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1-adrenoceptor effects in ventricular myocytes from
NE-treated guinea pigs
Cardiac Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, London SW3 6LY, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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It has been
suggested that there is a preferential coupling in heart muscle between
the inhibitory G protein (Gi) and the 
2-subtype of the -adrenergic receptor (-AR), since
pertussis toxin (which inactivates Gi) reveals latent
2-ARs in rat and mouse myocytes. We have previously
shown that guinea pigs treated with norepinephrine (NE) for 7 days have
myocytes that are desensitized to -AR-agonist stimulation, and that
pertussis toxin restores these responses. The purpose of the present
investigation was to determine whether pertussis toxin specifically
upregulated 2-ARs in myocytes from NE-treated guinea
pigs. The sole -AR subtype in control guinea pig myocytes was
confirmed as 1-AR by radioligand binding, single-cell
autoradiography, and concentration-response curves to isoproterenol in
contracting myocytes. In contrast, a minor pool of 2-ARs
was observed in rat myocytes by use of the same methods. NE treatment
decreased the maximum isoproterenol response (relative to high
Ca2+) from 0.89 ± 0.06 to 0.58 ± 0.08 (n = 7, P < 0.01) and the pD2 (
log
EC50) from 8.8 ± 0.2 to 7.5 ± 0.2 (n = 7, P < 0.01). Pertussis toxin treatment increased the
isoproterenol-to-Ca2+ ratio to 0.88 ± 0.04 (n = 6, P < 0.05) and the pD2 to 8.6 ± 0.3 (P < 0.01). This was not mediated by increases in either
number or function of 2-ARs. Gi is therefore
able to modulate 1-AR responses in guinea pig myocytes.
G proteins; rat; contraction; norepinephrine; radioligand binding
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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BOTH 1- AND
2-adrenergic receptors (ARs) can enhance
ventricular contraction, with the contribution from the
2-AR subtype being particularly prominent in human
myocardium (2, 6, 15). The relative contribution of the
2-AR subtype is increased in failing human heart (1),
where either subtype can produce a maximal inotropic response to
catecholamine stimulation (6). An important role of the
2-AR subtype has recently been suggested by the finding
that a polymorphism that reduces the activity of 2-ARs
is associated with greatly increased mortality in heart failure
patients (21).
Classically, it was thought that -ARs coupled to adenylate cyclase
via the stimulatory G protein, Gs. However, recent evidence has shown that dual coupling is a possibility, with -receptors shuttling between Gs and the inhibitory G protein(s) (e.g.,
Gi) (5). It has been proposed that there is a difference
between the two -subtypes in this effect, with 2-ARs
selectively coupling to Gi. Evidence for this includes data
in rat myocytes, where treatment with pertussis toxin (which
inactivates Gi) preferentially upregulated
2-AR responses (29), and from transgenic (TG) mice overexpressing the human 2-AR
(TG-2). Myocytes from these mice were shown
to have reduced or absent responses to -AR stimulation, even though
high Ca2+ or cAMP analogs could still increase contraction
(11, 28). Gi levels were increased in the myocardium, and
treatment of myocytes with pertussis toxin restored responses to
isoproterenol or zinterol through 2-AR receptors (28).
However, both we (11) and Xiao et al. (28) have observed that in
TG-2 mice, it was not only the response through the
overexpressed 2-AR that was lost when Gi was
increased but also that through the native 1-AR, which mediates the majority of the -AR effect in wild-type mice.
These observations may have relevance to the mechanism of -AR
desensitization in human heart failure. The loss of responsiveness to
-agonists is accompanied by a decrease in 1-AR but
not 2-AR number and an increase in Gi levels
and activity (9). We have shown that treatment of myocytes from failing
human heart with pertussis toxin restores -AR responses but have not
determined the subtype mediating the increase in contraction (3). It
may be that pertussis toxin has enhanced responses to the remaining 2-AR receptors. A parallel study on myocytes from guinea
pigs infused with norepinephrine to produce -AR desensitization has shown similar results, with pertussis toxin able to reverse completely the loss of response (3). Untreated guinea pig myocytes have little or
no contribution from the 2-AR subtype to the increased ventricular contraction produced by isoproterenol (6). However, the
possibility still exists that a pool of latent 2-ARs is
revealed by the removal of Gi, and that this accounts for
the reversal of desensitization after pertussis toxin treatment.
