AJP - Heart Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 278: H1807-H1814, 2000;
0363-6135/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ranu, H. K.
Right arrow Articles by Harding, S. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ranu, H. K.
Right arrow Articles by Harding, S. E.
Vol. 278, Issue 6, H1807-H1814, June 2000

Gi-dependent suppression of beta 1-adrenoceptor effects in ventricular myocytes from NE-treated guinea pigs

Hardeep K. Ranu, Judith C. W. Mak, Peter J. Barnes, and Sian E. Harding

Cardiac Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, London SW3 6LY, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

It has been suggested that there is a preferential coupling in heart muscle between the inhibitory G protein (Gi) and the beta 2-subtype of the beta -adrenergic receptor (beta -AR), since pertussis toxin (which inactivates Gi) reveals latent beta 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 beta -AR-agonist stimulation, and that pertussis toxin restores these responses. The purpose of the present investigation was to determine whether pertussis toxin specifically upregulated beta 2-ARs in myocytes from NE-treated guinea pigs. The sole beta -AR subtype in control guinea pig myocytes was confirmed as beta 1-AR by radioligand binding, single-cell autoradiography, and concentration-response curves to isoproterenol in contracting myocytes. In contrast, a minor pool of beta 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 beta 2-ARs. Gi is therefore able to modulate beta 1-AR responses in guinea pig myocytes.

G proteins; rat; contraction; norepinephrine; radioligand binding


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BOTH beta 1- AND beta 2-adrenergic receptors (ARs) can enhance ventricular contraction, with the contribution from the beta 2-AR subtype being particularly prominent in human myocardium (2, 6, 15). The relative contribution of the beta 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 beta 2-AR subtype has recently been suggested by the finding that a polymorphism that reduces the activity of beta 2-ARs is associated with greatly increased mortality in heart failure patients (21).

Classically, it was thought that beta -ARs coupled to adenylate cyclase via the stimulatory G protein, Gs. However, recent evidence has shown that dual coupling is a possibility, with beta -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 beta -subtypes in this effect, with beta 2-ARs selectively coupling to Gi. Evidence for this includes data in rat myocytes, where treatment with pertussis toxin (which inactivates Gi) preferentially upregulated beta 2-AR responses (29), and from transgenic (TG) mice overexpressing the human beta 2-AR (TG-beta 2). Myocytes from these mice were shown to have reduced or absent responses to beta -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 beta 2-AR receptors (28). However, both we (11) and Xiao et al. (28) have observed that in TG-beta 2 mice, it was not only the response through the overexpressed beta 2-AR that was lost when Gi was increased but also that through the native beta 1-AR, which mediates the majority of the beta -AR effect in wild-type mice.

These observations may have relevance to the mechanism of beta -AR desensitization in human heart failure. The loss of responsiveness to beta -agonists is accompanied by a decrease in beta 1-AR but not beta 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 beta -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 beta 2-AR receptors. A parallel study on myocytes from guinea pigs infused with norepinephrine to produce beta -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 beta 2-AR subtype to the increased ventricular contraction produced by isoproterenol (6). However, the possibility still exists that a pool of latent beta 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 beta -AR desensitization by pertussis toxin in myocytes from norepinephrine-treated guinea pigs was due to rescue of the beta 1-AR responses or uncovering of latent beta 2-AR. If the first option is correct, then the hypothesis that beta 2-ARs are uniquely modulated by Gi is contradicted. We have included data from radioligand binding and single-cell autoradiography to show the strong beta 1-AR dependency of guinea pig myocytes. These are compared with data from whole heart to confirm previous observations that beta 2-ARs in ventricular homogenates are largely associated with nonmyocyte cell types. Comparison is also made with rat myocytes, where a minor functional beta 2-AR component has been described. We show that guinea pig myocyte responses restored by pertussis toxin are mediated entirely by beta 1-AR, indicating that increased Gi is able to suppress effect on contraction mediated through either the beta 1- or the beta 2-AR subtypes.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 · h-1. 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.

