© 1996 American Heart Association, Inc.
Articles |
Adrenergic Modulation of Ultrarapid Delayed Rectifier K+ Current in Human Atrial Myocytes
From the Montreal Heart Institute Research Centre (Canada).
Correspondence to Stanley Nattel, MD, Montreal Heart Institute Research Centre, 5000 Belanger St, Montreal, Quebec, H1T 1C8, Canada.
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Abstract The ultrarapid delayed rectifier K+ current (IKur) in human atrial cells appears to correspond to Kv1.5 cloned channels and to play an important role in human atrial repolarization. Kv1.5 channels have consensus sites for phosphorylation by protein kinase A and C, suggesting possible modulation by adrenergic stimulation. The present study was designed to assess the adrenergic regulation of IKur in human atrial myocytes. Isoproterenol increased IKur in a concentration-dependent manner, with significant effects at concentrations as low as 10 nmol/L. The effects of isoproterenol were reversible by washout or by the addition of propranolol (1 µmol/L). Isoproterenol's effects were mimicked by the direct adenylate cyclase stimulator, forskolin, and by the membrane-permeable form of cAMP, 8-bromo cAMP. Isoproterenol had no effect on IKur when the protein kinase A inhibitor peptide, PKI(6-22)amide, was included in the pipette solution; in a separate set of experiments in which isoproterenol alone increased IKur by 45±9% relative to control, subsequent superfusion with isoproterenol in the presence of the protein kinase inhibitor H-7 failed to alter IKur. In contrast to isoproterenol, phenylephrine (in the presence of propranolol to block ß-adrenergic effects) induced a concentration-dependent inhibition of IKur, with significant effects observed at concentrations as low as 10 µmol/L. The inhibitory actions of phenylephrine were reversed by the addition of prazosin and prevented by coadministration with a highly selective inhibitor of protein kinase C, bisindolylmaleimide. These results indicate that ß-adrenergic stimulation enhances, whereas
-adrenergic stimulation inhibits,
IKur and suggest that these actions are mediated by protein
kinase A and protein kinase C, respectively. The modulation of
IKur by adrenergic influences is a potentially novel
control mechanism for human atrial repolarization and
arrhythmias.
Key Words: ion channels • cardiac arrhythmias • isoproterenol • phenylephrine • heart repolarization
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adrenergic influences on ion channels are potentially important regulators of cardiac function. ß-Adrenergic stimulation increases the probability of a variety of supraventricular1 2 and ventricular3 4 5 arrhythmias.
-Adrenergic stimulation may play a role in arrhythmias
related to ventricular ischemia and
reperfusion6 and exerts a positive inotropic action that
may be due to action potential prolongation caused by Ito
inhibition.7 8 The function of a wide variety of cardiac
ion channels can be modulated by adrenergic
stimulation.7 8 9 10 11 12 13 14 15
We have recently identified a novel K+ current in human
atrial myocytes,16 which shows delayed rectification but
activates at least two orders of magnitude faster than classic
cardiac delayed rectifiers.17 18 19 Because the novel current
activates much faster than either the "rapid" or
"slow" components of the classic delayed rectifier (or
IKr and IKs, respectively, after
Sanguinetti and Jurkiewicz20 21 ), we have proposed that it
be called the "ultrarapid delayed rectifier," or
IKur.16 IKur has
physiological and pharmacological properties that
distinguish it from IKr and IKs and that
strongly resemble those of Kv1.5 channels cloned from human
heart.22 23 24 The cDNA sequences coding for Kv1.5
channels22 24 possess multiple consensus sites for
phosphorylation by PKA and PKC. These protein kinases
are important parts of the signaling system that transduces ß- and
-adrenergic effects on the heart.12 25 Therefore,
we hypothesized that IKur, which appears to be
important in human atrial repolarization,16 might be
subject to regulation by adrenergic receptor stimulation.
The present experiments were designed to (1) determine
whether and how IKur is influenced by stimulation of
cardiac ß- or -adrenergic receptors and (2) evaluate the
potential nature of the signal transduction systems involved.
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Materials and Methods |
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Myocyte Isolation
Human atrial cells were isolated from specimens of right atrial appendage obtained from patients undergoing coronary bypass surgery. All specimens were grossly normal at the time of excision. Patients were free of heart failure and atrial arrhythmias. Upon excision, the samples were immediately placed in oxygenated, nominally Ca2+-free Tyrode's solution for transport to the laboratory. The time between excision and the beginning of laboratory processing was
5 minutes. The procedure
for obtaining the tissue was approved by the ethics committee of the
Montreal Heart Institute. Atrial myocytes were enzymatically isolated
by using a technique based on procedures described by Escande et
al.26 The tissue was chopped into cubic chunks (1
mm3) in nominally Ca2+-free Tyrode's solution
(36°C). The tissue was then placed in a 25-mL flask containing 10 mL
of the Ca2+-free solution, agitated by continuous bubbling
with 100% O2, and stirred with a magnetic bar.
