© 1996 American Heart Association, Inc.
Articles |
1-Adrenergic Inhibition of the ß-Adrenergically Activated Cl- Current in Guinea Pig Ventricular Myocytes
From the Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio.
Correspondence to Robert D. Harvey, Department of Physiology and Biophysics, Case Western Reserve University, 2109 Adelbert Rd, Cleveland, OH 44106-4970. E-mail rdh3@po.cwru.edu.
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Abstract
-Adrenergic receptor stimulation regulates the
activity of a number of different cardiac ion channels, including those
underlying one or more distinct Cl- conductances. The
whole-cell patch-clamp technique was used in the present
study to investigate the effects of -adrenergic stimulation on
the ß-adrenergically regulated Cl- current in
guinea pig ventricular myocytes. Neither
1-adrenergic receptor stimulation with
methoxamine (25 to 500 µmol/L) nor direct activation of
endogenous protein kinase C (PKC) with phorbol
12,13-dibutyrate (PDBu, 100 nmol/L) evoked a Cl-
current. On the contrary, the Cl- current
activated by 30 nmol/L isoproterenol was inhibited by
methoxamine, with an EC50 of 6.7±2.6 µmol/L, and
this response was blocked by prazosin, an 1-adrenergic
receptor antagonist. Prazosin also decreased the
EC50 for current activation by norepinephrine
from 53±7.1 to 18±3.8 nmol/L, demonstrating that the ability of this
endogenous neurotransmitter to activate the
Cl- current through ß-adrenergic receptor
stimulation is limited by its intrinsic ability to also
activate -adrenergic receptors. Methoxamine did
not inhibit the Cl- current evoked by either direct
activation of adenylate cyclase with forskolin or
inhibition of phosphodiesterase activity with
3-isobutyl-1-methylxanthine, indicating that -adrenergic
stimulation inhibits ß-adrenergic responses at a point upstream
of adenylate cyclase activation. Methoxamine also
did not inhibit the Cl- current activated by
histamine, suggesting that -adrenergic stimulation specifically
inhibits ß-adrenergic receptor–mediated responses. The
inhibitory effect of methoxamine was not mimicked
by PDBu, and it persisted in the presence of bisindolylmaleimide, a
selective PKC inhibitor. However, methoxamine
inhibition of the isoproterenol-activated
Cl- current was sensitive to pertussis toxin. These
results suggest that -adrenergic receptor stimulation inhibits
the ß-adrenergically activated Cl-
current, demonstrating a novel mechanism by which -adrenergic
receptors may regulate ion channel activity in the heart.
Key Words: phorbol 12,13-dibutyrate • acetylcholine • protein kinase C • isoproterenol • methoxamine
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Introduction |
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Stimulation of
-adrenergic receptors has been reported to regulate the activity
of a number of different cardiac ion channels,1 2
including those underlying one or more distinct Cl-
conductances. In rabbit atrial myocytes, -adrenergic stimulation
inhibits a swelling-induced Cl-
current.3 However, in guinea pig ventricular
myocytes, it has been reported that -adrenergic stimulation
actually activates a Cl-
conductance.4 Although the identity of the channel
responsible for the Cl- current activated by
-adrenergic stimulation has not been determined, an
alternatively spliced isoform of CFTR has been clearly demonstrated to
conduct a Cl- current that is activated by
ß-adrenergic stimulation acting through a PKA-dependent
mechanism.5 6 7 The question that then arises is whether
-adrenergic stimulation can activate the CFTR
Cl- current in cardiac myocytes. Unfortunately, very
little work has been conducted to determine exactly what effect, if
any, -adrenergic stimulation has on this current. One might
predict that -adrenergic stimulation would have a stimulatory
effect on the cardiac CFTR Cl- current, since it is
known to stimulate PKC activity in cardiac muscle,1 2 and
PKC activation has been reported to have a stimulatory effect on both
cardiac and epithelial CFTR Cl-
channels.8 9 10 11 Furthermore, phorbol esters have been
reported to activate a macroscopic Cl-
current in guinea pig and cat ventricular myocytes through
a PKC-dependent mechanism.4 12 13 However, despite the
observation that these whole-cell currents exhibit some properties
that are similar to the CFTR Cl- current, it has not
been determined whether PKC-dependent activation of CFTR
Cl- channels evokes a measurable macroscopic
Cl- current in cardiac myocytes.
In the present study, the whole-cell patch-clamp technique
was used to examine the effect of -adrenergic receptor
stimulation on the cardiac CFTR Cl- current in guinea
pig ventricular myocytes. Contrary to previous reports, it
is found that -adrenergic stimulation does not activate
any Cl- current in these cells. In fact,
-adrenergic stimulation actually inhibits the
ß-adrenergically activated cardiac CFTR
Cl- current. These data further demonstrate a
potentially important role for -adrenergic receptor stimulation
in the sympathetic regulation of cardiac ion channels.
