© 2000 American Heart Association, Inc.
Cellular Biology |
Overexpressed A1 Adenosine Receptors Reduce Activation of Acetylcholine-Sensitive K+ Current by Native Muscarinic M2 Receptors in Rat Atrial Myocytes
From Abteilung Zelluläre Physiologie, Ruhr-Universität Bochum, Bochum, Germany, and the Department of Molecular Pharmacology and Biological Chemistry (M.B.), Northwestern University Medical School, Chicago, Ill.
Correspondence to Dr Lutz Pott, Ruhr-Universität Bochum, Abteilung Zelluläre Physiologie, D-44780 Bochum, Germany. E-mail lutz.pott{at}ruhr-uni-bochum.de
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Abstract |
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Abstract—In adult rat atrial myocytes, muscarinic acetylcholine (ACh)-sensitive K+ current activated by a saturating concentration of adenosine (IK(ACh),(Ado)) via A1 receptors (A1Rs) amounts to only 30% of the current activated by a saturating concentration of ACh (IK(ACh),(ACh)) via muscarinic M2 receptors. The half-time of activation of IK(ACh),(Ado) on a rapid exposure to agonist was
4-fold longer than that of
IK(ACh),(ACh). Furthermore,
IK(ACh),(Ado) never showed fast
desensitization. To study the importance of receptor density for
A1R-IK(ACh),(Ado) signaling,
adult atrial myocytes in vitro were transfected with cDNA encoding for
rat brain A1R and enhanced green fluorescent
protein (EGFP) as a reporter. Whole-cell current was measured on days 3
and 4 after transfection. Time-matched cells transfected with only the
EGFP vector served as controls. In 30% of EGFP-positive cells
(group I), the density of IK(ACh),(Ado) was
increased by 72%, and its half-time of activation was reduced. Density
and kinetic properties of IK(ACh),(ACh) were
not affected in this fraction. In 70% of transfection-positive
myocytes (group II), the density of
IK(ACh),(ACh) was significantly reduced, its
activation was slowed, and the fast desensitizing component was lost.
Adenosine-induced currents were larger in group II than in
group I, their activation rate was further increased, and a fast
desensitizing component developed. These data indicate that in native
myocytes the amplitude and activation kinetics of
IK(ACh),(Ado) are limited by the expression
of A1R. Overexpression of A1R negatively
interferes with signal transduction via the muscarinic M2
receptor–linked pathway, which might reflect a competition of
receptors with a common pool of G proteins. Negative
interference of an overexpressed receptor with
physiological regulation of a target protein by a
different receptor should be considered in attempts to use receptor
overexpression for gene therapy.
Key Words: atrial myocytes • gene transfer • muscarinic receptors • adenosine receptors • K+ currents
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Myocytes of the atria and conductive tissue of the heart express a type of inwardly rectifying K+ channel that is composed in a heterotetrameric fashion of 2 different subunits (Kir3.1/Kir3.4, previously GIRK1/GIRK4).1 2 3 4 The classical pathway for its activation is initiated by binding of vagally released acetylcholine (ACh) to the muscarinic M2 receptor (M2R). Cardiac ACh-sensitive K+ (K(ACh)) channels represent the prototype of a G-protein–activated ion channel. Channels of this type with different subunit composition have been found more recently in neurons of various regions of the brain, where they are assumed to represent a major mechanism of synaptic inhibition,5 6 7 but also in other tissues or cell types.8 9 Kir3.x channels have in common that their opening probability is increased by direct interaction of their subunits with G-protein ß
subunits.10 In the heart,
apart from M2R, a number of different receptors,
including the adenosine (Ado) A1 receptor
(A1R) have been shown to converge via
Gi/Go on
K(ACh) channels.11 12 13 More
recently, coupling of native atrial K(ACh)