The purpose of the present study was to determine whether the reversal
of -AR desensitization by pertussis toxin in myocytes from
norepinephrine-treated guinea pigs was due to rescue of the 1-AR responses or uncovering of latent
2-AR. If the first option is correct, then the
hypothesis that 2-ARs are uniquely modulated by
Gi is contradicted. We have included data from radioligand binding and single-cell autoradiography to show the strong
1-AR dependency of guinea pig myocytes. These are
compared with data from whole heart to confirm previous observations
that 2-ARs in ventricular homogenates are largely
associated with nonmyocyte cell types. Comparison is also made with rat
myocytes, where a minor functional 2-AR component has
been described. We show that guinea pig myocyte responses restored by
pertussis toxin are mediated entirely by 1-AR,
indicating that increased Gi is able to suppress effect on
contraction mediated through either the 1- or the
2-AR subtypes.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Infusion of norepinephrine into guinea pigs.
Male Dunkin-Hartley guinea pigs weighing 300-500 g were
anesthetized with the use of 2% Hypnorm (0.5 ml/kg) and a local
injection of lidocaine hydrochloride (2 ml/kg). Osmotic minipumps
(model 2002, Alzet) containing l-norepinephrine
hydrochloride (Sigma) dissolved in saline and 1 mM ascorbate were
incubated in saline for 4 h at 37°C and implanted subcutaneously in
the neck. The mean output rate of the pumps was 0.97 ml/h, with a
concentration of norepinephrine (NE) such that the guinea pigs
received, over a period of 7 days, 900 µg · kg
1 · h1.
Weight-matched, sham-operated animals were given the same anesthesia and incision, but no minipump was implanted. The guinea pigs were fed
standard laboratory diet and water ad libitum.
Isolation of ventricular myocytes. Male Dunkin-Hartley guinea pigs or Sprague-Dawley rats were heparinized and killed by cervical dislocation, and the heart was rapidly excised and placed in ice-cold Krebs-Henseleit (KH) solution of the following composition (in mM): NaCl 119, KCl 4.7, MgSO4 0.94, KH2PO4 1.2, NaHCO3 25, glucose 11.5, and CaCl2 1; equilibrated to pH 7.4 with 95% O2-5% CO2.
A Langendorff perfusion method similar to that described previously (25) was used. The heart was perfused in a retrograde manner for 5 min with KH solution. An additional 5-min perfusion was carried out with a low-Ca2+ solution of the following composition (in mM): NaCl 120, KCl 5.4, MgSO4 5, pyruvate 5, glucose 20, taurine 20, HEPES 10, and nitrilotriacetic acid (NTA) 5, with a pH of 6.95 and Ca2+ added to give a final concentration of 12-14 mmol/l as measured with a Ca2+ electrode. This solution was equilibrated with 100% O2. A solution similar to the low-Ca2+ one but with no NTA and with 200 µmol/l added Ca2+ and enzymes was perfused through the heart. For rat, collagenase (1 mg/ml) and hyaluronidase (0.6 mg/ml) were perfused through the heart for 10 min. For guinea pig, a 1-min perfusion with Pronase was followed by 10 min with 0.3 mg/ml collagenase. The two ventricles were then separated, keeping the interventricular septum with the left ventricle; the right ventricle was discarded. After being chopped with scissors, the pieces were shaken at 35°C under 100% O2 for 5 min with the same enzyme-containing solution, strained through gauze (mesh size 300 µm), and incubated for an additional 5 min with enzyme. The supernatant was centrifuged at 400 g for 1 min at room temperature. Cells were washed and resuspended in the low-Ca2+ solution with no NTA and with Ca2+ at a concentration of 200 µmol/l (ES).Percoll gradients.
This was performed on myocyte suspensions before radioligand binding
studies to remove nonmyocyte cells. A myocyte suspension was layered on
10% Percoll in KH and then centrifuged at 400 g for 1 min; the
supernatant was removed, and the pellet was resuspended in a small
amount of ES and then layered on a second 10% Percoll gradient. The
subsequent pellet was washed twice with ES, and the final pellet of
myocytes was quick frozen in liquid nitrogen and then stored at
80°C.
Pertussis toxin treatment of myocytes. One milliliter of a myocyte suspension was shaken gently with 1 µg of pertusiss toxin and left to incubate at 35°C for 2 h. Parallel incubations at 35°C in the absence of toxin were performed as previously described (3). For individual myocytes, the efficiency of Gi inactivation was confirmed by challenge of the myocyte with 10 µmol/l adenosine in the presence of isoproterenol. Complete abolition of the inhibitory effect of adenosine was taken as evidence for loss of Gi function (3).
Radioligand receptor binding assay.