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Materials and Methods |
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Cell Isolation
Cells were isolated using a previously described method.14 Briefly, hearts excised from anesthetized adult Hartley guinea pigs of either sex were subject to coronary perfusion via the aorta with KHB containing (mmol/L) NaCl 120, KCl 4.8, CaCl2 1.5, MgSO4 2.2, NaH2PO4 1.2, NaHCO3 25, and glucose 11. The buffer's pH was maintained at 7.35 by bubbling with 95% O2/5% CO2 at 37°C. Immediately after removal, the heart was perfused with normal Ca2+-containing KHB for 5 minutes, followed by Ca2+-free KHB for an additional 5 minutes. The heart was then perfused with Ca2+-free KHB containing collagenase B (0.5 to 0.7 mg/mL, Boehringer Mannheim) for 45 minutes. After this perfusion, the right ventricle was removed, minced, rinsed free of collagenase, and reintroduced to Ca2+-containing KHB. Gentle trituration freed individual cells for use in patch-clamp experiments.
Data Acquisition and Analysis
Membrane currents were recorded using the whole-cell
configuration of the patch-clamp technique.15
Microelectrodes were pulled from borosilicate glass capillary tubing
(Corning 7052, Garner Glass) and had resistances between 0.5 and 1.5
M
. The small size of the currents measured did not necessitate
series resistance compensation. Currents were recorded using an
Axopatch 200 voltage-clamp amplifier (Axon Instruments) and an
IBM-compatible computer with a TL-1-125 interface and pCLAMP software
(Axon Instruments).
Changes in the Cl- conductance were monitored by applying 100-millisecond voltage steps to +50 mV once every 3 seconds. I-V relationships were measured by applying 100-millisecond voltage steps to test potentials from -120 to +50 mV. The Cl- current was defined as the difference current obtained by subtracting currents recorded in the absence from those recorded in the presence of drug(s). Current magnitude was determined by calculating the average current during a 15-millisecond span at the end of each 100-millisecond voltage step. Slope conductances were calculated by linear regression of the I-V relationship positive to the reversal potential. For dose-response relationships, data were fitted with weighting (1/variance) to a logistic equation using a nonlinear least-squares curve-fitting routine (SIGMAPLOT, Jandel Scientific Software). Changes in the delayed rectifier K+ current were monitored by applying 3-second voltage steps to +50 mV once every 20 seconds. The K+ current was defined as the time-dependent current elicited during the step to +50 mV.
Reversal of inhibitory responses after drug washout was
used to control for the possible contribution of rundown to the
apparent inhibitory effects of methoxamine.
Although currents typically returned to 
75% of their initial
magnitude, rundown could have caused a slight overestimation of
methoxamine's potency. Results are reported as mean±SE.
Statistical comparison of responses between groups of cells was
conducted by one-way ANOVA and the Bonferroni t test
(SIGMASTAT, Jandel Scientific Software).
Solutions
When Cl- current was studied, cells were
bathed in an external solution containing (mmol/L) NaCl 140, CsCl 5.4,
CaCl2 2.5, MgCl2 0.5, HEPES 5.5, and glucose
11; the pH was adjusted to 7.4 with NaOH. Unless otherwise noted, cells
were dialyzed with an internal solution containing (mmol/L) glutamic
acid 130, HEPES 10, EGTA 10, CaCl2 1,
tetraethylammonium chloride 20, MgATP 5,
and Tris-GTP 0.1; the pH was adjusted to 7.05 with CsOH. When these
solutions were used, the Iso-activated
Cl- current reversed at -45±0.79 mV (n=10),
which is near the predicted Cl- equilibrium potential
of -50 mV. When studying the delayed rectifier K+
current, cells were bathed in an external solution containing (mmol/L)
NaCl 140, KCl 5.4, CaCl2 2.5, MgCl2 0.5, HEPES
5.5, and glucose 11; the pH was adjusted to 7.4 with NaOH. Cells were
dialyzed with an internal solution containing (mmol/L) potassium
glutamate 120, HEPES 10, EGTA 10, CaCl2 1, KCl 20, MgATP 5,
and Tris-GTP 0.1; the pH was adjusted to 7.05 with KOH. The composition
of these internal solutions was calculated to result in a free
Ca2+ concentration of 
10 nmol/L.16 L-type
Ca2+ current was blocked by adding 1 µmol/L nisoldipine
(a gift from Miles Laboratories) to all external solutions.
Na+ and T-type Ca2+ channels were
inactivated by using a holding potential of -30 mV.
Cells were placed in a 0.5-mL chamber on the stage of an inverted
microscope, with control external solution flowing at an approximate
rate of 1 mL/min. Temperature for all experiments was maintained at
36°C to 37°C using a servo-controlled system.17
Except when calculating the reversal potential of the
Iso-activated Cl- current, data were not
adjusted to account for any junction potential.
Once the whole-cell configuration had been achieved, cells were positioned in front of a fast-flow system that allowed the external solution bathing a cell to be changed in <1 second.18 Most drugs were prepared as stock solutions so that the desired final concentration was achieved by 1:1000 dilution with the appropriate external solution. ACh (Research Biochemicals International), histamine (Sigma Chemical Co), Iso (Research Biochemicals International), methoxamine hydrochloride (Research Biochemicals International), NE (Research Biochemicals International), PTX (List Biological Laboratories, Inc), and propranolol (Sigma) were prepared in distilled water. BIS (Calbiochem), IBMX (Calbiochem), PDBu (Research Biochemicals International), and prazosin hydrochloride (Research Biochemicals International) were initially prepared in dimethyl sulfoxide (Sigma) and further diluted in water or external solution.
