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1- and
2-adrenergic receptors in
regulation of Cl
and
Ca2+ channels in guinea pig
ventricular myocytes
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The role of
1- and
2-adrenergic receptor
stimulation in modulating adenosine 3',5'-cyclic
monophosphate (cAMP)-regulated Cl
and
Ca2+ currents was investigated
with use of guinea pig ventricular myocytes. Activation of the
Cl current by the
nonselective -receptor agonist isoproterenol (Iso) was not affected
by the 2-receptor antagonist
ICI-118,551 (ICI), but it was blocked by the
1-receptor antagonist atenolol. The inability of 2-receptor
stimulation to activate the
Cl current was confirmed by
the lack of response to the selective 2-receptor agonists salbutamol
and zinterol. Responses to
2-adrenergic receptor
stimulation were also looked for in pertussis toxin (PTX)-treated myocytes because PTX increases the sensitivity of responses to Iso, and
PTX has been reported to increase the responsiveness to
2- but not
1-receptor stimulation. PTX
treatment increased the sensitivity of the
Cl current to activation by
Iso in the presence of ICI, indicating that PTX increases
1-receptor responsiveness. PTX
treatment also resulted in the ability of salbutamol to activate the
Cl current. However, the
response to salbutamol was blocked by atenolol but not by appropriate
concentrations of ICI, suggesting that salbutamol was activating
1-receptors. These results
indicate that PTX treatment increases the sensitivity to
1-receptor stimulation, without
affecting 2-responsiveness. To
verify that the lack of response to
2-receptor stimulation was not
unique to the Cl current,
the effects of 2-receptor
agonists on the L-type Ca2+
current were also examined. The
Ca2+ current was only affected by
high concentrations of zinterol or salbutamol, and such responses were
blocked by atenolol, but not by ICI, suggesting that activation of
1-receptors was involved. These
results indicate that 1- but
not 2-adrenergic receptor stimulation plays an important role in modulating the cAMP-regulated Cl and
Ca2+ currents in guinea pig
ventricular myocytes.
pertussis toxin-sensitive G protein; isoproterenol; salbutamol; zinterol; ICI-118,551
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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CATECHOLAMINES exert a number of physiologically
significant effects on the heart through the activation of
-adrenergic receptors and subsequent modification of both electrical
and mechanical properties of cardiac muscle. Responses to
-adrenergic receptor stimulation are generally attributed to a
signaling pathway that involves adenylate cyclase activation, adenosine
3',5'-cyclic monophosphate (cAMP) production, protein
kinase A stimulation, and protein phosphorylation. In the
heart, -adrenergic responses are most commonly associated with the
activation of 1-adrenergic receptors. Nevertheless, cardiac muscle also possesses a significant population of 2-adrenergic
receptors (2, 12, 15, 17, 22). Most studies investigating the role of
-adrenergic receptors in regulating cardiac contractility or ion
channel function have used combined
1- and
2-adrenergic-receptor agonists
such as isoproterenol (Iso) and have largely ignored the contributions of the individual receptor subtypes. Although some studies have examined the role that 1- and
2-adrenergic receptors may play in regulating contractile function, the results have not been consistent (14, 25). Even fewer studies have looked at the role of
-receptor subtypes in regulating electrical activity (1, 4, 19, 24,
25).