After 12 minutes, the chunks were reincubated in a similar solution
containing 150 to 300 U/mL collagenase (CLS II, Worthington
Biochemical) and 4 U/mL protease (type XXIV, Sigma Chemical Co) for 45
minutes. The supernatant was then removed and discarded. The chunks
were reincubated in a fresh solution with 150 to 300 U/mL
collagenase. Microscopic examination of the medium was
performed every 5 to 10 minutes to determine the number and quality of
the isolated cells. When the yield appeared to be maximal, the chunks
were suspended in a storage solution (for composition, see below) and
gently pipetted. The isolated myocytes were kept in the medium for at
least 1 hour before use. A small aliquot of the solution containing the isolated cells was placed in an open perfusion chamber (1 mL) mounted on the stage of an inverted microscope. Myocytes were allowed to adhere to the bottom of the dish for 5 to 10 minutes and were then superfused at 2 to 3 mL/min with Tyrode's solution. Experiments were conducted at room temperature (22°C to 24°C) in order to be able to observe the activation of IKur, which is too rapid to resolve at body temperature.16 Only quiescent cells showing clear cross striations were used.
Solutions and Drugs
The standard Tyrode's solution contained (mmol/L) NaCl 126.0,
KCl 5.4, MgCl2 1.0, CaCl2 1.0,
NaH2PO4 0.33, glucose 10.0, and HEPES 10.0, pH
adjusted to 7.4 with NaOH. For cell dissociation, Ca2+ was
omitted. The storage solution consisted of (mmol/L) KCl 20,
KH2PO4 10, glucose 10, glutamic acid (potassium
salt) 70, ß-hydroxybutyric acid 10, taurine 10, and EGTA 0.5,
along with 1.0% albumin, pH adjusted to 7.3 with KOH. The
pipette solution contained (mmol/L) KCl 20, potassium aspartate 110,
MgCl2 1.0, HEPES 10, EGTA 5.0, GTP 0.1, and
Mg2ATP 5.0, pH adjusted to 7.2 with KOH.
Isoproterenol, 8-bromo cAMP, and phenylephrine were
purchased from Sigma and freshly prepared as a 1 mmol/L or 10 mmol/L
(for phenylephrine) stock solution in distilled water.
Forskolin (Sigma) and bisindolylmaleimide (Sigma) were dissolved in
dimethyl sulfoxide as 1 mmol/L stock solutions. The specific PKA
inhibitor peptide, PKI (GIBCO Corp), and the relatively
nonspecific protein kinase inhibitor, H-7 (Sigma), were
prepared as a stock solution in distilled water before each experiment.
Stock solutions (1 mmol/L) of the ß- and -adrenergic receptor
antagonists propranolol (Sigma) and prazosin
(kindly supplied by Pfizer Pharmaceuticals) were prepared in distilled
water. 4-AP (Sigma) was prepared as a 1 mol/L stock solution, with pH
adjusted to 7.4 with the addition of 1N hydrochloric acid.
Data Acquisition and Analysis
Borosilicate glass electrodes (outer diameter, 1.0 mm) were
used, with tip resistances of 2 to 4 M
when filled with pipette
solution, and were connected to a patch-clamp amplifier (Axopatch
200, Axon Instruments). Command pulses were generated by a 12-bit
digital-to-analog converter controlled by pClamp software (Axon
Instruments). Recordings were low pass–filtered at 2 kHz,
and data were acquired by analog-to-digital conversion at a
maximum rate of 100 kHz (TL-1, DMA, Axon Instruments) and stored on the
hard disk of an IBM-compatible computer. In all of the cells studied,
junction potentials (2 to 10 mV) were compensated before the pipette
touched the cell. A tight seal was created with gentle suction, and
seals with resistances of <10 G were rejected. The cell membrane
was ruptured by additional suction to establish the whole-cell
configuration for voltage clamping.
Rs was electrically compensated to minimize the duration of
the capacitive transient. Rs along the clamp circuit was
estimated by dividing the time constant obtained by fitting the decay
of the capacitive transient by the calculated membrane capacitance
(obtained from the time integral of the capacitive response to a 5-mV
hyperpolarizing step from a holding potential of -60 mV). In 31
cells, the decay of the capacitive surge was fitted by a single
exponential having a time constant of 516±57 µs (cell capacitance,
69±7.7 pF) before Rs compensation. After compensation, the
capacitive time constant was reduced to 255±26 µs (cell capacitance,
66±7.6 pF). The initial Rs was 7.7±1.2 M, and
Rs was reduced to 4.3±0.9 M after compensation. For
most experiments, we selected cells lacking Ito,
which, as we have previously shown, have an IKur identical
to cells possessing Ito.16 In some
experiments, cells possessing Ito were studied, with a
prepulse (100 ms to +40 mV 10 ms before the test pulse) used to
inactivate Ito and isolate
IKur, as previously described.16 When
recordings shown in a figure were obtained with the use of a
prepulse, this is indicated in the voltage protocol shown in the figure
inset. Since identical results were obtained whether or not a prepulse
was used, the results obtained with both methods were collated for
analysis. In all cases, IKur was measured as the
current level at the end of a depolarizing pulse relative to the zero
current level. To account for variations in cell size, mean
IKur amplitudes were expressed in terms of current density
(absolute current amplitude divided by cell capacitance). Leak currents
were assessed by the response to 10-mV hyperpolarizing pulses from the
holding potential (-50 mV) and by changes in holding current at a
holding potential of -50 mV. Since the activation threshold of
IKur is -30 mV,16 changes in
IKur do not alter membrane conductance at -50 mV.