channels to Gsß has been
demonstrated.14 15 Regulation of various targets,
including K(ACh) channels, via
A1Rs is of particular interest in cardiac
physiology and pathophysiology because of its potential
clinical/therapeutic relevance. The A1R is being
discussed as a mediator of cardiac protection under various conditions,
such as heart failure16 and ischemia, which is
supported by the finding that overexpression by transfection in a cell
culture model17 or targeted transgenic overexpression in
mice increases myocardial resistance to
ischemia.18 This makes the
A1R a potential candidate for gene therapy. Atrial K(ACh) channels represent the most proximal target accessible to a direct measurement of a receptor–G protein pathway, providing a convenient and most sensitive online assay for receptor activation. In a number of studies, it has been demonstrated that in atrial myocytes whole-cell currents evoked by saturating concentrations of Ado are smaller than currents evoked by saturating concentrations of ACh and, on fast application of an agonist, are activated at a slower rate.19 20 21 This has been discussed to reflect the smaller number of A1Rs compared with M2Rs. So far, however, this has not been supported by experimental data, and it is conceivable that other factors are limiting the responsiveness of K(ACh) channels to A1R stimulation. In the present study, the expression level of A1Rs in adult rat atrial myocytes has been manipulated by transfection. The results to be presented confirm that the expression level of A1Rs represents a limiting factor for the activation of K(ACh) channels in native atrial myocytes. Surprisingly, in the majority of transfected myocytes, saturating ACh-evoked current was strongly reduced, pointing to a novel type of cross talk between 2 receptors converging on the same target.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation and Culture of Atrial Myocytes
Experiments were performed with the approval of the local ethics committee. Wistar-Kyoto rats of either sex (
200 g) were
anesthetized by intravenous injections of urethane
(1 g/kg). The chest was opened, and the heart was excised. A cannula
was inserted into the aorta and connected to a sterile device for
coronary perfusion at constant flow. Details of enzymatic
isolation of atrial myocytes and cell culture have been described
elsewhere.22
Solutions and Chemicals
For the patch-clamp measurements, an extracellular solution of
the following composition was used (mmol/L): NaCl 120, KCl 20,
CaCl2 2.0, MgCl2 1.0, and
HEPES/NaOH 10.0, pH 7.4. The solution for filling the patch-clamp
pipettes for whole-cell voltage clamp contained (mmol/L): potassium
aspartate 110, KCl 20, NaCl 10, MgCl2 1.0, MgATP
2.0, EGTA 2.0, GTP 0.01, and HEPES/KOH 10.0, pH 7.4. Standard chemicals
were from Merck. EGTA, HEPES, MgATP, Ado, GTP, and ACh iodide were from
Sigma.
Current Measurement
Membrane currents were measured by using whole-cell patch
clamps. Pipettes were fabricated from borosilicate glass and were
filled with the solution listed above (DC resistance 4 to 6 M
).
Currents were measured by means of a patch-clamp amplifier (List LM/EPC
7). Signals were analog-filtered (corner frequency 1 to 3 kHz),
digitally sampled at 5 kHz, and stored on a computer equipped with a
hardware/software package (ISO2, MFK) for voltage control and data
acquisition. Experiments were performed at ambient temperature (22°C
to 24°C). Cells were voltage-clamped at -90 mV, ie, negative to
K+ reversal potential, resulting in inward
K+ currents. Current-voltage (I/V)
relations were determined by means of voltage ramps from -120 mV to 60
mV at 200 mV/s. Rapid superfusion of the cells for application and
withdrawal of different solutions was performed by means of a
solenoid-operated flow system that permitted switching between up to 6
different solutions (half-time 
100 milliseconds).