In the present study, we chose to look specifically at the effects that
1- and
2-adrenergic receptor
stimulation have on cardiac ion channel function. -Adrenergic
stimulation, in general, affects a number of different ion channels,
including those responsible for the cAMP-regulated
Cl current and the L-type
Ca2+ current (8). The aims of this
study were to determine the relative significance of the role that
1- and
2-adrenergic receptor activation plays in regulating these ion channels in guinea pig ventricular myocytes, a commonly used model for studying cardiac electrophysiology. We find that the nonselective -adrenergic agonist
Iso regulates Cl and
Ca2+ currents solely through the
activation of 1-adrenergic
receptors. In fact, it appears that
2-receptor activation has no
effect on the activity of either of these channels.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Cell isolation. Single ventricular myocytes were isolated from adult Hartley guinea pigs with use of a modification of a previously described method (9). Hearts were quickly excised from anesthetized animals, and the coronary arteries were perfused via the aorta with a Krebs-Henseleit buffer (KHB). The KHB contained (in mM) 120 NaCl, 4.8 KCl, 1.5 CaCl2, 2.2 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose. The pH of the solution was maintained at 7.35 by bubbling with 95% O2-5% CO2 at 37°C. The heart was initially perfused with Ca2+-containing KHB for 5 min. The solution was then switched to Ca2+-free KHB for a further 5 min, after which time collagenase B was added (Boehringer Mannheim) to achieve a final concentration of 0.5-0.7 mg/ml. After 30-45 min of digestion the ventricles were cut down, minced, rinsed free of collagenase, and reintroduced to Ca2+-containing KHB. Single cells were obtained by gentle trituration of the tissue. Cells were used on the day of isolation only.
Measurement of membrane currents.
Membrane currents were recorded with the conventional whole cell
configuration of the patch-clamp technique (7). Microelectrodes were
pulled from borosilicate glass capillary tubing (Corning 7052, Garner
Glass) and had resistances between 0.5 and 1.5 M
when filled with
the following intracellular solution (in mM): 130 glutamic acid, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 10 ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 20 tetraethylammonium chloride (TEA), 5 MgATP, 0.1 tris(hydroxymethyl)aminomethane-GTP, 1 CaCl2; the pH was adjusted to 7.05 with CsOH. The control extracellular solution contained
(in mM) 140 NaCl, 5.4 CsCl, 2.5 CaCl2, 0.5 MgCl2, 5.5 HEPES, and 11 glucose;
pH was adjusted to 7.4 with NaOH. With intra- and extracellular
Cl concentrations 22 and
151.4 mM, respectively, the predicted
Cl equilibrium potential
was 
50 mV.
Experimental protocols.
The Cl current was isolated
by blocking all K+ channels with
Cs+- and/or TEA-containing
intra- and extracellular solutions. L-type Ca2+ channels were blocked by
adding 1 µM nisoldipine (Miles Laboratories) to all extracellular
solutions. Sodium channels were inactivated by using a holding
potential of 30 mV. The time courses of changes in
Cl conductance were
monitored by applying 100-ms voltage steps to +50 mV once every 3 s.
Current-voltage relationships were recorded by applying 100-ms voltage
steps from the holding potential of 30 mV to test potentials
from 120 to +50 mV in 10-mV increments. The
Cl current was defined as
the agonist-induced difference current determined by subtracting
currents recorded in the absence of drugs from those recorded in the
presence of drugs. Current magnitude was defined as the average current
measured over a 15-ms span at the end of each 100-ms voltage-clamp
step. Cl conductance was
calculated by linear regression of the current-voltage relationship
positive to the reversal potential.
80 mV. Sodium channels were
inactivated by applying a 50-ms prepulse to 30 mV immediately
before each test pulse. The time course of changes in
Ca2+ conductance were monitored by
applying a 75-ms test pulse to 0 mV once every 10 s. The magnitude of
the Ca2+ current was determined by
measuring the peak inward current recorded during the step to 0 mV.
In some experiments myocytes were incubated in KHB containing pertussis
toxin (PTX, 2 µg/ml) at 37°C for at least 3 h before being used
for voltage-clamp experiments. Effective PTX treatment was verified in
each cell by demonstrating the inability of 10 µM acetylcholine (ACh)
to inhibit the Iso-activated current (10). To minimize the possible
contribution of current rundown, only currents that returned to at
least 75% of their initial magnitude after washout of the antagonist
were included in this study. Results are reported as means ± SE.
Statistical comparisons were conducted using paired or unpaired
t-tests where indicated (SigmaStat,
Jandel Scientific Software).