Portions of membrane at a protein concentration of 20 µg/tube were incubated with [125I]iodocyanopindolol ([125I]ICYP; specific activity, 2,000 Ci/mmol) in the absence and presence of 200 mmol/l of isoproterenol (to define nonspecific binding) in 25 mmol/l Tris · HCl buffer (pH 7.4 at 37°C) containing 154 mmol/l NaCl and 1.1 mmol/l ascorbic acid (to prevent oxidation of isoproterenol). The density of beta 2-ARs was determined in the presence of 300 nmol/l CGP-20712A (a highly selective beta 1-antagonist), a concentration at which >99% of beta 1-ARs and <1% of beta 2-ARs are predicted to be occupied (8, 16, 20). The density of beta 1-ARs was analyzed by ICYP saturation binding in the presence of 50 nmol/l ICI 118,551 (a selective beta 2-antagonist), a concentration at which >99% of beta 2-ARs and <5% of beta 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 beta 1-AR, beta 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 (beta 1-AR, 300 nmol/l) or ICI 118,551 (beta 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 1- to beta 2-ARs was 46 to 54. In agreement with this, the high-affinity binding site for CGP-20712A (beta 1-AR) comprised 45-50% of total binding. Full displacement was not attained with CGP-20712A because of its extremely low affinity for the beta 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).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1.   A: displacement of [125I]iodocyanopindolol ([125I]ICYP) binding by specific antagonists CGP-20712A [beta 1-adrenergic receptor (beta 1-AR)] and ICI 118,551 (beta 2-AR) in membranes from guinea pig whole heart (each point shows mean ± SE of n = 4 preparations from 4 animals, with all determinations done in triplicate). B: displacement of [125I]ICYP binding by specific antagonists CGP-20712A (beta 1-AR) and ICI 118,551 (beta 2-AR) in membranes from guinea pig myocytes (each point shows mean ± SE of n = 4 preparations from 8 animals, with all determinations done in triplicate).

In contrast to whole heart, competition curves in myocyte membranes (Fig. 1B) were essentially monophasic. The Ki for CGP-20712A was 1.4 ± 0.1 nmol/l, close to that for the high-affinity site in whole heart, whereas that for ICI 118,551 was 110 ± 12 nmol/l, close to its low-affinity value in whole heart. This indicates that all receptors detected were of the beta 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 beta 1- to beta 2-AR ratio of 48 to 52. In agreement with this, the high-affinity binding site for CGP-20712A (beta 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 beta 2-ARs.

Single myocyte receptor autoradiography. In a further attempt to eliminate the contribution of other cell types to the beta -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 beta 2-AR-selective ICI 118,551, whereas almost all specific activity was removed with the beta 1-AR-selective CGP-20712A (Fig. 2). Rat myocytes showed significantly more displacement by ICI 118,551, indicating a larger proportion of beta 2-ARs on the ventricular myocytes.


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 2.   Grain density of images of single ventricular myocytes from guinea pig (G. pig) or rat heart. For each condition, 10 myocytes were measured from each of 3 rats or guinea pigs (120 myocytes in total for each species). Total [125I]ICYP binding was displaced by 50 nmol/l ICI 118,551 (to block beta 2-ARs), 300 nmol/l CGP-20712A (to block beta 1-ARs), or 200 µmol/l isoproterenol (to give nonspecific binding, NS). Results in presence of blockers are related to total grain density. Data were pooled for each animal so that points are means ± SE for n = 3. * P < 0.05 compared with guinea pig.

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 beta 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 beta 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).


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3.   Contractile responses to isoproterenol (Iso) in guinea pig ventricular myocytes. Sequential concentration-response curves were constructed on same myocytes in presence and absence of 50 nmol/l ICI 118,551 (ICI; to block beta 2-ARs), 300 nmol/l CGP-20712A (CGP; to block beta 1-ARs), or both. Responses are shown as percentages of maximum change in contraction amplitude. Data are means ± SE; n = no. of animals. A: myocytes in absence and presence of ICI 118,551 (n = 6). Maximum contraction amplitudes were 9.1 ± 1.3 for first curve and 8.7 ± 1.6% shortening for second curve. B: myocytes in absence and presence of CGP-20172A and then ICI + CGP (n = 4). Maximum amplitudes were 11.7 ± 1.0 for first curve, 10.9 ± 1.3 for second curve, and 9.7 ± 1.7% shortening for third curve. C: 3 consecutive concentration-response curves to isoproterenol in absence of antagonists. Maximum amplitudes were 9.5 ± 1.2 for first curve (n = 7), 8.6 ± 1.1 for second curve (n = 7), and 10.0 ± 1.4% shortening for third curve (n = 4). There was no significant decrease in maximum amplitude with time for any series of myocytes.