Transfection
After isolation, myocytes were cultured overnight to allow
attachment. For transfection, 2.5 µg/plate of the reporter
IRES–enhanced green fluorescent protein (EGFP)
(Clontech) and 2.5 µg/plate pSV-SPORT1-A1R
construct (rat brain A1R was kindly provided by
Dr A. Karschin, Göttingen. Germany) were used. The cDNA of the
EGFP was under CMV promoter control, and the A1R
cDNA was under SV40 promoter control. A1R cDNA
was ligated into the pSV-SPORT1 multiple cloning site by using 5'
EcoRI and 3' HindIII restriction enzymes. To
precomplex the DNA for each plate, constructs were incubated with 100
µL transfection medium (M199 without FCS and antibiotics) and 5 µL
Plus reagent (Life Technologies Inc) for 15 minutes at room
temperature. After incubation, 2 µL lipofectamine/plate (Life
Technologies Inc) was diluted in 100 µL transfection medium, mixed
with the Plus/DNA solution, and incubated in the same way before
dilution with transfection medium to a final volume of 1 mL. Myocytes
were washed with prewarmed PBS and incubated for 3 hours under normal
cell culture conditions with the transfection solution. Thereafter,
dishes were washed with PBS and incubated with medium M199 supplemented
with gentamycin and canamycin (20 mg/mL each) at 37°C and 5%
CO2.
Electrophysiological recordings were made on days 3 and 4 after transfection. Transfected cells were identified by epifluorescence of EGFP (excitation wavelength 470 nm).
Data Analysis
Amplitudes of currents were normalized to cell capacity to yield
current densities. The statistical significance of differences between
calculated means was evaluated by Student t test for
unpaired samples. A value of P<0.05 was considered to be
significant.
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Results |
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As shown previously, saturating concentrations of Ado (
100
µmol/L) elicited currents of smaller amplitude and slower activation
kinetics than currents elicited by ACh at saturating concentrations of
2 µmol/L.20 21 23 Moreover, rapid
desensitization, ie, an immediate and rapidly reversible decay in
agonist-evoked current, was present in most cells on application of
ACh. The mechanism underlying this acute desensitization at present
is not completely understood. It is generally accepted that it does not
reflect a receptor desensitization but occurs downstream, ie, either at
the G protein or the Kir3.1/3.4 channel.24 25 Fast
desensitization was never seen on challenging a native cell with Ado.
These differences are summarized in Figure 1
, which shows the range of ACh-sensitive
K+ current (IK(ACh))
activated by a saturating concentration of Ado
(IK(ACh),(Ado)) found in control myocytes.
Traces in Figure 1A are from a cell that produced a small
Ado-induced IK(ACh) in relation to the
response evoked by 2 µmol/L ACh (15%), whereas the traces in
Figure 1B represent the myocyte with a large response to
Ado (47%). The I/V relations of the agonist-evoked currents
(obtained by a ramp protocol) that identify the current changes as
IK(ACh) by their strongly inwardly
rectifying properties are shown in Figure 1C. In Figure 1D, the I/V curve of
IK(ACh),(Ado) (from Figure 1B) has
been scaled up to match the ACh-induced current at -100 mV. Both
curves are perfectly superimposable, in line with previous studies
demonstrating that identical current pathways are activated by
either agonist/receptor.19 20 Activation of an
additional current pathway by Ado, such as
IK(ATP), as described
previously,26 can be safely excluded in the
present experimental conditions. Any contamination by
simultaneously active IK(ATP)
gives rise to an I/V curve with much less pronounced inward
rectification, which can be clearly identified by subtraction
protocols.27 The mean amplitude of
IK(ACh),(Ado) normalized to
IK(ACh),(ACh) (which is
IK(ACh) activated by a saturating
concentration of ACh) in the same cell was 33% (14.3±2.13 versus
42.7±4.87 pA/pF, n=20). No significant differences were found between
time-matched nontransfected cells, cells transfected with the EGFP
vector only, and EGFP-negative cells in cultures exposed to both
vectors (data not shown).