In a few experiments, 100 to 500 µmol/L DIDS (Sigma) was added
directly to external solution to verify that it did not inhibit the
Iso-activated current. In experiments using prazosin, cells
were exposed to this 1-adrenergic receptor
antagonist for a period beginning at least 1 hour before
and continuing through completion of the patch-clamp experiments.
This ensured that prazosin binding had reached steady
state.19 In experiments using PDBu, bovine serum
albumin was added to the external solutions (0.1%) as a
carrier to ensure that the highly hydrophobic phorbol ester reached the
cells. In these experiments, albumin was also added to control
solutions to ensure that it was not the cause of any apparent response
to PDBu. Ascorbic acid (50 µmol/L) was added to all solutions
containing Iso or NE to prevent oxidative degradation.
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Results |
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1-Adrenergic Agonists Do Not Activate a
Cl- CurrentTo determine whether
1-adrenergic receptor
stimulation could activate the cardiac CFTR
Cl- current or any other Cl-
current, guinea pig ventricular myocytes were exposed to
various concentrations of methoxamine, a specific
1-adrenergic receptor agonist (Fig 1
).
There was no detectable effect on the Cl- conductance
of any cell exposed to either 25 µmol/L (n=9) or 500 µmol/L (n=8)
methoxamine. However, in each of these cells,
ß-adrenergic stimulation with 1 µmol/L Iso consistently
activated a time-independent current with properties
characteristic of the CFTR Cl- current found in
cardiac ventricular myocytes, including a reversal
potential near the equilibrium potential for Cl-,
outward rectification, and insensitivity to DIDS.6 7 14 20
This indicates that even though these Cl- channels
were present, they were unable to be activated by
1-adrenergic receptor stimulation. Similar results were
obtained when cells were exposed to 10 µmol/L NE, a combined - and
ß-adrenergic receptor agonist, in the presence of 1 to 10
µmol/L propranolol, a specific ß-adrenergic
receptor antagonist (n=3). These results suggest that
1-adrenergic receptor stimulation alone is not able to
activate a Cl- conductance in guinea pig
ventricular myocytes.
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I) obtained by subtracting currents
recorded under control conditions (a) from currents recorded in
the presence of 500 µmol/L methoxamine alone (b), in the
presence of 30 nmol/L Iso alone (c), and after Iso washout (d).
PKC Activation Does Not Induce a Cl-
Current
Since 1-adrenergic stimulation has been linked to
activation of PKC1 2 and phorbol esters have been reported
to activate a PKC-dependent Cl- current with
properties similar to the -adrenergically activated
Cl- current,4 we investigated the
ability of the phorbol ester PDBu to activate the cardiac CFTR
Cl- current. Consistent with the results
illustrated in Fig 1
, activation of PKC with PDBu did not induce a
Cl- current in any guinea pig ventricular
myocyte tested (n=10). In these experiments, each cell was exposed to
100 nmol/L PDBu for an average of 20 minutes. During this time, there
was no change in the background conductance of these cells. However, in
every instance, the cardiac CFTR Cl- current was
activated by subsequent exposure to 1 µmol/L Iso (Fig 2).
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To verify that exposure to phorbol ester resulted in activation of PKC,
we used the same protocol to monitor the effect of PDBu on the delayed
rectifier K+ current, which is well known to be increased
by activation of PKC.21 22 23 24 Unlike the lack of effect on
the Cl- channels in these cells, PDBu caused a
significant increase in the magnitude of this time-dependent
K+ current in five of six cells tested (Fig 3). These results suggest that the cardiac CFTR
Cl- current cannot be activated through a
PKC-dependent mechanism alone.
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1-Adrenergic Agonist Inhibits the ß-Adrenergically
Stimulated Cl- Current
Previous reports have indicated that PKC can stimulate CFTR
Cl- channel activity in epithelial cells but that the
magnitude of the response is small relative to the magnitude of the
response to activation by PKA.8 9 10 However, it has been
suggested that prior stimulation by PKC can augment the response of
PKA.9 10 Therefore, one might predict that a stimulatory
effect of -adrenergic stimulation could be seen more clearly as
a facilitation of the response to ß-adrenergic stimulation. To
test this possibility, we first exposed guinea pig
ventricular myocytes to 25 µmol/L methoxamine
followed by methoxamine plus 10 nmol/L Iso. This concentration
of Iso is near the EC50 for activation of the
Cl- current.18 Therefore, if
1-adrenergic stimulation does activate the
Cl- current by facilitating the response to
ß-adrenergic agonists, washing out methoxamine in the
continued presence of Iso might be expected to result in a decrease in
the magnitude of the Cl- current activated by
Iso. As before, exposure to methoxamine alone had no effect,
but subsequent exposure to Iso in the continued presence of
methoxamine activated a small Cl-
current. However, immediately after washout of methoxamine,
there was a significant increase, not a decrease, in the magnitude of
the Cl- current (Fig 4). The same
response was observed in six different cells.
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These results indicate that -adrenergic stimulation actually
inhibits ß-adrenergic activation of the current. Fig 5A demonstrates that this inhibition occurs in a
concentration-dependent manner. Fig 5B further illustrates the
concentration-dependent inhibition of Cl- current
activated by a maximally effective concentration of Iso. Cells
were first exposed to 30 nmol/L Iso, followed by Iso plus increasing
concentrations of methoxamine. The results indicate that
methoxamine can inhibit the Iso-activated current
in a concentration-dependent manner with an EC50 of
6.7±2.6 µmol/L.