In rat myocytes the pattern was slightly different. Although ICI 118,551 alone produced little shift in the isoproterenol concentration-response curves (Fig. 4A), the presence of CGP-20712A revealed a biphasic response (Fig. 4B). The first portion of the response was reduced by the further addition of ICI 118,551, with the pD2 decreased from 6.4 ± 0.4 to 5.2 ± 0.4 (n = 8, P < 0.01; Fig. 4B). This indicates a minor beta 2-AR-mediated component of contraction revealed when the beta 1-ARs were blocked. Once again, the three time-matched controls were superimposable (Fig. 4C).


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 4.   Contractile responses to isoproterenol in rat ventricular myocytes. Sequential concentration-response curves were constructed on the same myocytes in presence and absence of 50 nmol/l ICI 118,551 (to block beta 2-ARs), 300 nmol/l CGP-20712A (to block beta 1-ARs), or both. Responses are shown as percentages of maximum change in contraction amplitude. Data are means ± SE; n = no. of animals. A: myocytes in absence and presence of ICI 118,551 (n = 5). Maximum contraction amplitudes were 12.9 ± 1.2 for first curve and 12.3 ± 1.7% shortening for second curve. B: myocytes in absence and presence of CGP-20172A and then ICI + CGP (n = 6-8). Maximum amplitudes were 14.3 ± 1.4 for first curve, 10.7 ± 1.1 for second curve, and 11.7 ± 1.7% shortening for third curve. C: 3 consecutive concentration-response curves to isoproterenol in absence of antagonists (n = 7). Maximum amplitudes were 11.4 ± 1.2 for first curve, 11.2 ± 1.9 for second curve, and 10.8 ± 2.1% shortening for third curve. There was no significant decrease in maximum amplitude with time for any series of myocytes.

Zinterol is a beta 2-AR-selective agonist and has been used to show that contractile effects on cardiac myocytes are beta 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 beta 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 beta 1-ARs predominate, it appears that the majority of the effect of zinterol is mediated through the beta 1-AR subtype.


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5.   Contractile responses to zinterol in rat ventricular myocytes. Sequential concentration-response curves were constructed on same myocytes in presence and absence of 50 nmol/l ICI 118,551 (to block beta 2-ARs) or 300 nmol/l CGP-20712A (to block beta 1-ARs). Responses are shown as percentages of maximum first zinterol response (Zint 1). Data are means ± SE; n = no. of animals. A: myocytes in absence and presence of ICI 118,551 (n = 4). B: myocytes in absence and presence of CGP-20712A (n = 4).

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 beta 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 beta 2-AR number, since there was no increase in the number or proportion of beta 2-ARs detected by radioligand binding (Fig. 7). (For this series the beta 1-AR number was determined by displacement of beta 2-AR binding with 50 nmol/l ICI 118,551.) In fact, pertussis toxin treatment slightly decreased the total beta -AR number: this may be due to the lability of beta -ARs during the 2-h incubation, since a parallel incubation without toxin had a similar effect (data not shown).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 6.   Contractile responses to isoproterenol in myocytes from sham-operated guinea pigs: control (Con), untreated (circles, n = 7) or pertussis toxin (PTX) treated (squares, n = 6). Sequential concentration-response curves were constructed on same myocytes in absence (solid line and symbols) and presence (dashed line and open symbols) of 50 nmol/l ICI 118,551. Responses are shown as percentages of maximum (max) change in contraction amplitude. Data are means ± SE; n = no. of animals.