Frozen guinea pig or rat whole heart was pulverized under liquid
nitrogen with a pestle and mortar and resuspended in 10 vol of 25 mmol/l Tris · HCl buffer (pH 7.4) containing 0.32 M
sucrose at 4°C. Pellets of rat left ventricular myocytes were
resuspended in the same buffer and homogenized with a Polytron
homogenizer (Kinematica, Basel, Switzerland) at a setting of 6 in 30-s
bursts. The homogenate was centrifuged at 1,000 g for 10 min at
4°C to remove unhomogenized debris; the supernatant was then
centrifuged at 40,000 g for 20 min at 4°C, and the
resulting pellet was washed with 25 mmol/l Tris · HCl
buffer (pH 7.4 at 4°C) and recentrifuged at the same speed. The
final homogenate was resuspended in 25 mmol/l
Tris · HCl buffer (pH 7.4 at 37°C) and then quick
frozen in liquid nitrogen and stored at 80°C without loss of
binding characteristics. For the whole heart and myocytes comparison, membrane isolation and the subsequent competition curves were all
carried out on the same day.
2-ARs was determined in the presence of 300 nmol/l
CGP-20712A (a highly selective 1-antagonist), a
concentration at which >99% of 1-ARs and <1% of
2-ARs are predicted to be occupied (8, 16, 20). The
density of 1-ARs was analyzed by ICYP saturation binding
in the presence of 50 nmol/l ICI 118,551 (a selective
2-antagonist), a concentration at which >99% of
2-ARs and <5% of 1-ARs are predicted
to be occupied. Incubation was carried out at 37°C for 120 min,
which was found to be optimal for specific binding. Incubations were performed in triplicate. The incubation was terminated by rapid vacuum
filtration through GF/C glass-fiber filters (Whatman, Clifton, NJ) with
the use of a cell harvester (model M-24, Brandel). Each filter was
rapidly washed with 3 × 5 ml ice-cold 25 mmol/l
Tris · HCl buffer (pH 7.4). The filters were counted
in a auto gamma counting system (model 5550; Packard Instruments,
Downers Grove, IL) at an efficiency of 80%. Protein concentration was
determined by the method of Lowry et al. (22), with bovine serum
albumin used as the standard.
Single-cell receptor autoradiography.
A drop of the myocyte suspension was fixed with 2%
paraformaldehyde for 2 h, washed, and centrifuged onto slides with the use of a Shandon cytospin-3; it was stored at 80°C until
required for use. The cell density was adjusted so that cells formed a monolayer and were well separated. After thawing, slides were preincubated in incubation buffer (IB; 25 mmol/l
Tris · HCl, pH 7.4, at 37°C with 154 mmol/l NaCl
and 1.1 mmol/l ascorbic acid) with 0.25% polypeptide (Sigma) for 10 min. Slides were then incubated with IB and 25 pmol/l ICYP for 2 h to
determine total binding, or in the presence of 50 nmol/l ICI 118,551, 300 nmol/l CGP-20712A, or 200 µmol/l isoproterenol for
1-AR, 2-AR, or nonspecific binding, respectively. Labeled sections were then washed for 2 × 15 min in ice-cold 25 mmol/l Tris · HCl, pH
7.4, with a final rinse in ice-cold distilled water for 15 s, and dried
in a stream of cold dehumidified air. The slides were dipped directly
into Ilford K-5 emulsion (1:1 dilution with distilled water) and then
left to dry for 3-4 h and placed into light-tight boxes with
desiccant for 7 days at 4°C. They were developed for 5 min using
D19 developer, placed in an Ilford stop bath for 30 s, fixed for 5 min
in Hypam fixer, and rinsed under running water for 1 h. Slides were
counterstained with hemotoxylin to identify myocytes.
Measurement of contraction. Myocytes in suspension were placed into a Perspex bath with a glass floor on the stage of an inverted microscope. After 5 min to allow settling, they were superfused with KH solution equilibrated with 95% O2-5% CO2 and maintained at 32°C by a feedback system. Cells were electrically stimulated at 0.5 Hz with the use of a bipolar pulse via platinum electrodes along the sides of the bath. Pulse width for each one-half of the stimulation was 0.1-0.5 ms. Continuous measurement of contraction amplitude and rates of shortening and relaxation were carried out as described by Harding et al. (10). A television system and length-detection device were used to display cell length and rate of change of cell length continuously to a chart recorder. The scanning rate of the camera was 50 or 100 Hz, and spatial resolution was 1 in 256 or 512.