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-Adrenergic Receptor–Mediated Inhibition
Next, we investigated whether the inhibitory response
to methoxamine was due to activation of
1-adrenergic receptors. To answer this question, we
compared the response of methoxamine on the
Iso-activated Cl- current in the absence
(Fig 6A) and presence (Fig 6B) of 1 µmol/L prazosin,
an antagonist of 1-adrenergic receptors. The
results demonstrate that in the absence of prazosin, 25 µmol/L
methoxamine rapidly inhibited the Cl- current
activated by 30 nmol/L Iso by 69±4.9% (n=17), and this effect
was readily and completely reversible. However, in cells exposed to
prazosin, methoxamine inhibited the current by only 29±3.2%
(n=15). Prazosin significantly attenuated the response to
methoxamine (P<.05), consistent with the
idea that methoxamine inhibition of the
Iso-activated current is mediated through activation of
1-adrenergic receptors.
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In the heart, many responses to 1-adrenergic receptor
stimulation are mediated by PTX-sensitive G proteins.1 2
To determine whether PTX-sensitive G proteins are involved in
methoxamine's effect, additional experiments were conducted
using myocytes incubated in external solution containing 2 µg/mL PTX
for a period of 2 to 5 hours. Since it is well documented that
muscarinic receptor stimulation inhibits the
ß-adrenergically activated Cl-
current via a PTX-sensitive G protein,25 26 only cells
that exhibited complete blockade of the response to 1 µmol/L ACh were
included in the present study. Fig 6C shows that PTX treatment of
the cells abolished 1-adrenergic–induced inhibition
of the Iso-activated Cl- current. In the
PTX-treated cells, 25 µmol/L methoxamine resulted in only
5.2±1.7% (n=5) inhibition of the Cl- current
activated by 30 nmol/L Iso. PTX significantly attenuated the
response to methoxamine (P<.05), consistent
with the idea that methoxamine inhibition of the
Iso-activated Cl- current is coupled to a
PTX-sensitive G protein.
Specific Inhibition of ß-Adrenergic Responses
To determine whether methoxamine-induced inhibition of
the Cl- current might be due to a direct effect on
Cl- channel function or perhaps an indirect effect on
the second messenger pathway involved in Cl- channel
activation, we examined the response to methoxamine when the
Cl- current was activated independent of
ß-adrenergic receptor stimulation. We found that 100 µmol/L
methoxamine had no effect on the Cl- current
induced by direct activation of adenylate cyclase with 1
µmol/L forskolin. In the presence of forskolin plus
methoxamine, the Cl- conductance was
103±5.5% of that measured in the presence of forskolin alone (n=3).
Similar results were obtained when the Cl- current
was activated by inhibition of phosphodiesterase activity with
75 to 100 µmol/L IBMX. In the presence of IBMX plus 100 µmol/L
methoxamine, the Cl- conductance was
98.5±3.7% of that measured in the presence of IBMX alone (n=4). This
suggests that -adrenergic receptor stimulation does not inhibit
Cl- channels directly and that the
inhibitory effect occurs at a point before the activation
of adenylate cyclase.
We also found that 100 µmol/L methoxamine did not inhibit the
Cl- current activated by 450 nmol/L histamine
(Fig 7). In the presence of histamine plus
methoxamine, the Cl- conductance was
101±5.6% of that measured in the presence of histamine alone (n=7).
Other than acting through H2-histaminergic receptors,
histamine activates the Cl- current via the
same cAMP-dependent pathway used by ß-adrenergic agonists.
Therefore, these results suggest that -adrenergic inhibition of
the Iso-activated Cl- current may be due
to direct inhibition of the ß-adrenergic receptor.
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PKC-Independent Inhibition
As previously stated, 1-adrenergic receptor
stimulation is frequently associated with PKC
activation,1 2 and it has been demonstrated that
-adrenergic receptor stimulation inhibits a
swelling-activated Cl- current in rabbit
atrial cells3 through a PKC-dependent mechanism.
Therefore, we investigated the potential role of PKC in
methoxamine's inhibition of Iso-stimulated current. One
approach was to determine whether activation of PKC with PDBu could
mimic the effect of -adrenergic receptor stimulation. Fig 8A shows that 100 nmol/L PDBu did not mimic the effect
of methoxamine on the Iso-activated current. Even
after exposure to PDBu for up to 20 minutes, there was no inhibition of
the current. In fact, there appeared to be a slight increase in the
current. Similar results were obtained in eight separate experiments.
The Cl- conductance measured in the presence of 30
nmol/L Iso plus 100 nmol/L PDBu was 121±9.8% of that measured in the
presence of this maximally effective concentration of Iso alone (n=4).
The Cl- conductance measured in the presence of 10
nmol/L Iso plus 100 nmol/L PDBu was 129±7.3% of that measured in the
presence of this submaximally effective concentration of Iso alone
(n=4).