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It is evident from Figure 1
that apart from the smaller
amplitude, the Ado-induced current is characterized by its slower onset
kinetics. In the present experimental conditions, this was not
limited by the time course of the rise in agonist concentration seen by
the cell under study. It rather reflects a genuine property of the
A1R-mediated response, as demonstrated in Figure 2, which shows that the rising phases of
inward IK(ACh),(Ado) evoked by 2 different
but highly saturating concentrations of Ado (0.1 mmol/L and
10 mmol/L) are identical (Figure 2A). If it is assumed that
the rates of the reactions subsequent to receptor-agonist binding are
not different for the M2R and
A1R, the rate of rise of
IK(ACh) should be dependent on the number
of ligated receptors, which is given by a simple law of mass action. In
the case of saturating concentrations of either agonist, the difference
in activation rates thus should reflect the difference in receptor
densities. If this assumption is valid, it should be possible to mimic
the response to a saturating concentration of Ado with regard to its
amplitude and activation time course by activating a smaller number of
M2Rs by using a lower concentration of ACh.
Figure 2B shows current recordings from a cell in which
the response to 1 mmol/L Ado could be almost perfectly mimicked by
0.3 µmol/L ACh, a sub-EC50 concentration,
with regard to both its amplitude and its onset kinetics. Taken
together, these findings demonstrate that
IK(ACh),(Ado) does not have intrinsically
slower onset kinetics than does
IK(ACh),(ACh). The close correlation of
amplitude and onset kinetics and its independence of the individual
receptor support the notion that in the case of
A1Rs, both parameters are limited by
receptor density.
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In a total of 75 EGFP-positive double-transfected cells (25 cultures
from 10 animals), currents evoked by ACh (10 µmol/L) and Ado
(100 µmol/L) were compared. In this population, 2 groups were
defined by the ratio
IK(ACh),(Ado)/IK(ACh),(ACh).
In group I (28 cells), the ratio
IK(ACh),(Ado)/IK(ACh),(ACh)
was also <1, as in all nontransfected and mock-transfected cells, but
was significantly larger (P<0.001) than in the control
group, whereas in group II (47 cells), this ratio was reversed.
Representative examples of current recordings
from individual cells of both groups are illustrated in Figure 3. In Figure 3A, the amplitude of
IK(ACh),(Ado) was 0.63 that of
IK(ACh),(ACh), whereas in Figure 3B, IK(ACh),(Ado) was larger than
IK(ACh),(ACh) by a factor of 2.24. The
Ado-induced current in Figure 3B displayed distinct rapid
desensitization, which was absent in control cells and group
I20 21 (compare Figures 1 and 2). The
summarized data in Figure 3C demonstrate that in group I, the
density of IK(ACh),(Ado) (24.6±3.7 pA/pF)
was significantly larger than in the control cells
(P<0.02). There was no significant difference in the
densities of IK(ACh),(ACh) between this
group and control cells (P=0.49). Transfection-positive
cells of group II were characterized by a larger density of
IK(ACh),(Ado) (34.9±1.9 pA/pF) compared
with control cells and transfected cells of group I. Surprisingly, in
group II, the density of IK(ACh),(ACh)
(18.8±2.2 pA/pF) was significantly smaller (P<0.002) than
in control myocytes (42.7±4.87 pA/pF) and transfected cells
(P<0.002) of group I (48.6±7.3 pA/pF).
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As shown in previous studies21 22 (see also Figures 1 and 2), there is a direct relation between the
amplitude and activation rate of IK(ACh),
which is independent of the activating receptor. Correspondingly, the
changes in densities of IK(ACh),(Ado) and
IK(ACh),(ACh) in myocytes overexpressing
A1Rs were paralleled by changes in the
activation rate. As shown in Figure 4, the increase in density of IK(ACh),(Ado) in
transfected cells was paralleled by a decrease in half-time, which
was more pronounced in group II. Moreover, in this group compared with
controls, a significant (P<0.001) increase in the half-time
of IK(ACh),(ACh) was observed.