Drugs and chemicals. Most drugs were prepared as stock solutions so that the desired final concentration was achieved by 1:1,000 dilution with the control extracellular solution. ACh, Iso, and atenolol (all from Research Biochemicals), ICI-118,551 (ICI; Tocris Cookson), salbutamol (Sigma Chemical), and PTX (List Biological Laboratories) were prepared in distilled water. Zinterol hydrochloride (kindly supplied by Bristol-Myers, Evansville, IN) was initially prepared in dilute NaOH. Nisoldipine was prepared as a stock solution in polyethylene glycol (Sigma Chemical) and diluted 1:2,000 in extracellular solution. Ascorbic acid (50 µM) was added to all solutions to prevent oxidative degradation of Iso.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Iso activates Cl current via
1-adrenergic receptors.
Iso activates both 1- and
2-adrenergic receptors. To
determine whether both receptor subtypes contribute to the effect of Iso in guinea pig ventricular myocytes, we looked to see if Iso was
able to activate the cAMP-regulated
Cl current in the presence
of selective 1- and
2-receptor antagonists. First,
we wanted to determine whether Iso is able to elicit responses through
activation of 2-receptors. We
therefore looked at the effect that acute exposure to 100 nM ICI had on
the response to Iso. This concentration of ICI selectively blocks
2-mediated responses (5, 15,
17). Cells were first exposed to 1 µM Iso to activate the otherwise
absent time-independent, outwardly rectifying, cAMP-regulated
Cl current. Subsequent
exposure to ICI in the continued presence of Iso had no effect (Fig.
1). In nine separate experiments, the Cl conductance measured in
the presence of 100 nM ICI plus 1 µM Iso was 97 ± 2.5% of that
found in the same cell in the presence of 1 µM Iso alone.
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current through the
stimulation of 1-adrenergic
receptors. We further investigated the ability of Iso to activate the
Cl current via
1-receptors. For these
experiments, myocytes were exposed to 100 nM ICI for at least 30 min
before, as well as throughout the duration of the voltage-clamp
experiments. In the presence of the
2-antagonist, Iso was able to
activate the Cl current
(Fig. 2), and the response to Iso was
blocked by atenolol, a selective
1-receptor antagonist. In the
presence of ICI, 1 µM atenolol attenuated the
Cl current activated by 30 nM Iso by 96 ± 2.4% (n = 7), and
10 µM atenolol inhibited the current activated by 1 µM Iso by 88 ± 5.1% (n = 4). These
concentrations of atenolol selectively block
1-adrenergic receptors (5, 13,
18).
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2-receptor stimulation
contributes very little to Iso responses in guinea pig ventricular
myocytes. To test this conclusion further, we looked for a response to
Iso in the presence of atenolol. For these experiments, cells were
exposed to 10 µM atenolol for at least 30 min before as well as
throughout the duration of the voltage-clamp experiments. In the
presence of the 1-antagonist, Iso was unable to elicit a significant response, even though direct activation of adenylate cyclase with 3 µM forskolin clearly
demonstrated that Cl
channels were present (Fig. 3). This
concentration of forskolin is maximally effective, activating the
Cl current to the same
level as a maximally effective concentration of Iso (10, 11). In nine
separate experiments, the magnitude of the response measured in the
presence of atenolol plus Iso was 9.3 ± 5.3% of that elicited by
forskolin. Any response to Iso that was observed was not blocked by 100 nM ICI. These results further support the idea that in guinea pig
ventricular myocytes, responses to Iso are mediated solely through
1-receptor activation.
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Do 2-receptor agonists
activate the cAMP-regulated Cl
current?
Although Iso was unable to elicit a response by activating
2-receptors, that does not
preclude the possibility that a more effective
2-agonist could activate the
Cl current. To test this
possibility, we investigated the effect of salbutamol, a selective
2-receptor agonist (3, 6).
Responses to salbutamol were compared with the effect of a maximally
stimulating concentration of Iso in the same cell. Using this protocol,
we found that 10 µM salbutamol elicited little or no response, even though Iso was clearly able to activate the
Cl current (Fig.