The selection criteria described previously (10) were used to ensure that a population of cells with similar functional properties was used for contraction studies. Cells used were rod shaped with clear sarcomeres and no blebs or granulations. They had sarcomere lengths not less than 1.70 µm. In 1 mM Ca2+ they were quiescent or had spontaneous contractile waves at not more than two per minute. They had a stable contraction amplitude and diastolic length at 0.5 Hz; exposure to 8 mM Ca2+ resulted in an increase in contraction amplitude and rates of contraction and relaxation, which rapidly recovered on return to 1 mM Ca2+. Cells that satisfied these criteria were subjected to cumulative concentration-response experiments, using isoproterenol or zinterol; each concentration of agonist was left in contact for 10 min. Maximum contraction was judged to have occurred either when there was no increase in contraction amplitude between successive concentrations of isoproterenol or when phasic contractions were observed. The subtype-selective antagonist CGP-20712A (1-AR, 300 nmol/l) or ICI 118,551 (2-AR, 50 nmol/l) was then
superfused over the myocytes for 30 min, and the concentration-response
curves were repeated on the same cell in the continued presence of the
antagonist. For the third curve, both antagonists were included: it is
not possible to wash away the initial antagonist in the time-scale of
these experiments. Time-matched controls were performed by repeating
the isoproterenol concentration-response curves three times at 30-min intervals.
Data analysis. The experimental data are given as means ± SE. The significance of difference was tested by paired or unpaired Student's t-test; P < 0.05 was considered to be statistically significant. Parameters (dissociation constant and maximal binding capacity) of [125I]ICYP binding were obtained in individual experiments by using the computer programs EBDA and LIGAND. Competition curves were analyzed with the use of a nonlinear least-squares regression program (Inplot version 4; Graphpad, San Diego, CA).
Materials. Pyruvate, taurine, NTA, hyaluronidase (type I-s), Pronase (protease type XXIV), and isoproterenol-HCl were obtained from Sigma Chemical (Poole, UK). Collagenase was either type II (Worthington) from Cambridge Bioscience (Cambridge, UK) or Sigma Type V.
All other agents used for KH and low-Ca2+ solutions were obtained from BDH (Poole, UK). AristaR-grade KCl and glucose were used for low-Ca2+ solutions. All other reagents were AnalaR grade. AnalaR water (BDH) was used for low-Ca2+ solutions. Milli Q water was used for other solutions. CGP-20712A (1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino-3-[4-(1-methyl-4-trifluoromethyl-2-imidazoyl)]phenoxy]- 2-propanol methanesulphonate) was obtained from Ciba-Geigy (Basel, Switzerland), and ICI 118,551 ([±]-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol) was obtained from Zeneca (Macclesfield, UK). Zinterol was generously provided by Bristol-Myers Squibb. [125I]ICYP (2,000 Ci/mmol) was obtained from Amersham.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Radioligand binding: competition experiments.
In membranes from whole guinea pig heart, competition curves
for displacement of ligand by CGP-20712A and ICI 118,551 were biphasic
(Fig. 1A). The
percentages of high- and low-affinity binding sites for ICI 118,551 were 54 and 46%, respectively (n = 4). This indicates that for
guinea pig whole heart membrane, the proportion of 1- to
2-ARs was 46 to 54. In agreement with this, the
high-affinity binding site for CGP-20712A (1-AR)
comprised 45-50% of total binding. Full displacement was not
attained with CGP-20712A because of its extremely low affinity for the
2-receptor. For ICI 118,551, the inhibition constant
(Ki) for the high-affinity binding
site (0.88 ± 0.03 nmol/l) was 115 times less than that for the
low-affinity binding site (101 ± 25 nmol/l). For CGP-20712A, the
Ki for the high-affinity binding site (0.35 ± 0.02 nmol/l) was 3,060 times less than that for the low-affinity
binding site (1,071 ± 126 nmol/l). These selectivity ratios are in
agreement with those previously reported (8, 16, 20).
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1-AR subtype.
Experiments in rat whole heart and isolated myocytes showed a similar
pattern. The percentages of high-affinity and low-affinity binding
sites for ICI 118,551 were 52 and 48%, respectively (n = 4),
giving a 1- to 2-AR ratio of 48 to 52. In
agreement with this, the high-affinity binding site for CGP-20712A
(1-AR) comprised 40-50% of total binding. In
myocytes the proportions of high-affinity and low-affinity binding
sites for ICI 118,551 were 5 and 95%, respectively (n = 4),
indicating a minor pool of 2-ARs.
Single myocyte receptor autoradiography.
In a further attempt to eliminate the contribution of other cell types
to the -AR pool, autoradiographs were taken of single cells layered
onto a slide in such a way that individual myocytes were spatially
separate. After binding to radioligand, in the presence and absence of
the specific antagonists ICI 118,551, CGP-20712A, and isoproterenol at
the same concentrations used above, slides were exposed
to emulsion. The grain density around individual myocytes (positively
identified by their rod shape) was counted to give the amount of
radioligand remaining bound. For guinea pig myocytes, there was
relatively little decrease in grain density with the
2-AR-selective ICI 118,551, whereas almost all specific
activity was removed with the 1-AR-selective CGP-20712A
(Fig. 2). Rat myocytes showed significantly
more displacement by ICI 118,551, indicating a larger proportion of
2-ARs on the ventricular myocytes.