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A second approach used to investigate the potential role of PKC in
mediating the inhibitory response to
1-adrenergic stimulation was to determine whether the
response to methoxamine was inhibited by BIS, a highly specific
inhibitor of PKC.27 Fig 8B shows that 300
nmol/L BIS, a concentration 100-fold greater than its
Ki for inhibition of PKC, did not prevent
inhibition of the Iso-activated Cl-
current by 25 µmol/L methoxamine. Similar results were
obtained in a total of five separate experiments using 0.3 to 3
µmol/L BIS. Results from both types of experiment described in Fig 8
indicate that -adrenergic inhibition of the
ß-adrenergically activated Cl- current
does not involve PKC.
-Adrenergic Component of the NE Response
NE is the endogenous sympathetic agonist regulating
cardiac function, and despite its ability to act at both - and
ß-adrenergic receptors, its net effect on L-type Ca2+
channels, delayed rectifier K+ channels, and CFTR
Cl- channels is a potent stimulation.28
This is presumably mediated via ß-adrenergic receptor activation.
We addressed the question of whether activation of
1-adrenergic receptors significantly contributes to the
NE response by comparing the concentration dependence of the response
to NE in the presence and absence of prazosin. Under control
conditions, NE activated the Cl- current in a
concentration-dependent manner, with an EC50 of 53±7.1
nmol/L. However, in cells pretreated with 1 µmol/L prazosin, the
EC50 for Cl- current activation decreased
to 18±3.8 nmol/L (Fig 9). This indicates that the
ability of NE to activate the Cl- current
through ß-adrenergic receptor stimulation is significantly
(P<.001) limited by its intrinsic ability to also
activate -adrenergic receptors.
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Does
-Adrenergic Stimulation Activate
Cl- Channels?In the present study,
-adrenergic receptor stimulation
failed to activate a Cl- current in guinea
pig ventricular myocytes. This contrasts with the earlier
work of Walsh,4 in which NE (10 µmol/L), in the presence
of propranolol (1 µmol/L), appeared to activate a
current that reversed near the predicted Cl-
equilibrium potential. Although the identity of this current is not
clear, it is unlikely to be conducted by the same channels that are
activated by ß-adrenergic stimulation, since the
NE-induced current exhibited a linear I-V relationship under conditions
in which CFTR Cl- currents are known to be outwardly
rectifying.29 It is equally unclear why this current was
not activated by either methoxamine or NE plus
propranolol in our own experiments. Because it was
originally suggested that -adrenergic stimulation
activates this current through a PKC-dependent
mechanism,4 one possible explanation for the lack of a
stimulatory response to methoxamine in the present study
might be that PKC was not being effectively activated. It is
known that receptor activation of some isoforms of PKC is
Ca2+ dependent.30 However, in the present
experiments, cells were dialyzed with an internal solution that
buffered internal Ca2+ at a level that should have
permitted activation of PKC.22 Moreover, another test of
whether -adrenergic stimulation of PKC should induce a current
in these cells was to directly activate PKC with 100 nmol/L
PDBu. Although there was still no Cl- current
produced, we were able to demonstrate that endogenous PKC
was being activated under these conditions by showing that PDBu
did have a stimulatory effect on the delayed rectifier K+
current. This suggests that the lack of an -adrenergically
activated current is not likely to be explained by dialysis of
endogenous PKC from the cell into the patch pipette. The inability to activate a Cl- current with phorbol esters also appears to contrast with earlier studies, in which it was reported that PDBu could activate a macroscopic Cl- current in guinea pig ventricular myocytes.4 12 However, in those experiments in which a Cl- current was observed, it was necessary to dialyze the cells with exogenous PKC. Phorbol ester stimulation of endogenous PKC has been shown to stimulate single Cl- channel activity in the same type of cells,11 but whether such an effect could produce a measurable macroscopic current was not determined. Our results suggest that activation of endogenous PKC alone is unable to activate a macroscopic Cl- current in guinea pig ventricular myocytes. Stimulation of endogenous PKC activity has been reported to produce a measurable macroscopic Cl- current in cat ventricular myocytes,13 but the identity of the Cl- channel responsible has not been determined. Therefore, the ability of phorbol esters alone to activate a macroscopic Cl- current in cat ventricular myocytes, but not guinea pig ventricular myocytes, may reflect a species-dependent difference in the expression of PKC-sensitive Cl- channels.
1-Adrenergic Inhibition of Cardiac CFTR
Cl- Current
The present study demonstrates that methoxamine, an
1-adrenergic receptor agonist, inhibited the
Iso-activated Cl- current in a
concentration-dependent manner, with an EC50 of 6.7
µmol/L. Iso-activated Cl- current was
also inhibited by phenylephrine in a
concentration-dependent manner, with an EC50 of 13
µmol/L.31 Phenylephrine is an agonist at
1-adrenergic receptors, but it can also activate
ß-adrenergic receptors at high concentrations.28
That is the reason we used methoxamine, a specific
1-adrenergic receptor agonist, in the present study.
If methoxamine were acting as a partial agonist or
antagonist at ß-adrenergic receptors,32
we would not have been able to prevent its inhibitory
response with the -adrenergic receptor antagonist
prazosin.