Leak subtraction was not used; cells showing significant leak currents
after membrane rupture, or in which significant leak currents developed
over the course of an experiment, were rejected.
Paired and unpaired Student's t tests were used for single statistical comparisons between two group means. ANOVA was used when more than two group means were involved in a comparison. Values of P<.05 were considered to indicate statistical significance. Group data are expressed as mean±SEM. Nonlinear curve fitting was performed with Marquardt's procedure.
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Results |
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Effects of Isoproterenol on IKur
Panels A through C of Fig 1
show typical
IKur recordings and illustrate the effects of
isoproterenol in a representative human atrial cell.
The control currents (Fig 1A) show the rapid activation, slow and minor
inactivation at positive potentials, and small tail currents typical of
IKur.16 Isoproterenol (1 µmol/L) increased
both IKur step and tail currents (Fig 1B). The
isoproterenol-induced currents (obtained by digital subtraction of
control recordings from those in the presence of isoproterenol,
Fig 1C; note change in current scale for subtracted current) are all
time dependent, showing rapid activation and tail currents upon
repolarization. No time-independent component, as might have been
expected from an action on the cAMP-dependent Cl-
current13 14 or other background currents, was seen.
Results similar to those shown in Fig 1A through 1C were obtained in a
total of 10 cells.
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Fig 1D
shows the average current density-voltage relation for
IKur in the absence and presence of 1 µmol/L
isoproterenol in 10 cells. Isoproterenol significantly increased
IKur at all test potentials positive to -20 mV. The
effect of isoproterenol was reversed by 20 minutes of washout (open
triangles in figure) and did not show significant voltage dependence
(ANOVA). Fig 1E shows an analysis of the concentration
dependence of isoproterenol's effects on IKur. Results
were obtained by analyzing the current elicited by depolarizations to 0
mV in the absence of isoproterenol and then in the presence of
different concentrations of the drug in six cells exposed to all drug
concentrations. Statistically significant increases began at an
isoproterenol concentration of 10 nmol/L and reached a maximum of
38±5% at a concentration of 10 µmol/L.
Panels A and B of Fig 2 show a
representative experiment studying the effect of
isoproterenol (1 µmol/L) on the reversal of tail currents.
Isoproterenol increased IKur tail current but did not alter
the reversal potential. In five cells, isoproterenol increased the tail
current at -40 mV from 57±28 to 84±34 pA (P<.05)
without altering the tail reversal potential (average, -73±5 and
-75±4 mV [P=NS] before and after isoproterenol,
respectively). The reversal potential of isoproterenol-induced tail
current averaged -76±3 mV.
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Despite increasing tail currents, ISO did not alter the reversal
potential. Similar results were obtained in a total of five cells. TP
indicates test potential. C, Voltage dependence of activation based on
analysis of tail currents (normalized to maximum value) after
150-ms activating pulse to voltages indicated, followed by a 60-ms
repolarization to -30 mV (delivered at 0.5 Hz). Symbols are
experimental data from 10 cells in the absence (
) of 1 µmol/L ISO, and the lines are the best-fit
Boltzmann functions. D, Activation time constants of
IKur, as obtained in six cells with the voltage
protocol shown in panel C by fitting the activation at each voltage
with a monoexponential function.
To determine the effects of isoproterenol on the voltage
dependence of IKur activation, we analyzed the
voltage dependence of IKur tail currents with the protocol
shown in the inset of Fig 2C, which shows mean normalized tail current
amplitudes in 10 cells. The curves shown are the best-fit Boltzmann
distribution equations of the following form:
I/Imax=1/{1+exp[(V-V50)/k]}, where
I is tail current at an activating voltage V, Imax is tail
current at an activating voltage of +50 mV, V50 is voltage
that produces 50% of maximal activation, and k is a slope constant.
When data from each experiment were fitted by this equation,
V50 averaged -2.9±0.2 mV under control conditions
and -5.6±1.9 mV after the addition of isoproterenol
(P=NS, paired t test). The slope constant
averaged 8.2±0.8 mV under control conditions and 8.1±0.7 mV after
isoproterenol (P=NS versus control). An analysis of
the activation time dependence of IKur is shown in Fig 2D.