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Myocyte contraction studies.
After an initial concentration-response curve to isoproterenol, guinea
pig myocytes were challenged with isoproterenol in the presence of ICI
118,551 (50 nmol/l) or CGP-20712A (300 nmol/l), and for some cells a
third curve was constructed in the presence of both blockers. ICI
118,551 produced little shift in the curve, indicating that
2-ARs had not mediated the original response (Fig.
3A). The pD2
(log EC50) was 9.1 ± 0.3 for the first compared with 9.0 ± 0.1 for the second curve (n = 6, P = not significant). CGP-20712A produced a marked shift of over three
log units: this is consistent with an action at a pure
1-AR subtype. The pD2 was decreased from 8.8 ± 0.1 to 5.4 ± 0.2 (n = 4, P < 0.001; Fig. 3B). Addition of ICI 118,551 to the CGP-20712A caused no
further shift (pD2 5.3 ± 0.2, n = 4; Fig.
3B). The three successive curves performed as time-matched
controls showed little shift, with pD2 values of 8.7 ± 0.1, 8.6 ± 0.2, and 8.4 ± 0.2 for the first, second, and third
curves, respectively (Fig. 3C).
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2-AR-mediated component of contraction revealed
when the 1-ARs were blocked. Once again, the three
time-matched controls were superimposable (Fig. 4C).
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2-AR-selective agonist and has been used
to show that contractile effects on cardiac myocytes are
2-AR mediated. We used zinterol as the agonist for the
competition curves in the hope that this might amplify the effects of
the small 2-AR population in the rat myocytes. However,
the concentration-response curve to zinterol was not significantly
shifted by ICI 118,551, whereas CGP-20712A produced a large and
significant displacement of the curve (Fig.
5, A and B). When
1-ARs predominate, it appears that the majority of the
effect of zinterol is mediated through the 1-AR subtype.
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Pertussis toxin on myocytes from control guinea pigs.
Treatment with pertussis toxin increased the sensitivity of
myocytes from control (sham operated) guinea pigs to isoproterenol (Fig. 6). The pD2 value was
increased from 8.8 ± 0.2 (n = 7) to 9.9 ± 0.2 (n = 6, P < 0.01). However, this effect was not mediated by
increased coupling of 2-ARs, since ICI 118,551 did not
shift the concentration-response curve significantly in
pertussis-treated cells (pD2 9.7 ± 0.1, n = 6;
Fig. 6), nor was there evidence for upregulation of 2-AR
number, since there was no increase in the number or proportion of
2-ARs detected by radioligand binding (Fig.
7). (For this series the
1-AR number was determined by displacement of
2-AR binding with 50 nmol/l ICI 118,551.) In fact,
pertussis toxin treatment slightly decreased the total -AR number:
this may be due to the lability of -ARs during the 2-h incubation,
since a parallel incubation without toxin had a similar effect (data
not shown).
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Desensitization of the -AR response in myocytes from
NE-treated guinea pigs.
Seven days of infusion of NE produced a robust -AR
desensitization, as shown previously (3, 23, 26, 27). The
concentration-response curve to isoproterenol in myocytes from
NE-treated guinea pigs was right shifted and depressed compared with
those from control animals (Fig. 8, cf Fig.
6). The pD2 value was decreased from 8.8 ± 0.2 to 7.5 ± 0.2 (n = 7, P < 0.01), and the
isoproterenol-to-Ca2+ ratio (maximum response to
isoproterenol compared with that for high Ca2+ in the same
cell) was reduced from 0.89 ± 0.06 to 0.58 ± 0.08 (n = 7, P < 0.01). The isoproterenol concentration-response curves were too depressed to repeat in the presence of blockers, so that the
functional contribution of 2-ARs could not be assessed.
Unexpectedly, there was no decrease in the number of -ARs in
NE-treated compared with control guinea pigs (Fig. 7).
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Pertussis toxin on myocytes from NE-treated guinea pigs.
Pertussis toxin treatment significantly reversed the -AR
desensitization in myocytes from NE-treated guinea pigs (Fig. 8) as
previously described (3). The isoproterenol-to-Ca2+ ratio
was restored to 0.88 ± 0.04 (P < 0.05, n = 6), and
the pD2 increased to 8.6 ± 0.3 (P < 0.01).