Although the inhibitory effect of methoxamine was
significantly attenuated in the presence of prazosin, it was not
completely blocked. If it is assumed that methoxamine and
prazosin are acting competitively at a single type of receptor and that
prazosin binding to the receptor was given enough time to reach
equilibrium, the antagonist should cause a parallel shift
of the methoxamine concentration-response relationship to
the right. Extrapolating our results obtained with 25 µmol/L
methoxamine in the presence and absence of 1 µmol/L prazosin
would correlate with an increase of the EC50 for
methoxamine to 70 µmol/L. Based on the above assumptions,
Schild analysis predicts that the apparent affinity
(Kb) of prazosin for this receptor is 100
nmol/L. This is higher than the 1-nmol/L affinity
(Ki) of prazosin for cardiac
1-adrenergic receptors determined by competitive
radioligand binding studies.33 The discrepancy
could be explained if our assumptions about the conditions being at
equilibrium or prazosin acting as a competitive antagonist
were not true. Alternatively, the actual affinity of prazosin for the
1-receptor subtype involved in this response may be
lower than expected. Our calculated values are similar to those of Duan
et al,3 who demonstrated that 2 µmol/L prazosin shifted
the EC50 for 1-adrenergic inhibition of the
swelling-activated Cl- current by
phenylephrine in rabbit atrial cells from 61 to 635
µmol/L. This correlates with a Kb for prazosin
of 200 nmol/L.
Another possible explanation for the low apparent affinity for prazosin
could be that the response to methoxamine is mediated by
2-adrenergic receptors. In agreement with this
suggestion, prazosin can also act as an antagonist at
2B- and 2C-receptors, with a
Ki of 30 and 60 nmol/L,
respectively.34 Furthermore, methoxamine has been
reported to produce responses in rat kidney that are consistent
with activation of presynaptic
2-receptors.35 However, there is little if
any evidence for the existence of postsynaptic
2-receptors in cardiac muscle.1 In the
guinea pig heart specifically, prazosin binds with high affinity to a
single class of sites that have only an extremely low affinity for
yohimbine,36 an antagonist with high affinity
for all subtypes of 2-receptor.34 More
detailed pharmacological studies will be necessary to conclusively
demonstrate that methoxamine inhibition of ß-adrenergic
responses is due to the activation of 1-adrenergic
receptors and determine the specific subtype (1A or
1B) of receptor involved.
In addition to prazosin, PTX also affected the response to
methoxamine. There have been reports that PTX influences the
sensitivity to ß-adrenergic receptor stimulation. However, PTX
has been shown to block several responses mediated by
1-adrenergic receptor stimulation in cardiac myocytes,
including inhibition of the background K+ conductance and
activation of the Na+-K+ pump in guinea pig
ventricular cells37 as well as inhibition of
the swelling-activated Cl- current in
rabbit atrial myocytes.3 Although not all
1-adrenergic responses in cardiac muscle are PTX
sensitive, many that involve the 1B subtype are mediated
by an inhibitory G protein (Gi), which is PTX
sensitive.33 This suggests that the
methoxamine-induced inhibition of ß-adrenergic
responses may be mediated by this subtype of
1-adrenergic receptor.
1-Adrenergic stimulation has been reported to reduce
intracellular cAMP.38 Such an effect could explain the
response to methoxamine described in the present study. In
fact, 1-adrenergic stimulation would then be expected to
antagonize ß-adrenergic stimulation of any channel regulated by
cAMP, not just the Cl- current. Consistent
with this, the 1-adrenergic antagonist
prazosin has been reported to increase the magnitude of the L-type
Ca2+ current in response to NE39 in rat
ventricular myocytes. We have also observed that the
1-adrenergic agonist methoxamine can inhibit Iso
stimulation of the Ca2+ current as well as the delayed
rectifier K+ current in guinea pig ventricular
myocytes (L.M. Oleksa and R.D. Harvey, unpublished data, 1995). Buxton
and Brunton38 have suggested that
1-adrenergic stimulation decreases cAMP levels by
inhibiting phosphodiesterase activity. Although this is
consistent with our finding that methoxamine had no
effect on the Cl- current activated with
IBMX, such a mechanism does not explain why 1-adrenergic
stimulation did not inhibit the Cl- current
activated by forskolin or histamine. These observations do
agree with the work of Barrett et al,40 who suggested that
1-adrenergic receptor stimulation does not involve
inhibition of phosphodiesterase activity but, rather, that it inhibits
the synthesis of cAMP through a PTX-sensitive mechanism. Our results go
further to suggest that the effect is due specifically to inhibition of
the ß-adrenergic receptor, since methoxamine, at a
concentration that was high enough to completely inhibit the
Iso-activated Cl- current, had no effect
on the histamine-activated current.
Although specific inhibition of ß-adrenergic responses is a
novel effect of 1-adrenergic stimulation, it is not yet
clear whether this is an effect mediated via a PTX-sensitive G protein
or an effect on a PTX-sensitive signaling cascade. This question
remains to be answered. Many responses to 1-adrenergic
receptor stimulation are also linked to activation of PKC. This
includes 1-adrenergic inhibition of the
swelling-activated Cl-
current.3 However, methoxamine inhibited the
ß-adrenergically stimulated Cl- current in a
PKC-independent manner. This indicates that the effects of
1-adrenergic stimulation on the cardiac CFTR
Cl- current and the swelling-induced
Cl- current are mediated by distinctly different
mechanisms.