4). In five separate experiments, the
response to 10 µM salbutamol was only 3.0 ± 3.0% of that
elicited by 30 nM Iso. Similar results were obtained when the
concentration of salbutamol was increased to 1 mM
(n = 4). We also tested the response to zinterol, another
2-selective agonist (1, 14, 16,
23-25), using the same protocol. Again, very little current was
elicited by 2-receptor
stimulation. The response to 10 µM zinterol was 6.6 ± 2.2% of
that elicited by 30 nM Iso in the same cell
(n = 5). Increasing the
concentration of zinterol to either 100 µM or 1 mM did not increase
the relative magnitude of the response (n = 7). These
results suggest that under normal conditions, 2-receptor activation is unable to activate the cAMP-regulated
Cl current in guinea pig
ventricle myocytes.
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Exposure to PTX alters response to -receptor
agonists.
We have previously found that exposure of cells to PTX increases the
sensitivity of the Cl
current to -receptor activation. PTX treatment decreased the concentration of Iso necessary to cause half-maximal activation of the
Cl current from 5.0 to 1.4 nM (10). Recent studies have suggested that
2- but not
1-adrenergic receptors are
functionally coupled to a PTX-sensitive G protein in rat ventricular
myocytes (24). This finding led us to speculate that PTX treatment
might be unmasking an Iso-induced
2-response in guinea pig
ventricular myocytes. To test this hypothesis we investigated the
specific effects of PTX treatment on responses to
1- and
2-receptor stimulation, respectively.
1-adrenergic responses were
studied in cells preincubated in ICI-containing solution, as described
previously. We then compared the magnitude of the response to a near-
threshold concentration of Iso (3 nM) to that of a maximally
stimulating concentration of Iso (30 nM) in the same cell. This
protocol was conducted in control cells and PTX-treated cells (Fig.
5). In cells that were not treated with
PTX, 3 nM Iso elicited a current that was 27 ± 7.9%
(n = 6) of the current activated by 30 nM Iso. The relative magnitude of the current activated by 3 nM Iso is
consistent with previously published data (10, 27). However, in
PTX-treated cells, the current elicited by 3 nM Iso was 82 ± 6.8%
(n = 7) of the current activated by 30 nM Iso. This indicates that PTX treatment significantly (P < 0.001, unpaired
t-test) increased the sensitivity of
these cells to 1-adrenergic
receptor stimulation. A comparison of Fig. 5A to Fig.
5B illustrates the increase in
sensitivity of the Cl
current to 3 nM Iso caused by PTX.
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channels by
2-adrenergic receptors is
similarly influenced by PTX, we compared the magnitude of the response
elicited by 10 µM salbutamol to that elicited by 30 nM Iso in the
same cell. This protocol was conducted with control and PTX-treated
cells. Consistent with the response illustrated in Fig. 4, salbutamol
elicited very little current in cells that were not PTX treated (Fig.
6A). In
cells that had been incubated in PTX, however, exposure to 10 µM
salbutamol resulted in a significant response (Fig.
6B). The magnitude of the
Cl current activated by
salbutamol in PTX-treated cells was 73 ± 12% of that activated by
Iso (n = 7).
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current in guinea pig
ventricular myocytes treated with PTX suggests that the sensitivity to
2-receptor stimulation had been
increased. To verify that the response to salbutamol was actually due
to activation of 2-receptors,
we looked to see if the salbutamol-activated current could be blocked
by 2- but not
1-receptor antagonists. In
three different PTX-treated cells, we found that 100 nM ICI, the
selective 2-antagonist, did not
inhibit the response to 10 µM salbutamol (Fig.
7A).
However, complete inhibition of the current activated by salbutamol was
observed in four different cells when the concentration of ICI was
increased to 10 µM (Fig. 7B).
Because this concentration of ICI is reported to act at
1-receptors (15), we examined
the effect of the selective
1-receptor antagonist atenolol
on the current activated by salbutamol. In five separate experiments,
1-10 µM atenolol completely inhibited the current elicited by 10 µM salbutamol in cells exposed to PTX (Fig.
7C). These results strongly suggest
that 2-receptor stimulation is not able to activated the
Cl current under control
conditions, and the Cl
current elicited by salbutamol in PTX-treated cells can be attributed to activation of 1-receptors
alone.
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Do 2-receptor agonists
activate L-type
Ca2+ current?