The activation of IKur at various voltages was fitted by a
monoexponential function, as previously
described.16 Results from six cells are summarized in the
figure and indicate that IKur activation became faster at
more positive voltages but that isoproterenol did not alter the
activation time course.
Fig 3 shows the effect of propranolol on
isoproterenol-induced increases in IKur in a
representative cell. Step current was stable before the
addition of isoproterenol and increased over time to reach a steady
value 10 minutes after the onset of isoproterenol exposure (Fig 3A).
The addition of propranolol reversed the increases caused
by isoproterenol, despite continued exposure to the latter, with step
current approaching control values 7 minutes after the addition of
propranolol. Representative
recordings obtained under each condition are shown in Fig 3B.
In four cells studied with this protocol, the mean amplitude of
IKur before the administration of isoproterenol was
5.8±1.2 pA/pF. Isoproterenol increased IKur to 7.3±1.4
pA/pF (P<.01), and the addition of propranolol
returned the current amplitude to 6.1±1.3 pA/pF (P<.05
versus isoproterenol alone; P=NS versus value before
isoproterenol).
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Effects of Forskolin and 8-Bromo cAMP on
IKur
To determine the potential role of cAMP in mediating
isoproterenol's effects on IKur, we studied changes
produced by forskolin, which activates adenylate
cyclase independently of ß-adrenergic receptors, and by the
cell membrane–permeable form of cAMP, 8-bromo cAMP. Fig 4 shows the effects of forskolin on IKur. In
the example shown, a prepulse was used (as described in "Materials
and Methods") to inactivate Ito and isolate
IKur. The addition of 3 µmol/L forskolin to the
superfusate for 10 minutes (Fig 4B) substantially increased
IKur relative to control conditions (Fig 4A). The
forskolin-induced difference current (Fig 4C; note change in
current scale) shows that the drug-induced current was time
dependent, with tail currents upon repolarization. Mean
current-voltage relations from five cells (Fig 4D) indicate that
forskolin increased IKur over the entire range of voltages,
with no statistically significant voltage-dependent action
(ANOVA).
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Fig 5 illustrates the effects of 8-bromo cAMP. Compared
with control recordings (Fig 5A), recordings obtained
10 minutes after the addition of 50 µmol/L 8-bromo cAMP (Fig 5B) show
an increase in IKur step and tail currents. The
drug-sensitive current (Fig 5C) was time dependent and showed rapid
activation. Mean data from five cells (Fig 5D) indicate that 8-bromo
cAMP increased IKur in a significant and
voltage-independent fashion that was qualitatively similar to the
actions of isoproterenol and forskolin illustrated in Figs 1, 2, and 4.
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Potential Role of PKA in Mediating Isoproterenol
Effects
To examine whether PKA may be involved in
isoproterenol-induced increases in IKur, we
applied isoproterenol in the presence of PKA inhibitors.
The specific PKA inhibitor peptide, PKI, was included in
the pipette solution at a concentration of 50 nmol/L (20 times the
Kd for PKA, 2.3 nmol/L),27 and
isoproterenol was then added. Panels A through C of Fig 6 show representative tracings from a
typical experiment. After 20 minutes of dialysis with the
PKI-containing pipette solution, the amplitude of IKur is
unaffected (Fig 6B) compared with the control condition (Fig 6A). In
the presence of PKI, isoproterenol (1 µmol/L) fails to alter
IKur (Fig 6C). Mean data from five cells (Fig 6D) indicate
that 20 minutes of dialysis with PKI-containing pipette solution did
not alter IKur and that in the presence of PKI,
isoproterenol had no effect on the current. As a positive control, we
evaluated the effect of PKI on isoproterenol-induced increases in
ICa. The latter was elicited by 250-ms depolarizing pulses
from -50 to +10 mV in five cells studied without PKI in the
pipette and in five studied with PKI in the pipette. Cells from
individual preparations were studied in a paired fashion. In the
absence of PKI, isoproterenol increased ICa from
-5.1±1.5 to -11.5±2.1 pA/pF (P<.01). When PKI
was included in the pipette, ICa averaged -5.5±1.4
pA/pF before and -5.4±1.3 pA/pF after isoproterenol
administration.
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Although PKI is an effective and specific inhibitor of
PKA,27 it has to be included in the pipette solution,
making it difficult to compare isoproterenol effects before and after
the inhibition of PKA. Therefore, we performed additional experiments
with the protein kinase inhibitor H-7. Although H-7
inhibits both PKA and PKC, it is a more potent inhibitor of
PKA.28 IKur was first studied in the absence
of isoproterenol and then after 10 minutes of superfusion with 1
µmol/L isoproterenol. Isoproterenol was then washed out, and in cells
used for studying H-7, the latter was added to the
superfusate at a concentration of 5 µmol/L. Finally, the
cell was once more exposed for 10 minutes to 1 µmol/L isoproterenol,
in the presence or absence of H-7. Repeated exposure to isoproterenol
in the absence of H-7 produced very consistent effects in six
cells (Fig 7A). In contrast, when the second exposure to
isoproterenol occurred in the presence of H-7, no effect was seen in
five cells (Fig 7B).