The physiological importance of the
inhibitory effect that -adrenergic stimulation has
on ß-adrenergic responses is clearly demonstrated by the
increased sensitivity to NE that is observed in the presence of
prazosin. It indicates that -adrenergic receptor stimulation
significantly limits the net ß-adrenergic response produced by
this endogenous sympathetic agonist. This may
represent a mechanism for fine tuning sympathetic responses. It
is also likely to be of pathological significance during
ischemia, when 1-adrenergic receptor levels are
significantly increased.41
In summary, the results of the present study show that (1) neither
1-adrenergic receptor stimulation nor direct activation
of PKC alone induces a macroscopic Cl- current in
guinea pig ventricular myocytes, (2)
1-adrenergic stimulation actually inhibits the
ß-adrenergically activated CFTR Cl-
current in these cells, and (3) the
1-adrenergic–induced inhibition is directed
specifically at the ß-adrenergic receptor through a
PKC-independent mechanism. These data describe a regulatory role for
1-adrenergic receptor stimulation and a novel mechanism
through which ß-adrenergically activated ion channels are
modulated in the heart.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant from the National Institutes of Health (HL-45141), an Established Investigatorship from the American Heart Association (Dr Harvey), and a postdoctoral fellowship from the Northeast Ohio Affiliate of the American Heart Association (Dr Hool). The authors would also like to thank C.M. Luca for conducting preliminary experiments related to this project.
Received October 10, 1995; accepted February 26, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Fedida D, Braun AP, Giles WR.
1-Adrenoceptors in myocardium: functional
aspects and transmembrane signaling mechanisms. Physiol
Rev. 1993;73:469-487.
2.
Terzic A, Pucéat M, Vassort G, Vogel SM.
Cardiac 1-adrenoceptors: an overview.
Pharmacol Rev. 1993;45:147-175. [Medline]
[Order article via Infotrieve]
3.
Duan D, Fermini B, Nattel S. -Adrenergic
control of volume-regulated Cl- currents in
rabbit atrial myocytes: characterization of a novel ionic regulatory
mechanism. Circ Res. 1995;77:379-393.
4. Walsh KB. Activation of a heart chloride current during stimulation of protein kinase C. Mol Pharmacol. 1991;40:342-346. [Abstract]
5.
Horowitz B, Tsung SS, Hart P, Levesque PC, Hume
JR. Alternative splicing of CFTR Cl- channels
in heart. Am J Physiol.. 1993;264:H2214-H2220.
6. Bahinski A, Nairn AC, Greengard P, Gadsby DC. Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes. Nature. 1989;340:718-721. [Medline] [Order article via Infotrieve]
7.
Harvey RD, Hume JR. Autonomic regulation of a
chloride current in heart. Science. 1989;244:983-985.
8.
Berger HA, Travis SM, Welsh MJ. Regulation of
the cystic fibrosis transmembrane conductance regulator
Cl- channel by specific protein kinases and protein
phosphatases. J Biol Chem. 1993;268:2037-2047.
9. Tabcharani JA, Chang X-B, Riordan JR, Hanrahan JW. Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene. Nature. 1991;352:628-631.[Medline] [Order article via Infotrieve]
10.
Chang X-B, Tabcharani JA, Hou Y-X, Jensen TJ, Kartner
N, Alon N, Hanrahan JW, Riordan JR. Protein kinase A (PKA) still
activates CFTR chloride channel after mutagenesis of all 10 PKA
consensus phosphorylation sites. J
Biol Chem. 1993;268:11304-11311.
11.
Collier ML, Hume JR. Unitary chloride channels
activated by protein kinase C in guinea pig
ventricular myocytes. Circ
Res. 1995;76:317-324.
12.
Walsh KB, Long KJ. Properties of a protein
kinase C–activated chloride current in guinea pig
ventricular myocytes. Circ
Res. 1994;74:121-129.
13.
Zhang K, Barrington PL, Martin RL, Ten Eick RE.
Protein kinase–dependent Cl- currents in feline
ventricular myocytes. Circ
Res. 1994;75:133-143.
14.
Harvey RD, Clark CD, Hume JR. Chloride current
in mammalian cardiac myocytes: novel mechanism for autonomic regulation
of action potential duration and resting membrane potential.
J Gen Physiol. 1990;95:1077-1102.
15. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85-100.[Medline] [Order article via Infotrieve]
16. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 1988;157:378-416. [Medline] [Order article via Infotrieve]
17. Datyner NB, Gintant GA, Cohen IS. Versatile temperature controlled tissue bath for studies of isolated cells using an inverted microscope. Pflugers Arch. 1985;403:318-323. [Medline] [Order article via Infotrieve]
18.
Zakharov SI, Harvey RD. Altered
ß-adrenergic and muscarinic response of CFTR
Cl- current in dialyzed cardiac myocytes.
Am J Physiol. 1995;268:H1795-H1802.
19.
Rugevics C-U, Schumann HJ.
1-Adrenoceptors in guinea-pig heart ventricles:
[3H]prazosin and [125I]IBE 2254 are
bound
specifically to a receptor population with high affinity and low
density. Arch Int Pharmacodyn Ther. 1987;289:234-250. [Medline]
[Order article via Infotrieve]
20. Harvey RD. Effects of stilbenedisulfonic acid derivatives on the cAMP-regulated chloride current in cardiac myocytes. Pflugers Arch. 1993;422:436-442. [Medline] [Order article via Infotrieve]
21.
