23 24 26 and
those of a stretch-induced Cl- current in rabbit atrial
cells.25 The reversal potential of ICl.swell
responded to changes in [Cl-] gradient in a fashion
consistent with a Cl--selective current and with
the same slope factor for [Cl-]o dependence
as ICl.b. Exposure to hyperosmotic superfusate
substantially reduced ICl.b. ICl.b and
ICl.swell were inhibited by -adrenoceptor agonists in a
quantitatively similar fashion, and -adrenergic inhibition of either
was PKC dependent. These findings suggest that both currents are
carried by the same underlying volume-sensitive Cl-
channel.
Comparison With Previous Observations of
1-Adrenoceptor Modulation of Ion Currents
Recent reports have indicated that 1-adrenergic
stimulation suppresses several K+ currents, including
Ito,6 7 8 9 52 IK1, and
IKACh.10 11 In their studies on rat
ventricular myocytes, Ravens and
colleagues6 52 reported that 1-adrenergic
stimulation inhibited both the 4-AP–sensitive transient outward
current and the 4-AP–insensitive sustained current.6 52
In rabbit atrium, the sustained current activated by depolarization is
resistant to 4-AP and has properties suggesting a potentially
important contribution from ICl.b.21 In the
present study, we have found that 1-adrenoceptor
stimulation inhibits both ICl.b and
ICl.swell, at concentrations very similar to those
found to inhibit K+ current in other
studies.6 7 8 9 52 This is, to our knowledge, the first report
of -adrenergic inhibition of a cardiac Cl- current.
Subtype Selectivity of 1-Adrenoceptor Effects
on ICl.swell
McGrath and Wilson53 suggested the existence of two
subtypes of 1-adrenoceptors based on differences in
agonist affinity. Han et al54 and Minneman55
classified 1-adrenoceptors into 1A and
1B subtypes, based on tissue responses to various
agonists and antagonists and on competitive ligand-binding
studies with WB 4101 and CEC. The 1A subtype has a high
affinity for the competitive antagonist WB 4101 and is not
inactivated by the alkylating agent CEC. The 1B subtype
has a 20- to 1500-fold lower affinity for WB 4101 and is potently
inhibited by CEC.54 55 56 Additional
1A-selective antagonists have been
developed, including 5MU,57 58 a serotonin
antagonist, and (+)-niguldipine,59 60 a
dihydropyridine Ca2+ channel
blocker. It has been suggested that 5MU is currently the best
antagonist for identifying
1A-receptor–mediated responses.61
Recently, two additional subtypes of -adrenoceptors,
1C and 1D, have been identified on
the basis of molecular cloning.62 63 The
1D-receptor differs from the 1A-receptor
clone by two amino acids and, in contrast to the latter, is sensitive
to inhibition by CEC.62 The 1C-receptor
shows exquisite and equal sensitivity to inhibition by (+)-niguldipine
and 5MU, whereas the 1A-receptor is >10 times as
sensitive to 5MU as it is to (+)-niguldipine.63
In the present study, phenylephrine-induced
inhibition of ICl.swell was unaffected by CEC at high
concentrations but was inhibited by (+)-niguldipine and 5MU, showing
particular sensitivity [>10-fold greater than that to
(+)-niguldipine] to the latter agent. These observations suggest the
involvement of an 1A type of receptor. Both
1A- and 1B-receptors have been shown to
exist in canine ventricle on the basis of radioligand and
electrophysiological studies.64 The
1A-receptor–mediated system is involved in the
induction of abnormal automaticity in canine cardiac Purkinje fibers
exposed to ischemic conditions.65 66 67 This system
is PTX sensitive66 67 and causes an increase in
longitudinal resistance consistent with a decrease in membrane
conductance.67 It would be interesting to explore the
possible activation of volume-sensitive Cl- currents by
ischemia in canine Purkinje fibers, which could be amenable to
-adrenergic inhibition by the system described in the present
study.