2-ARs. ICI 118,551 did not shift the concentration-response curve to isoproterenol
significantly in pertussis toxin-treated myocytes from NE-treated
guinea pigs (Fig. 8), nor was there any increase in the proportion of
2-AR as determined by radioligand binding (Fig. 7). Once
again, toxin treatment decreased the total number of -ARs.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Inactivation of Gi by pertussis toxin significantly
reversed the functional -AR desensitization in myocytes from
NE-treated guinea pigs. The maximum response to isoproterenol was
completely restored to control levels, and the EC50
decreased 10-fold. This was not due to either an upregulation or
increased coupling of 2-ARs. The guinea pig myocytes had
no detectable 2-AR by radioligand binding and no
functional 2-AR-mediated responses either before or
after treatment with pertussis toxin. -AR coupling was also increased by pertussis toxin in myocytes from control guinea pigs, with
concentration-response curves to isoproterenol shifted leftward by an
order of magnitude. Once again, this was without significant increases
in 2-AR number or function. Removal of Gi
can therefore increase the function of 1-ARs without
increasing their number. This strongly suggests that Gi
does not couple exclusively to the 2-AR but that it can
couple to the 1-AR also.
Comparison of guinea pig whole heart with myocytes washed through
Percoll confirms a previous study (7) showing that the 2-ARs detected in whole heart membranes are not from
myocytes but are from other cell types including fibroblasts,
endothelial cells, and smooth muscle cells. Until recently it was
thought that rat also lacked 2-ARs, since two studies
had reported that 2-AR binding in rat ventricle was also
localized to nonmyocyte components (4, 7). However, Xiao and Lakatta
(30) showed an increase in contraction in rat myocytes with zinterol
that was antagonized by ICI 118,551. This finding was reproduced by Kuztensov et al. (18). In contrast, a careful study by Kitagawa et al. (17), using full concentration-response curves to isoproterenol in the presence and absence of CGP-20712A and ICI 118,551, was unable
to detect 2-AR binding or cyclase stimulation in rat
myocyte membrane. We show a small functional effect of
2-AR on contraction in rat myocytes, revealed only when
1-ARs are blocked. The 2-AR binding
component was difficult to detect through competition radioligand
binding studies, being <5% of total, but was evident from
autoradiography of the single myocytes.
Surprisingly, zinterol was not able to mediate increases in contraction
through this minor 2-AR pool in rat myocytes.
Contractile responses to zinterol were abolished by the
1-AR but not the 2-AR antagonist. This
confirms previous work (19), also on isolated rat myocytes, where
zinterol acted mainly through 1-ARs even after pertussis
treatment. Zinterol is a 2-AR-selective partial agonist
often used to provide evidence that an effect is 2-AR
mediated in cardiac studies. Partial agonists are, by definition, less
active at the receptor, and therefore reductions in receptor number
will often abolish the action of partial agonists while the response to
full agonists is still evident. It may be that zinterol does not have
sufficient efficacy to produce a response through the minor
2-AR pool but is nonselective enough to stimulate the
much larger 1-AR population. Our results indicate that a response to zinterol should not be used as sole evidence that an effect
is 2-AR mediated without the concurrent use of specific antagonists.
Further evidence that inhibition of Gi can affect
1-AR function comes from a study of chloride channels in
guinea pig myocytes, where pertussis toxin enhanced the effect of
-agonist stimulation without apparent 2-AR
involvement (12). How can the results on guinea pigs myocytes be
reconciled with previous observations that pertussis toxin treatment
uncovered a latent pool of 2-AR in rat myocytes (29)? It
may be that when both 1- and
2-AR are present together, then the 2-ARs
preferentially couple to Gi. Alternatively, Gi
may simply produce a tonic inhibition of -AR coupling by a mechanism
unrelated to the receptors themselves. Removal of Gi
therefore increases stimulation of cAMP through any
Gs-coupled receptor. The effects observed on the
2-ARs are particularly strong only because this receptor
subtype mediates a submaximal response initially. Removal of
Gi therefore increases the maximum effect as well as
increases the sensitivity of the receptors. An example of this effect
is seen with the guinea pig myocytes in the present study. For the
desensitized cells, the maximum response to isoproterenol was increased
and the EC50 was decreased. For the control cells, the
EC50 was decreased but the maximum was unaffected, probably
because it was limited by the maximum contraction of the cell.