Previous studies investigating the effect of zinterol on the L-type
Ca2+ current in rat heart have
shown that the 2-receptor
agonist can increase the peak Ca2+
current amplitude (1, 25). To rule out the possibility that the lack of
responsiveness to 2-receptor
stimulation in our experiments might represent a unique property of the
Cl current, we examined the
effect of the 2-receptor
agonist zinterol on the Ca2+
current in guinea pig ventricular myocytes. In nine separate cells, 10 µM zinterol increased the basal
Ca2+ current by an average of 96 ± 17%. This increase in basal
Ca2+ current is similar to that
caused by the same concentration of zinterol in rat ventricular
myocytes (25). We also investigated the effect of receptor subtype
selective antagonists on the zinterol-activated Ca2+ current. As shown in Fig.
8, the
Ca2+ current response to 10 µM
zinterol was inhibited by 10 µM atenolol, whereas 100 nM ICI had no
effect. In six experiments, 1-10 µM atenolol attenuated the
Ca2+ current activated by zinterol
by 92 ± 5.0%. The order in which cells were exposed to either
antagonist was randomized, with no difference in the results. To ensure
that the inhibition induced by atenolol was due to antagonism at
1-receptors and not a
nonspecific effect of the drug on the
Ca2+ channel, we examined the
effect of 10 µM atenolol on the basal Ca2+ current. In the presence of
atenolol, the magnitude of the
Ca2+ current was 99.2 ± 0.5%
of that recorded under control conditions (n = 6). Similarly, ICI alone had
little effect. The magnitude of the
Ca2+ current recorded in the
presence of 100 nM ICI was 98 ± 0.7% that recorded under control
conditions (n = 5). We also examined the effect of a lower concentration of zinterol on the
Ca2+ current because the drug has
been reported to act at
1-receptors at higher
concentrations, and we wanted to ensure that the concentration of the
agonist was selective for
2-receptors (16). In four experiments, 100 nM zinterol had no effect on the basal current.
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1-receptors.
2-receptor activation does not
appear to stimulate either the
Ca2+ current or the
Cl current in guinea pig
ventricular myocytes.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Iso acts predominantly via
1 receptors.
Although radioligand binding assays have demonstrated the existence of
2-adrenergic receptors in the
cardiac tissue of a number of mammalian species, including guinea pigs
(15, 17, 22), very few studies have investigated the functional role of
this receptor subtype in the regulation of cardiac ion channels. Most
studies investigating -adrenergic regulation of cardiac ion channel
function have used the combined
1- and
2-receptor agonist Iso without
addressing the roles of the individual receptor subtypes. Using
selective antagonists, we investigated the effects of Iso on ion
channel regulation, initially looking at its effects on the
cAMP-regulated Cl current.
Our results suggest that Iso acts exclusively through the activation of
1-receptors. This conclusion is
supported by the finding that acute exposure to ICI did not inhibit the
current activated by Iso (Fig. 1). In addition, Iso activated the
Cl current in the presence
of the selective 2-receptor
antagonist ICI (Fig. 2) but not when cells had been preincubated in the
1-receptor antagonist atenolol
(Fig. 3). Furthermore, exposure to the selective 2-receptor agonists salbutamol
and zinterol did not elicit a response.
2-receptor stimulation has no
significant effect on the activity of the cAMP-regulated
Cl current are in contrast
to previous reports demonstrating that zinterol is able to enhance the
L-type Ca2+ current in rat
ventricular myocytes (25). Although we were able to observe an effect
of salbutamol and zinterol on the
Ca2+ current under control
conditions and the Cl
current in PTX-treated guinea pig ventricular myocytes, such responses
were most likely due to activation of
1-receptors because they could
be blocked by atenolol or high concentrations of ICI but not
concentrations of ICI that should have selectively blocked 2-receptor-mediated responses.
These results suggest that there may be distinct species-dependent
differences in the effect that 2-adrenergic receptor
stimulation has on ion channel function. The inability of
2-receptor stimulation to
affect ion channel function in guinea pig ventricular myocytes may also
reflect tissue specific differences within the same species because
2-receptor stimulation has also
been reported to have positive chronotropic effects in guinea pig
atrial tissue (22).