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The above experiments indicate that ß-adrenergic stimulation
increases IKur in a way that is reversible upon washout,
can be inhibited by propranolol, can be mimicked by
interventions that increase intracellular cAMP activity independently
of the ß-adrenergic receptor, and can be blocked by
inhibitors of PKA. We then turned our attention to
potential effects of -adrenergic receptor stimulation on
IKur.
Effects of Phenylephrine on
IKur
To study -adrenergic modulation of
IKur, we used the -adrenergic selective
agonist phenylephrine, administered along with
propranolol (1 µmol/L) to prevent actions due to
collateral ß-adrenergic receptor stimulation. Fig 8 shows representative
recordings in the absence (Fig 8A) and presence (Fig 8B) of
phenylephrine in a human atrial cell lacking
Ito. Phenylephrine reduced the amplitude of
IKur, with a drug-sensitive difference current
(Fig 8C) that was time dependent, showed rapid activation, and had
small tail currents. Mean data for the effects of 100 µmol/L
phenylephrine in seven cells are shown in Fig 8D. The drug
reduced IKur amplitude at all activation voltages, in a
voltage-independent fashion. An analysis of the
concentration dependence of phenylephrine action on
IKur in six cells is shown in Fig 8E. Small but
statistically significant effects were noted at concentrations as low
as 10 µmol/L, and drug actions continued to increase at
concentrations up to 500 µmol/L. Thus, phenylephrine
inhibits IKur in a concentration-dependent and
voltage-independent fashion.
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Phenylephrine did not alter the voltage dependence of
IKur in five cells (Fig 9A). The time
dependence of IKur activation was analyzed by
fitting activation upon depolarization from -50 mV by a
single-exponential function. Exposure to 100 µmol/L
phenylephrine failed to alter the activation kinetics of
IKur in six cells (Fig 9B).
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) for IKur (mean±SEM, n=6),
obtained as described in Fig 2D
To confirm that phenylephrine's effect is mediated
by -adrenergic receptor stimulation, we studied the ability of
the 1-selective antagonist
prazosin29 to reverse phenylephrine's
actions. Fig 10 shows results from a
representative cell. Ten minutes of exposure to
phenylephrine (100 µmol/L, Fig 10B) substantially
inhibited IKur relative to predrug control (Fig 10A). The
subsequent addition of prazosin (1 µmol/L) reversed the
phenylephrine-induced inhibition (Fig 10C), with
current in the presence of prazosin and phenylephrine not
significantly different from control values obtained before the
addition of phenylephrine. Similar results were obtained in
all five cells studied. In these cells, mean IKur current
density was significantly reduced by phenylephrine (Fig 10D), an effect significantly inhibited by prazosin.
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Effects of a PKC Inhibitor on the Response to
Phenylephrine
To assess the potential role of PKC in the
1-adrenergic receptor–mediated decrease in
IKur, we determined the change in
phenylephrine action produced by the highly selective PKC
inhibitor bisindolylmaleimide.30 Cells were
first exposed to phenylephrine alone to quantify the
response in the absence of a PKC inhibitor and then
reexposed to phenylephrine in the presence of 50 nmol/L
bisindolylmaleimide. Fig 11 shows recordings
obtained under each condition in a representative cell.
Control currents obtained upon depolarization for 200 ms to a variety
of test potentials are shown in Fig 11A. After 10 minutes of exposure
to 100 µmol/L phenylephrine, IKur was
inhibited (Fig 11B). Phenylephrine-sensitive currents
(Fig 11C) show the typical properties of IKur. After 20
minutes of phenylephrine washout and 10 minutes of
superfusion with bisindolylmaleimide (Fig 11D), currents returned to
control values. The addition of 100 µmol/L phenylephrine
for 10 minutes in the presence of bisindolylmaleimide (Fig 11E) failed
to alter IKur, as illustrated by the difference
currents (Fig 11F) between results obtained in the presence of
bisindolylmaleimide alone and those in the presence of
phenylephrine and bisindolylmaleimide.
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Fig 12 shows mean data from cells studied with the
protocol illustrated in Fig 11, with the second
phenylephrine exposure occurring in the absence (Fig 12A,
five cells) or presence (Fig 12B, six cells) of bisindolylmaleimide.
Although repeated exposures to 100 µmol/L phenylephrine
in the absence of the protein kinase inhibitor (Fig 12A)
produced consistent inhibitory effects, when the
second exposure to phenylephrine occurred in the presence
of bisindolylmaleimide, no significant phenylephrine effect
was seen (Fig 12B).