Walsh KB, Kass RS. Regulation of a heart
potassium channel by protein kinase A and C. Science. 1988;242:67-69.
22.
Yazawa K, Kameyama M. Mechanism of
receptor-mediated modulation of the delayed outward potassium
current in guinea-pig ventricular myocytes.
J Physiol (Lond). 1990;421:135-150.
23. Tohse N, Kameyama M, Sekiguchi K, Shearman MS, Kanno M. Protein kinase C activation enhances the delayed rectifier potassium current in guinea-pig heart cells. J Mol Cell Cardiol. 1990;22:725-734. [Medline] [Order article via Infotrieve]
24.
Tohse N, Nakaya H, Kanno M.
1-Adrenoceptor stimulation enhances the delayed
rectifier K+ current of guinea pig ventricular
cells through the activation of protein kinase C.
Circ Res. 1992;71:1441-1446.
25.
Harvey RD, Hume JR. Autonomic regulation of
delayed rectifier K+ current in mammalian heart involves G
proteins. Am J Physiol. 1989;257:H818-H823.
26.
Hwang T-C, Horie M, Nairn AC, Gadsby DC. Role of
GTP-binding proteins in the regulation of mammalian cardiac chloride
conductance. J Gen Physiol. 1992;99:465-489.
27.
Toullec D, Pianetti P, Coste H, Bellevergue P,
Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F,
Duhamel L, Charon D, Kirilovsky J. The bisindolylmaleimide GF
109203x is a potent and selective inhibitor of protein
kinase C. J Biol Chem. 1991;266:15771-15781.
28. Hartzell HC. Regulation of cardiac ion channels by catecholamines, acetylcholine and second messenger systems. Prog Biophys Mol Biol. 1988;52:165-247. [Medline] [Order article via Infotrieve]
29.
Overholt JL, Hobert ME, Harvey RD. On the
mechanism of rectification of the isoproterenol-activated
chloride current in guinea-pig ventricular
myocytes. J Gen Physiol. 1993;102:871-895.
30. Huang K-P. The mechanism of protein kinase C activation. Trends Neurosci. 1989;12:425-432. [Medline] [Order article via Infotrieve]
31. Oleksa LM, Luca CM, Harvey RD. Alpha-adrenergic inhibition of the cardiac CFTR Cl- current. Circulation. 1995;92(suppl I):I-576. Abstract.
32. Hoffman BB, Lefkowitz RJ. Catecholamines, sympathomimetic drugs, and adrenergic receptor antagonists. In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Gilman AG, eds. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 9th ed. New York, NY: McGraw-Hill Publishing Co; 1995:199-248.
33. Hwa J, De Young MB, Perez DM, Graham RM. Autonomic control of myocardium: alpha-adrenoceptor mechanisms. In: Shepherd J, Vatner SF, eds. The Nervous Control of the Heart. Cambridge, Mass: Harvard Academic Press; 1996.
34. Bylund DB, Eikenberg DC, Hieble JP, Langer SZ, Lefkowitz RJ, Minneman KP, Molinoff PB, Ruffolo RR Jr, Trendelenburg U. International union of pharmacology nomenclature of adrenoceptors. Pharmacol Rev. 1994;46:121-136. [Medline] [Order article via Infotrieve]
35.
Bohmann C, Schollmeyer P, Rump LC.
Methoxamine inhibits noradrenaline release through
activation of 1- and 2-adrenoceptors in
rat isolated kidney: involvement of purines and
prostaglandins. Naunyn Schmiedebergs Arch
Pharmacol. 1993;347:273-279. [Medline]
[Order article via Infotrieve]
36. Karliner JS, Barnes P, Hamilton CA, Dollery CT. Alpha 1-adrenergic receptors in guinea pig myocardium: identification by binding of a new radioligand, (3H)-prazosin. Biochem Biophys Res Commun. 1979;90:142-149. [Medline] [Order article via Infotrieve]
37. Shah A, Cohen SI, Rosen MR. Stimulation of cardiac alpha receptors increases Na/K pump current and decreases GK via a pertussis toxin-sensitive pathway. Biophys J. 1988;54:219-225. [Medline] [Order article via Infotrieve]
38.
Buxton IL, Brunton LL. Action of the cardiac
alpha 1-adrenergic receptor: activation of cyclic AMP
degradation. J Biol Chem. 1985;260:6733-6737.
39.
Boutjdir M, Restivo M, Wei Y, El-Sherif N.
1- and ß-adrenergic interactions on L-type calcium
current in cardiac myocytes. Pflugers Arch. 1992;421:397-399. [Medline]
[Order article via Infotrieve]
40. Barrett S, Honbo N, Karliner JS. Alpha1-adrenoceptor-mediated inhibition of cellular cAMP accumulation in neonatal rat ventricular myocytes. Naunyn Schmiedebergs Arch Pharmacol. 1993;347:384-393. [Medline] [Order article via Infotrieve]
41. Kurz T, Yamada KA, DaTorre SD, Corr PB. Alpha 1-adrenergic system and arrhythmias in ischaemic heart disease. Eur Heart J. 1991;12(suppl F):88-98.
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