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The data presented so far lend support to the notion that the
sensitivity of IK(ACh) to Ado in native
myocytes is limited by the density of A1Rs. In
cells with an apparent high expression level of
A1Rs, there is a substantial reduction in
IK(ACh),(ACh) that is concomitant with a
slowing of activation and a loss in rapid desensitization. These
changes are similar to those produced by long-term treatment of atrial
myocytes with the muscarinic agonist carbachol, causing desensitization
or downregulation of M2Rs, ie, a loss in density
of functional receptors.22 Whereas changes in
M2R density result in substantial changes in
EC50 values for muscarinic
agonists,22 28 no significant difference in
EC50 values was found between control myocytes
and those with a decreased density of
IK(ACh),(ACh) (data not shown). This
suggests that the reduction in responsiveness to ACh is not caused by a
reduced density of M2Rs but is reflecting a novel
type of cross talk between 2 different 7-helix receptors. We
hypothesized that one of the downstream signaling elements shared by
the 2 receptors, ie, either the G-protein or the channel complex,
becomes limiting for the M2R if the
A1R is overexpressed. This could be the
consequence of preformed complexes of receptor and G-protein molecules
or receptor, G-protein, and channel complexes.29
Whereas the existence of stable complexes with G proteins has been
demonstrated for a couple of receptors, including the
A1R,30 tight precoupling of
G-protein and channel complexes is unlikely. Previous studies have
provided evidence that various intrinsic and heterologously expressed
receptors in atrial myocytes converge on the same population of
K(ACh) channels.11 19 31
Simultaneous stimulation of 2 different receptors
consistently resulted in a nonadditive activation of
macroscopic current, which, because of fast desensitization, was
smaller than the peak current activated by the agonist that
yielded a saturating response. As shown in Figure 5, this also applies to the overexpressed
A1R. In this representative cell,
saturating stimulation of M2R resulted in a
current of 30% of peak IK(ACh),(Ado).
Activation of IK(ACh),(Ado) in addition to
IK(ACh),(ACh) resulted in a total peak
current that was smaller (by 28%) than
IK(ACh),(Ado) in the absence of muscarinic
stimulation. Both the nonadditivity of the currents activated
by both agonists and the heterologous nature of rapid desensitization
clearly suggest that both receptors have access to the entire
population of K(ACh) channels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The classic receptor that controls K(ACh) channels in the heart is the M2R, which confers vagal activity to inhibition of important physiological parameters, such as cardiac frequency and atrioventricular nodal conduction. Coupling of A1R to a K+ current in the heart was first described by Belardinelli and Isenberg.32 The underlying current pathway was subsequently identified as IK(ACh) by Kurachi et al.19 Apart from the M2R and the A1R, a few other heptahelical receptors have been found to converge on atrial IK(ACh), such as endothelin-A receptors,12 33 sphingolipid receptors,11 somatostatin receptors,34 and a few others that, so far, have not been studied in macroscopic (whole-cell) measurements. The physiological significance of activating IK(ACh) via these receptors to date is far from being understood. As for the function of activation of this current by Ado, it is assumed that it serves as a metabolic feedback mechanism, limiting heart rate and force of contraction (eg, during high sympathetic activity). Ado via A1Rs and A3 receptors is considered a major mediator of protection against ischemia-induced injury in the heart.35 36 It appears to play a key role in ischemic preconditioning.37 In this context, the A1R is being discussed and probed as a potential target of a gene therapeutic approach.17 18 In consideration of this background, a detailed knowledge of how manipulation of the expression level of A1R in a cardiac myocyte affects other aspects of normal cellular function is desirable. The major results of the present study are that (1) transfection of adult rat atrial myocytes with a vector containing the cDNA encoding for a rat brain A1R increases the amplitude and speed of activation of IK(ACh) induced by Ado, and (2) in the majority of successfully transfected myocytes in which the expression level of A1R was assumed to be particularly high, sensitivity of IK(ACh) to ACh via M2R was significantly reduced.