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Experiments With the Perforated-Patch Technique
A response of IKur to adrenergic stimulation was seen
in cells from 29 (67%) of 43 hearts. When a response was seen in a
given heart, it was consistently detectable in cells from that
preparation. No patient features (age, drug therapy, previous
infarction) predicted responsiveness. These findings suggest that cell
isolation and/or dialysis with pipette solution may have limited the
response of IKur to adrenergic stimulation. Because of the
possibility that our results could have been affected by dialysis of
cell contents, additional experiments were performed with the nystatin
perforated-patch technique. Access to the cell interior was
obtained with the use of nystatin (300 µg/mL) in the patch pipette,
resulting in a mean Rs after compensation of 5.9±1.2 M
in nine cells. IKur was measured upon depolarization from
-50 to +40 mV, before and after 1 µmol/L isoproterenol
(five cells) or before and after 100 µmol/L phenylephrine
(four cells). Mean current density was increased from 8.4±2.4 to
11.2±3.2 pA/pF by isoproterenol (P<.01), a mean increase
of 37±4%. Phenylephrine decreased IKur
density from 11.7±2.7 to 8.6±2.0 pA/pF (P<.01), a
decrease of 26±4%. These changes were similar to those observed with
tight-seal patch clamp, as described above.
Specificity of IKur Modulation
The kinetic properties of isoproterenol- and
phenylephrine-sensitive current strongly resemble those
of IKur. To determine more precisely whether the adrenergic
effects observed in the present study are mediated by changes in
IKur, we exploited the strong selectivity of 4-AP as
an IKur blocker.16 IKur was first
recorded under control conditions, and then 200 µmol/L 4-AP (a
concentration that reduces IKur by >90%16 )
was added to the superfusate, and the current was
recorded again. 4-AP was then washed out until the current
amplitude returned to control values, and isoproterenol (1 µmol/L) or
phenylephrine (100 µmol/L) was added in the absence of
4-AP. The latter was then added at a concentration of 200 µmol/L
during continued superfusion with isoproterenol or
phenylephrine. Typical results are shown in Fig 13. Under control conditions (Fig 13A), 200 µmol/L
4-AP virtually eliminated time-dependent step and tail currents. In
the presence of isoproterenol, step and tail currents were increased,
but the addition of 4-AP once again eliminated both time-dependent
components. 4-AP–sensitive currents were obtained by digital
subtraction of currents in the presence of 4-AP and those recorded
in its absence. As shown in Fig 13C, isoproterenol strongly increased
the 4-AP–sensitive component. In three cells studied in this fashion,
isoproterenol increased the 4-AP–sensitive step current at +20 mV from
237±47 to 345±65 pA (P<.05). The 4-AP–resistant
current was 153±12 and 193±7 pA before and after isoproterenol,
respectively (P=NS). A corresponding experiment with
phenylephrine is shown in Fig 13D through 13F. Once again,
4-AP virtually eliminated time-dependent current under control
conditions (Fig 13D). Phenylephrine inhibited the
time-dependent component, while not altering current recorded
in the presence of 4-AP (Fig 13E). Phenylephrine inhibition
of the 4-AP–sensitive component is clear in Fig 13F. In three cells,
phenylephrine reduced the 4-AP–sensitive step current at
+20 mV from 238±33 to 168±32 pA (P<.05), without altering
the 4-AP–resistant component (131±21 pA before and 155±24 pA
after phenylephrine, P=NS). These experiments
indicate that the adrenergic modulation studied in the present work
was quite specific for the 4-AP–sensitive component,
IKur.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have demonstrated that ß- and
1-adrenergic receptor stimulation modulates
IKur in human atrial myocytes. The effects of
ß-adrenergic receptor stimulation can be mimicked by
interventions that increase intracellular cAMP activity, and the
actions of either receptor system can be prevented by inhibiting
the appropriate protein kinase (cAMP-dependent PKA for the
ß-adrenergic receptor and PKC for the -adrenergic
receptor).
Comparison With Previous Studies of Adrenergic Actions on Other
K+ Channels
Adrenergic modulation is well known to modulate the properties of
a variety of K+ channels. ß-Adrenergic stimulation
increases the magnitude of classic
IK.9 10 31 32 33 Differing results have been
presented with respect to changes in the voltage dependence of
IK; some studies have found that ß-adrenergic
stimulation has no effect on the voltage dependence of
IK,31 32 whereas others have reported a
hyperpolarizing shift of the activation curve.9 We found
that isoproterenol enhances the magnitude of IKur in a
voltage-independent fashion, while causing a slight (and
statistically nonsignificant) negative voltage shift in the activation
curve.