The lack of any obvious
2-adrenergic effect on ion
channel activity would also seem to contradict previous reports that
2-receptor stimulation affects
contractility of the guinea pig heart (26). However, it is difficult to
compare the effects of -adrenergic stimulation on the regulation of
ion channel activity with its effects on contractility because the
latter is affected by cAMP-dependent changes in sarcoplasmic reticulum
Ca2+-adenosinetriphosphatase
activity and myofilament Ca2+
sensitivity, in addition to changes in
Ca2+ channel function (8, 20).
Although responses to both 1- and 2-adrenergic receptor
stimulation are associated with the production of cAMP, the level of
cAMP produced within different cellular fractions is distinctly
different for each receptor subtype (26).
PTX increases sensitivity of
1-receptors but not
2-receptors.
It has been suggested that in addition to activating adenylate cyclase
through a stimulatory G protein,
2-receptor stimulation may
activate a second, parallel signaling pathway that has an opposite, or
inhibitory, effect (24). Evidence that
2-adrenergic receptor
stimulation activates inhibitory as well as stimulatory pathways is
supported by the report that PTX-treatment increases the sensitivity of
2- but not
1-mediated responses in rat
ventricular myocytes. This suggested to us that
2-receptor stimulation might be
expected to produce a stimulatory effect on ion channel function in
guinea pig myocytes that are treated with PTX. Consistent with this
hypothesis, we observed that PTX-treatment increased the sensitivity of
the Cl current to
activation by Iso. However, we found that the increased sensitivity to
Iso was not due to the unveiling of a stimulatory 2 response.
2-agonists elicited
responses in guinea pig myocytes treated with PTX, those responses
could be blocked by atenolol, a selective
1-receptor antagonist, but not
by appropriate concentrations of ICI, a selective
2-receptor antagonist. Thus
changes in the sensitivity to Iso in PTX-treated myocytes can be
explained by an increase in
1-responsiveness alone.
Although our results do not rule out the possibility that PTX treatment
can affect 2-responses, they
clearly illustrate that PTX treatment can increase
1-responsiveness in guinea pig
ventricular myocytes. This might be interpreted to mean that in this
preparation, 1-receptors are
also coupled to an inhibitory signaling pathway via a PTX-sensitive G
protein. However, a more parsimonious interpretation might be that the stimulatory pathway to which
1-adrenergic receptors are
coupled is under the tonic influence of a PTX-sensitive G
protein-mediated inhibitory signaling mechanism. A likely candidate is
the pathway coupled to
A1-adenosine receptors and
M2-muscarinic receptors because
activation of either one can antagonize -adrenergic-stimulated adenylate cyclase activity via a PTX-sensitive G protein (8, 21).
Physiological significance.
Evidence for the presence of 2-adrenergic receptors in
guinea pig myocardium was not found in the present study. However, radioligand binding studies have reported that ~12-20% of the -receptor population in guinea pig ventricular myocytes is of the
2-subtype (15, 17, 22). Our
results suggest that they are not functionally coupled to the
cAMP-dependent regulation of ion channels. The observation that
1- and
2-adrenergic receptor stimulation can have distinctly different effects on electrical activity is important because it may help explain previous reports indicating that 1-adrenergic
stimulation is much more likely to be arrhythmogenic (4, 19).
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ACKNOWLEDGEMENTS |
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This work was supported by the National Heart, Lung, and Blood Institute Grant HL-45141, an Established Investigatorship from the American Heart Association (R. D. Harvey), and a Postdoctoral Fellowship from the Northeast Ohio Affiliate of the American Heart Association (L. C. Hool).
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FOOTNOTES |
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Address for reprint requests: R. D. Harvey, Dept. of Physiology and Biophysics, Case Western Reserve Univ., 2109 Adelbert Rd., Cleveland, OH 44106-4970.
Received 21 February 1997; accepted in final form 4 June 1997.
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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I) obtained by subtracting
currents recorded under control conditions
(a) from currents recorded in
presence of 1 µM Iso (b), Iso and
100 nM ICI (c), after washout of Iso
and ICI (d), and again in presence
of 1 µM Iso (e).