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 7.   Total beta -AR and specific beta 1-AR no. determined by saturation binding of [125I]ICYP in absence and presence of 50 nmol/l ICI 118,551. Untreated or PTX-treated myocytes were from sham-operated (n = 6) or norepinephrine (NE)-treated (n = 6-7) guinea pigs. Bmax, maximal binding capacity.

Desensitization of the beta -AR response in myocytes from NE-treated guinea pigs. Seven days of infusion of NE produced a robust beta -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 beta 2-ARs could not be assessed. Unexpectedly, there was no decrease in the number of beta -ARs in NE-treated compared with control guinea pigs (Fig. 7).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 8.   Contractile responses to isoproterenol, relative to maximum Ca2+ in the same cell, in ventricular myocytes from NE-treated guinea pigs: control, untreated (circles, n = 7) or PTX treated (squares, n = 6). Sequential concentration-response curves were constructed on same myocytes in absence (solid line and symbols) and presence (dashed line and open symbol) of 50 nmol/l ICI 118,551. Responses are shown as percentages of maximum change in contraction amplitude. Data are means ± SE; n = no. of animals.

Pertussis toxin on myocytes from NE-treated guinea pigs. Pertussis toxin treatment significantly reversed the beta -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).

The increase in sensitivity to isoproterenol was not due to upregulation or revealing of latent beta 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 beta 2-AR as determined by radioligand binding (Fig. 7). Once again, toxin treatment decreased the total number of beta -ARs.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Inactivation of Gi by pertussis toxin significantly reversed the functional beta -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 beta 2-ARs. The guinea pig myocytes had no detectable beta 2-AR by radioligand binding and no functional beta 2-AR-mediated responses either before or after treatment with pertussis toxin. beta -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 beta 2-AR number or function. Removal of Gi can therefore increase the function of beta 1-ARs without increasing their number. This strongly suggests that Gi does not couple exclusively to the beta 2-AR but that it can couple to the beta 1-AR also.

Comparison of guinea pig whole heart with myocytes washed through Percoll confirms a previous study (7) showing that the beta 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 beta 2-ARs, since two studies had reported that beta 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 beta 2-AR binding or cyclase stimulation in rat myocyte membrane. We show a small functional effect of beta 2-AR on contraction in rat myocytes, revealed only when beta 1-ARs are blocked. The beta 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 beta 2-AR pool in rat myocytes. Contractile responses to zinterol were abolished by the beta 1-AR but not the beta 2-AR antagonist. This confirms previous work (19), also on isolated rat myocytes, where zinterol acted mainly through beta 1-ARs even after pertussis treatment. Zinterol is a beta 2-AR-selective partial agonist often used to provide evidence that an effect is beta 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 beta 2-AR pool but is nonselective enough to stimulate the much larger beta 1-AR population. Our results indicate that a response to zinterol should not be used as sole evidence that an effect is beta 2-AR mediated without the concurrent use of specific antagonists.

Further evidence that inhibition of Gi can affect beta 1-AR function comes from a study of chloride channels in guinea pig myocytes, where pertussis toxin enhanced the effect of beta -agonist stimulation without apparent beta 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 beta 2-AR in rat myocytes (29)? It may be that when both beta 1- and beta 2-AR are present together, then the beta 2-ARs preferentially couple to Gi. Alternatively, Gi may simply produce a tonic inhibition of beta -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 beta 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 beta 1- and beta 2-ARs, and their coupling to Gi, is important in relation to the failing human heart. Both beta 1- and beta 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 beta 2-AR is phosphorylated by cAMP-dependent protein kinase (protein kinase A), were shown to shuttle the beta 2-AR away from Gs and toward Gi. It might therefore be predicted that conditions in the failing human heart are heavily biased towards beta -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 beta -ARs could well have unexpected consequences. With the current debate about the use of selective vs. nonselective beta -blockers in heart failure, it becomes increasingly important to predict the consequence of stimulation through the various subtypes.


    ACKNOWLEDGEMENTS

We thank Peter O'Gara for excellent technical assistance.


    FOOTNOTES

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.