-Adrenergic stimulation, which we found to inhibit
IKur, has been found to inhibit several other
K+ currents, including
Ito,8 9 34
IK1,35 and
IKACh.36 37 Fedida et al34 found
that 100 µmol/L methoxamine reduced Ito in rabbit
atrial myocytes by 30%, a change of the same order as the effect
that we noted for 100 µmol/L phenylephrine and
IKur. The same group observed a lack of effect of
-adrenergic stimulation on the voltage dependence of current
activation, similar to our findings with IKur. Ravens et
al38 studied the actions of -adrenergic receptor
stimulation on Ito of rat ventricular myocytes
and found that the sustained component of the current is
inhibited by -adrenergic stimulation. There is evidence that the
sustained current in rat ventricular myocytes is carried by
a delayed rectifier current with activation kinetics similar to
IKur,39 so the observations of Ravens
et al38 may be analogous to our findings in the
present study. Preliminary findings of
phenylephrine-induced inhibition of human
IKur have also been presented by Van Wagoner and
Lamorgese.40 At least one K+ current, the
classic delayed rectifier in guinea pig ventricle, can be enhanced by
-adrenergic stimulation,41 indicating the
potential diversity of -adrenergic effects on
K+ currents.
Signal Transduction of Adrenergic Actions
Protein kinases are potentially important components of the signal
transduction system for adrenergic stimulation. ß-Adrenoceptor
stimulation is well known to activate PKA by elevating
intracellular concentrations of cAMP,12 whereas
-adrenergic receptor stimulation activates
PKC.12 25 On the other hand, there is evidence that both
ß-adrenergic stimulation42 and -adrenergic
stimulation43 can produce
physiological actions that are independent of PKA
and PKC, respectively. In the present study, we provide evidence
for a central role of cAMP-dependent PKA in the mediation of
ß-adrenergic effects on IKur and for a role of PKC in
mediating -adrenergic inhibition of the same current.
Phosphorylation by PKA and PKC produces similar effects
on some ion channels. For example, IK in guinea pig
ventricular myocytes is enhanced by activation of PKA and
PKC,44 as are Cl- currents in guinea
pig13 14 45 46 and feline47
ventricular myocytes. There is evidence that PKA and PKC
may act on the same Cl- channel in the latter
systems,46 47 although this issue is not yet fully
resolved. On the other hand, -adrenergic stimulation acts via
PKC to inhibit ICl.swell,48 an action
opposite to the reported ability of ß-adrenergic stimulation to
augment ICl.swell.49 In the present study,
we found that ß-adrenergic stimulation and -adrenergic
stimulation had opposite effects on IKur. In view of the
ability of inhibition of PKA and PKC to prevent the actions of
isoproterenol and phenylephrine, respectively, our results
suggest that adrenergically induced phosphorylation by
PKA and PKC has opposite effects on IKur.
Novel Aspects and Potential Importance
IKur is a recently described K+ current
that appears to play an important role in human atrial
repolarization.16 An understanding of its
physiological and pharmacological regulation is
important in order to improve our appreciation for the factors that can
alter human atrial repolarization and thereby govern the occurrence of
atrial arrhythmias in humans. Such arrhythmias,
particularly atrial fibrillation, are the most common form of
arrhythmia requiring therapy50 and currently
present important therapeutic problems.51 The
autonomic nervous system plays a potentially important role in
determining the occurrence of atrial arrhythmias.2
The present article is the first, to our knowledge, to report the
adrenergic regulation of IKur. We found that ß- and
-adrenergic receptor stimulation have significant and
opposite effects on IKur. Since IKur is a
current that activates rapidly, shows little inactivation, and
is of substantial amplitude, adrenergically mediated changes in
IKur could produce significant alterations in human atrial
repolarization that could importantly affect the likelihood of
reentrant atrial arrhythmias, like atrial fibrillation.
Several lines of evidence point toward the possibility that IKur is the physiological counterpart of Kv1.5 channels encoded by cDNA cloned from the human heart.16 24 Since the deduced amino acid sequence of human Kv1.5 channels22 24 has several consensus cites for PKA and PKC, our results may bear on the potential physiological importance of these sites in the regulation of native ion channel function. It would therefore be very interesting to know the effects of PKA- and PKC-mediated phosphorylation on the function of Kv1.5 channels expressed in model systems. Recent studies have shown that PKC-mediated phosphorylation inhibits currents carried by a transient outward channel clone from human hearts (Kv1.4).52 ß-Adrenergic stimulation and PKA-mediated phosphorylation have been shown to increase K+ current through a rat atrial K+ channel clone closely related to human Kv1.5 channels.53
Potential Limitations
The activation of IKur is so rapid at normal body
temperatures that its time dependence is very difficult, if not
impossible, to resolve.16 To resolve the
time-dependent activation of IKur, it is
necessary to study the current at lower temperatures. Therefore, we
conducted all of the present experiments at room temperature. This
allowed us to determine that isoproterenol- and
phenylephrine-sensitive currents have the kinetic
properties of IKur and to exclude changes in
time-independent currents. On the other hand, adrenergic changes in
some other currents, such as classic IK, are reduced
at lower temperatures.44 Therefore, we may have
underestimated the extent of adrenergic effects on IKur
that can occur under physiological conditions.