An understanding of the relative effects of the 1- and
2-ARs, and their coupling to Gi, is important in
relation to the failing human heart. Both 1- and
2-ARs produce increases in contraction on the same
ventricular myocyte (6), both probably acting through increases in cAMP
(14). Responses through both subtypes are desensitized compared with
nonfailing heart, although by different mechanisms, and the activity
and amount of Gi is raised (9). Desensitizing conditions,
under which the 2-AR is phosphorylated by cAMP-dependent
protein kinase (protein kinase A), were shown to shuttle
the 2-AR away from Gs and toward
Gi. It might therefore be predicted that conditions in the
failing human heart are heavily biased towards -AR-Gi
coupling. Because Gi has been shown to modulate both the
Na+/H+-exchanger (13) and the
Na+/Ca2+-exchanger (24) in cardiac muscle,
increased coupling to Gi via -ARs could well have
unexpected consequences. With the current debate about the use of
selective vs. nonselective -blockers in heart failure, it becomes
increasingly important to predict the consequence of stimulation
through the various subtypes.
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ACKNOWLEDGEMENTS |
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We thank Peter O'Gara for excellent technical assistance.
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FOOTNOTES |
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H. Ranu was supported by British Heart Foundation Grant PG/94018.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. E. Harding, Cardiac Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse St., London SW3 6LY, UK (E-mail: sian.harding{at}ic.ac.uk).
Received 21 May 1999; accepted in final form 2 December 1999.
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REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Bristow, MR,
Ginsburg R,
Umans V,
Fowler M,
Minobe W,
Rasmussen R,
Zera P,
Menlove R,
Shah P,
Jamieson S,
and
Stinson EB.
1- and 2-Adrenergic receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective 1-receptor down-regulation in heart failure.
Circ Res
59:
297-309,
1986
2.
Brodde, O.
1- and 2-adrenoceptors in the human heart: properties, function, and alterations in chronic heart failure.
Pharmacol Rev
43/2:
203-235,
1991[ISI][Medline].
3.
Brown, LA,
and
Harding SE.
The effect of pertussis toxin on -adrenoceptor responses in isolated cardiac myocytes from noradrenaline-treated guinea pigs and patients with cardiac failure.
Br J Pharmacol
106:
115-122,
1992[ISI][Medline].
4.
Buxton, ILO,
and
Brunton LL.
Direct analysis of -adrenergic receptor subtypes in intact adult ventricular myocytes of the rat.
Circ Res
56:
126-132,
1985
5.
Daaka, Y,
Luttrell LM,
and
Lefkowitz RJ.
Switching of the coupling of the 2-adrenergic receptor to different G proteins by protein kinase A.
Nature
390:
88-91,
1997[Medline].
6.
Del Monte, F,
Kaumann AJ,
Poole-Wilson PA,
Wynne DG,
and
Harding SE.
Coexistence of functioning 1- and 2-adrenoceptors in single myocytes from human ventricle.
Circulation
88:
854-863,
1993
7.
Freissmuth, M,
Hausleithner V,
Nees S,
Bock M,
and
Schutz W.
Cardiac ventricular 2-adrenoceptors in guinea pigs and rats are localized on the coronary endothelium.
Naunyn Schmiedebergs Arch Pharmacol
334:
56-62,
1986[ISI][Medline].
8.
Hall, JA,
Kaumann AJ,
and
Brown MJ.
Selective 1-adrenoceptor blockade enhances positive inotropic responses to endogenous catecholamines mediated through 2-adrenoceptors in human atrial myocardium.
Circ Res
66:
1610-1623,
1990
9.
Harding, SE,
Brown LA,
Wynne DG,
Davies CH,
and
Poole-Wilson PA.
Mechanisms of -adrenoceptor desensitisation in the failing human heart.
Cardiovasc Res
28:
1451-1460,
1994[ISI][Medline].
10.
Harding, SE,
Vescovo G,
Kirby M,
Jones SM,
Gurden J,
and
Poole-Wilson PA.
Contractile responses of isolated rat and rabbit myocytes to isoproterenol and calcium.
J Mol Cell Cardiol
20:
635-647,
1988[ISI][Medline].
11.
Heubach, JF,
Trebess I,
Wettwer E,
Himmel HM,
Michel MC,
Kaumann AJ,
Koch WJ,
Harding SE,
and
Ravens U.
L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac 2-adrenoceptor.
Cardiovasc Res
42:
173-182,
1999
12.
Hool, LC,
and
Harvey RD.
Role of 1- and 2-adrenergic receptors in regulation of Cl and Ca2+ channels in guinea pig ventricular myocytes.
Am J Physiol Heart Circ Physiol
273:
H1669-H1676,
1997
13.
Jiang, T,
and
Steinberg SF.
2-Adrenergic receptors enhance contractility by stimulating HCO3-dependent intracellular alkalinization.
Am J Physiol Heart Circ Physiol
273:
H1044-H1047,
1997
14.