Intracellular Signaling Mechanisms Underlying -Adrenergic
Modulation of ICl.swell
Stimulation of 1-adrenoceptors leads to various
biochemical responses, including enhanced Ca2+
influx, phospholipase C and A2 activation, and changes in
intracellular cyclic nucleotide
levels.2 55 68 69 In the heart, the most extensively
documented signaling responses to 1-adrenoceptor
stimulation are mediated via phospholipase C–induced hydrolysis of
phosphatidylinositol 4,5-diphosphate, giving rise to a variety of
potential second messengers, including inositol 1,4,5-tris-phosphate
and 1,2-diacylglycerol,49 69 70 71 which is thought to be an
endogenous activator of PKC.68 69
PKC has been implicated in the modulation of ion channel function in
numerous studies.44 Many of the signaling mechanisms
mediating -adrenoceptor–induced responses are coupled by G
proteins, at least two of which are sensitive to inhibition by
PTX.48
PTX-sensitive G proteins, which may appear with
development,72 mediate the effects of
1-adrenergic stimulation on abnormal automaticity in
canine Purkinje fibers66 67 and the positive chronotropic
response to agonists in rat hearts.72 PKC produces
action potential changes similar to those caused by methoxamine
in guinea pig papillary muscles,12 and phospholipase C
produces positive chronotropic responses in canine Purkinje fibers
similar to those resulting from -adrenergic
stimulation.73 In contrast, -adrenergic inhibition of a
variety of K+ currents in rabbit atrial myocytes is
insensitive to PTX and agents that inhibit PKC
activity.9 11 In the present study,
1-adrenergic inhibition of Cl- current
was found to be sensitive to inhibition by PTX and interventions
that suppress PKC function, making this system a candidate to account
for some of the electrophysiological effects of
-adrenergic activation. Our findings are consistent with
studies in which PKC activation caused a reduction in Cl-
currents in a variety of noncardiac preparations.74 75 76
Limitations
Both ICl.b and ICl.swell are inhibited by
1-agonists, which raises a potential problem. Since in
many cells ICl.swell is composed of both a basal
(ICl.b) and swelling-induced component, the observed
changes in total Cl- current represent the sum of
-adrenergic effects on each. To have studied each selectively in
each cell would have been prohibitively difficult, requiring exposure
to control solutions, multiple drug concentrations, hypotonic
solutions, and then the same drug concentrations again. We have
presented extensive evidence (in Figs 1 through 7 and Fig 12),
discussed above, suggesting that ICl.b and
ICl.swell are due to the same volume-sensitive anion
conductance and justifying the use of ICl.swell as an index
of mechanisms of -adrenergic regulation of volume-sensitive
Cl- current.
The response of ICl.b and ICl.swell in rabbit
atrial myocytes to -adrenergic and PKC stimulation that we observed
is opposite the response to the Cl- current stimulation
noted in feline and guinea pig ventricular myocytes by
Zhang and colleagues30 31 and Walsh and
Long.28 29 Recently published work by Zhang et
al31 suggests that PKC and protein kinase A may act on the
same set of Cl- channels to elicit Cl-
current in feline ventricular myocytes. Rabbit atrium lacks
transcripts for the cystic fibrosis transmembrane conductance
regulator77 and
ICl.cAMP,78 which appears to be the
target for PKC activation of Cl- current.31
This may explain the apparently simple inhibitory effect of
1-adrenergic activation on ICl.b and
ICl.swell that we observed. Under conditions that cause
cell swelling in tissues that express both ICl.swell and
ICl.PKC, a more complex response to
1-adrenergic activation, including both stimulation of
ICl.PKC and inhibition of ICl.swell,
might occur. This question remains to be addressed in future studies.
Walsh and Long29 cite unpublished results suggesting that
in guinea pig ventricular myocytes, which manifest
ICl.PKC, ICl.swell is absent.
The response of ICl.b to hypertonic superfusate raises the question of whether cell swelling or cell stretch occurred during cell isolation, resulting in the activation of ICl.swell, which then appeared as a background current at the onset of whole-cell recording. An alternative explanation of these findings is that rabbit atrial cells have a background Cl- current that is sensitive to cell volume (and possibly cell stretch), and can be enhanced by exposure to hypotonic media (which cause cell swelling) or suppressed by exposure to hypertonic media (which cause cell shrinkage). If this were the case, the volume-sensitive Cl- current could modulate cell function in response to either an increase or a decrease in cell volume.
We obtained concentration-response curves by exposing each cell to five
or six drug concentrations over the steep portion of the concentration
response curve. Since saturating concentrations were not studied in all
cells, there is some uncertainty about the precise maximum inhibition
caused by -agonists, introducing some uncertainty in the calculation
of EC50 values. These uncertainties are relatively small
and do not alter the qualitative differences that were seen among
various -receptor antagonists.
Conclusions
We have found that 1-adrenergic activation inhibits
the volume-regulated Cl- currents ICl.b and
ICl.swell in rabbit atrial myocytes by a PTX-sensitive
PKC-dependent mechanism. This is, to our knowledge, the first
demonstration of the inhibition of Cl- current by an
-adrenergic mechanism in the heart. It contrasts with the
stimulatory effect of PKC on Cl- current previously
described in ventricular tissues from the cat and guinea
pig and indicates the potential complexity of the -adrenergic
regulation of cardiac Cl- currents. The inhibition of
ICl.b by 1-adrenergic activation could
contribute to some of the electrophysiological
effects of the latter, and -adrenergic inhibition of
ICl.swell may play a role in settings, such as acute
myocardial ischemia and heart failure, in which
catecholamine concentrations are increased and cell
swelling and/or stretch can activate ICl.swell.