The functional importance of adrenergic modulation of IKur
is difficult to establish with certainty, since a variety of currents
that flow during the plateau are affected by adrenergic
stimulation.6 7 8 9 10 11 12 13 14 15 However, IKur appears to be
an important repolarizing current, and 50% inhibition of
IKur prolongs atrial action potential duration by a mean of
60%.16 Isoproterenol increased IKur by means
of up to 45% (see Fig 7), whereas maximal inhibition by
phenylephrine was in the range of 30% (Fig 8E), changes of
the order previously shown to influence repolarization.16
Although ß-adrenergic stimulation can produce larger percentage
changes in ICa and IK, these currents
show rapid inactivation and slow activation, respectively, which may
limit the absolute magnitude of ß-adrenergic change in
repolarizing current that they mediate. Other repolarizing currents,
such as Ito, are inhibited by -adrenergic
stimulation.7 8 Clearly, the role of various ionic
currents in mediating changes in repolarization caused by adrenergic
influences remains an important and unresolved issue.
One of the difficulties in studying protein kinase mediation of drug actions is the limited availability of highly specific inhibitor compounds that can be used as probes. Bisindolylmaleimide is a highly selective inhibitor of PKC.30 Although the protein kinase inhibitory peptide that we used is a specific inhibitor of PKA,27 the fact that it has to be applied by intracellular dialysis makes it very difficult to ascertain the response to isoproterenol superfusion before and after PKA inhibition. Therefore, we performed complementary experiments in which the response to isoproterenol was assessed before and after exposure to H-7. The latter compound inhibits both PKA and PKC, with an IC50 for PKA (3 µmol/L) that is about half the IC50 for PKC (6 µmol/L).28 At a concentration that was almost double the IC50 for PKA and below the IC50 for PKC, H-7 completely prevented the response to 1 µmol/L isoproterenol in cells that had demonstrated a mean 45% increase in response to the drug before H-7 infusion. PKC inhibition is unlikely to have participated in the inhibition of isoproterenol action by H-7 because (1) PKI, a specific PKA inhibitor, also prevented the effect of isoproterenol on IKur; (2) the concentration of H-7 used is below the IC50 for PKC; (3) the inhibition of basal PKC activity by bisindolylmaleimide did not alter IKur; (4) ß-adrenergic stimulation is not known to enhance PKC activity; and (5) if PKC activity were enhanced by isoproterenol, an inhibitory effect on IKur would have been expected, like that of phenylephrine, rather than the stimulatory effect seen. We attempted to perform similar experiments with H-89, a more selective membrane-permeable PKA inhibitor, but found that the drug has direct effects on ionic currents (including IKur and Ito) that preclude its use as a probe for PKA-mediated changes in IKur. The results obtained with H-7, along with those obtained with the peptide PKA inhibitor, point to a central role for PKA as a mediator of ß-adrenergic actions on IKur.
Forskolin has been shown to have direct open-channel blocking
effects on voltage-dependent K+ channels in PC12
cells.54 IC50 in that study was in the range
of 30 µmol/L, 10 times the concentration we studied in the
present experiments. Any direct effect of forskolin on
IKur mediated by this type of action would be expected to
be small at the concentrations we used and to offset (rather than
contribute to) the stimulatory effects we observed (Fig 4).
Cell isolation methods can affect the expression of ionic currents, particularly IKr and IKs, which are much more easily detected when arterial perfusion is used for delivery of cell-isolation enzymes.55 In the present study, cells were isolated by the "chunk" method, which along with the study temperature (25°C) probably explains the absence of IKr or IKs in our recordings. This limitation prevented any direct comparison between adrenergic effects on IKur and those on IKr or IKs in the present experiments.
Conclusions
We have demonstrated that IKur in human atrial
myocytes is subject to regulation by both ß- and -adrenergic
receptor stimulation, which have opposite actions on current magnitude.
ß-Adrenergic effects are mediated by cAMP-dependent protein kinase,
whereas -adrenergic effects appear to be mediated by PKC. These
results indicate a potentially novel mechanism for sympathetic nervous
system control of human atrial repolarization, adrenergic modulation of
IKur.
|
Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
This study was supported by grants from the Medical Research Council of Canada, the Quebec Heart Foundation, and the Fonds de Recherche de l'Institut de Cardiologie de Montréal, Montreal, Quebec, Canada. Dr Li is a research scholar of the Fonds de la recherche en santé du Québec. The authors thank Johanne Doucet and Guylaine Nicol for technical support and Carolyn Gillis and Luce Bégin for secretarial assistance.
|
Footnotes |
|---|
Previously published as preliminary results in abstract form (Circulation. 1994;90[pt 2]:I-526).
Received April 19, 1995; accepted January 25, 1996.
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References |
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