Kaumann, AJ,
Bartel S,
Molenaar P,
Sanders L,
Burrell K,
Vetter D,
Hempel P,
Karczewski P,
and
Krause E-G.
Activation of 2-adrenergic receptors hastens relaxation and mediates phosphorylation of phospholamban, troponin I, and C-protein in ventricular myocardium from patients with terminal heart failure.
Circulation
99:
65-72,
1999
15.
Kaumann, AJ,
Hall JA,
Murray KJ,
Wells FC,
and
Brown MJ.
A comparison of the effects of adrenaline and noradrenaline on human heart: the role of 1- and 2-adrenoceptors in the stimulation of adenylate cyclase and contractile force.
Eur Heart J
10:
29-37,
1989.
16.
Kaumann, AJ,
and
Lemoine H.
2-Adrenoceptor-mediated positive inotropic effect of adrenaline in human ventricular myocardium.
Naunyn Schmiedebergs Arch Pharmacol
335:
403-411,
1987[ISI][Medline].
17.
Kitagawa, Y,
Adachi-Akahane S,
and
Nagao T.
Determination of -adrenoceptor subtype on rat isolated ventricular myocytes by use of highly selective -antagonists.
Br J Pharmacol
116:
1635-1643,
1995[ISI][Medline].
18.
Kuznetsov, V,
Pak E,
Robinson RB,
and
Steinberg SF.
2-Adrenergic receptor actions in neonatal and adult rat ventricular myocytes.
Circ Res
76:
40-52,
1995
19.
Laflamme, MA,
and
Becker PL.
Do 2-adrenergic receptors modulate Ca2+ in adult rat ventricular myocytes?
Am J Physiol Heart Circ Physiol
274:
H1308-H1314,
1998
20.
Lemoine, H,
and
Kaumann AJ.
Regional differences of 1- and 2-adrenoceptor-mediated functions in feline heart. A 2-adrenoceptor-mediated positive inotropic effect possibly unrelated to cyclic AMP.
Naunyn Schmiedebergs Arch Pharmacol
344:
56-69,
1991[ISI][Medline].
21.
Liggett, SB,
Wagoner LE,
Craft LL,
Hornung RW,
Hoit BD,
and
McIntosh TC.
The Ile164 2-adrenergic receptor polymorphism adversely affects the outcome of congestive heart failure.
J Clin Invest
102:
1534-1539,
1998[ISI][Medline].
22.
Lowry, OH,
Rosenbrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with the Folin phenol reagent.
J Biol Chem
193:
265-270,
1951
23.
Money-Kyrle, AR,
Davies CH,
and
Harding SE.
The role of cyclic AMP in the frequency-dependent changes of contraction of guinea pig cardiomyocytes.
Cardiovasc Res
37:
532-540,
1998
24.
Schubert, B,
VanDongen AM,
Kirsch GE,
and
Brown AM.
-Adrenergic inhibition of cardiac sodium channels by dual G-protein pathways.
Science
245:
516-519,
1989
25.
Vescovo, G,
Jones SM,
Harding SE,
and
Poole-Wilson PA.
Isoproterenol sensitivity of isolated cardiac myocytes from rats with monocrotaline-induced right-sided hypertrophy and heart failure.
J Mol Cell Cardiol
21:
1047-1061,
1989[ISI][Medline].
26.
Wynne, DG,
del Monte F,
and
Harding SE.
Cyclic AMP levels in ventricular myocytes from noradrenaline-treated guinea pigs.
Eur J Pharmacol
310:
235-242,
1996[ISI][Medline].
27.
Wynne, DG,
Poole-Wilson PA,
and
Harding SE.
Incomplete reversal of -adrenoceptor desensitisation by cyclic nucleotide phosphodiesterase inhibition in isolated cardiac myocytes from patients with cardiac failure and noradrenaline-treated guinea pigs.
Br J Pharmacol
109:
1071-1078,
1993[ISI][Medline].
28.
Xiao, RP,
Avdonin P,
Zhou YY,
Cheng H,
Akhter SA,
Eschenhagen T,
Lefkowitz RJ,
Koch WJ,
and
Lakatta EG.
Coupling of 2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.
Circ Res
84:
43-52,
1999
29.
Xiao, RP,
Ji X,
and
Lakatta EG.
Functional coupling of the 2-adrenoceptor to a pertussis toxin-sensitive G protein in cardiac myocytes.
Mol Pharmacol
47:
322-329,
1995[Abstract].
30.
Xiao, R-P,
and
Lakatta EG.
1-Adrenoceptor stimulation and 2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca2+ and Ca2+ current in single rat ventricular cells.
Circ Res
73:
286-300,
1993
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