It has previously been demonstrated in guinea pig20 and
rat21 atrial myocytes that availability of
IK(ACh) via A1R is
less than via M2R. This difference is larger in
the adult rat compared with the adult guinea pig, in which the fraction
of current available to Ado amounted to 60% of
IK(ACh),(ACh).20 This
species difference on the cellular level coincides with the effects of
Ado on sinus rate and nodal conduction in perfused intact
hearts.38
The present data clearly support the notion that the expression level of the A1R is limiting the amplitude of IK(ACh) that can be activated via this receptor. This interpretation is supported by the following results: (1) In a given cell, inward IK(ACh) evoked by a saturating concentration of Ado can be perfectly mimicked by application of a nonsaturating concentration of ACh. (2) Transfection with a vector containing the cDNA encoding for an A1R results in ACh-evoked currents of larger amplitude and faster activation kinetics and a decreased EC50. In the majority of A1R-overexpressing myocytes, currents evoked by saturating concentrations of Ado have the same properties as ACh-induced currents in native cells with regard to amplitude, activation kinetics, and fast desensitization. In a previous study, we have shown that for ACh-induced currents in guinea pig atrial myocytes, opposite changes in all these parameters were observed after a reduction in functional M2Rs that was due to long-term desensitization induced by treatment of cultures with carbachol.22 (It should be noted that long-term desensitization is different from the acute type of desensitization of the present study.) On the other hand, an increase in sensitivity to ACh with time had been observed in cultured myocytes; this observation was interpreted to reflect recovery from physiological desensitization caused by tonic vagal activity in the intact animal.28 These effects were studied for the M2R pathway only. Nevertheless, these studies demonstrated that reduction in the density of functional receptors (M2Rs) converging on K(ACh) channels by long-term desensitization has effects on IK(ACh),(ACh) that are opposite the effects on IK(ACh),(Ado) induced by A1R transfection.
The transfection-positive myocytes could be separated into 2 groups. In both groups, maximum IK(ACh),(Ado) was increased. However, 70% of these cells not only showed this increase but also showed a substantial decrease in IK(ACh),(ACh). Because the transfection rates in the cell type under study in terms of EGFP-positive cells were low (as a rule, <5%), comparisons of expression levels of A1Rs and/or M2Rs with use of immunoblots are not feasible. Even if transfection rates were higher, standard blotting techniques would not provide information on the differences in the expression levels of the 2 receptors in individual cells or in the 2 groups of transfected cells. Therefore, it is not possible to correlate different sensitivities of individual cells in a culture dish to Ado and ACh with expression of the receptor proteins. The reduction in M2R-evoked current could result from a competition of A1Rs and M2Rs (and possibly other heptahelical receptors) for a common G-protein pool. In such a case, availability of the coupling G protein might become a rate-limiting factor for activation of G-protein–regulated inwardly rectifying K+ channels. This interpretation would be in line with the increasing body of evidence that receptors together with G protein and effector molecules might be associated in functional clusters or microdomains, respectively.29 39 Although the Kir3.x subunits possess a motif for the binding of heterotrimeric G proteins,40 stable preformed arrays of all 3 signaling components are unlikely, because all Gi/Go-coupled receptors studied so far have access to the same population of channels. It is conceivable, however, that via precoupled receptor–G-protein complexes, the overexpressed A1R could cause depletion of G-protein molecules available for other receptors competing for the same pool. Alternatively, it is conceivable that overexpression of A1Rs somehow negatively interferes with the expression of M2Rs. An example of such a dysregulation of gene expression by increasing the dosage of another gene has been demonstrated recently for 2 neuronal K+ channels in a transgenic model.41 To obtain more information about this issue, further experiments using simultaneous manipulation of expression levels of receptors and GK (Gi/Go) are required. Independent of the precise mechanism, our data clearly indicate that in attempts of targeted overexpression of a receptor protein for therapeutic purposes, the potential benefit might be impaired by a loss of responsiveness to other receptors converging on the same signaling pathway.
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Acknowledgments |
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This study was supported by Fischer Stiftung und DFG (Po212-9/1). We thank Anke Galhoff for excellent technical assistance.
Received December 1, 1999; accepted January 17, 2000.
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