    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. beta 1- and beta 2-Adrenergic receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1-receptor down-regulation in heart failure. Circ Res 59: 297-309, 1986[Abstract/Free Full Text].

2.   Brodde, O. beta 1- and beta 2-adrenoceptors in the human heart: properties, function, and alterations in chronic heart failure. Pharmacol Rev 43/2: 203-235, 1991[Web of Science][Medline].

3.   Brown, LA, and Harding SE. The effect of pertussis toxin on beta -adrenoceptor responses in isolated cardiac myocytes from noradrenaline-treated guinea pigs and patients with cardiac failure. Br J Pharmacol 106: 115-122, 1992[Web of Science][Medline].

4.   Buxton, ILO, and Brunton LL. Direct analysis of beta -adrenergic receptor subtypes in intact adult ventricular myocytes of the rat. Circ Res 56: 126-132, 1985[Abstract/Free Full Text].

5.   Daaka, Y, Luttrell LM, and Lefkowitz RJ. Switching of the coupling of the beta 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 beta 1- and beta 2-adrenoceptors in single myocytes from human ventricle. Circulation 88: 854-863, 1993[Abstract/Free Full Text].

7.   Freissmuth, M, Hausleithner V, Nees S, Bock M, and Schutz W. Cardiac ventricular beta 2-adrenoceptors in guinea pigs and rats are localized on the coronary endothelium. Naunyn Schmiedebergs Arch Pharmacol 334: 56-62, 1986[Web of Science][Medline].

8.   Hall, JA, Kaumann AJ, and Brown MJ. Selective beta 1-adrenoceptor blockade enhances positive inotropic responses to endogenous catecholamines mediated through beta 2-adrenoceptors in human atrial myocardium. Circ Res 66: 1610-1623, 1990[Abstract/Free Full Text].

9.   Harding, SE, Brown LA, Wynne DG, Davies CH, and Poole-Wilson PA. Mechanisms of beta -adrenoceptor desensitisation in the failing human heart. Cardiovasc Res 28: 1451-1460, 1994[Web of Science][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[Web of Science][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 beta 2-adrenoceptor. Cardiovasc Res 42: 173-182, 1999[Abstract/Free Full Text].

12.   Hool, LC, and Harvey RD. Role of beta 1- and beta 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[Abstract/Free Full Text].

13.   Jiang, T, and Steinberg SF. beta 2-Adrenergic receptors enhance contractility by stimulating HCO-3-dependent intracellular alkalinization. Am J Physiol Heart Circ Physiol 273: H1044-H1047, 1997[Abstract/Free Full Text].

14.   Kaumann, AJ, Bartel S, Molenaar P, Sanders L, Burrell K, Vetter D, Hempel P, Karczewski P, and Krause E-G. Activation of beta 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[Abstract/Free Full Text].

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 beta 1- and beta 2-adrenoceptors in the stimulation of adenylate cyclase and contractile force. Eur Heart J 10: 29-37, 1989.

16.   Kaumann, AJ, and Lemoine H. beta 2-Adrenoceptor-mediated positive inotropic effect of adrenaline in human ventricular myocardium. Naunyn Schmiedebergs Arch Pharmacol 335: 403-411, 1987[Web of Science][Medline].

17.   Kitagawa, Y, Adachi-Akahane S, and Nagao T. Determination of beta -adrenoceptor subtype on rat isolated ventricular myocytes by use of highly selective beta -antagonists. Br J Pharmacol 116: 1635-1643, 1995[Web of Science][Medline].

18.   Kuznetsov, V, Pak E, Robinson RB, and Steinberg SF. beta 2-Adrenergic receptor actions in neonatal and adult rat ventricular myocytes. Circ Res 76: 40-52, 1995[Abstract/Free Full Text].

19.   Laflamme, MA, and Becker PL. Do beta 2-adrenergic receptors modulate Ca2+ in adult rat ventricular myocytes? Am J Physiol Heart Circ Physiol 274: H1308-H1314, 1998[Abstract/Free Full Text].

20.   Lemoine, H, and Kaumann AJ. Regional differences of beta 1- and beta 2-adrenoceptor-mediated functions in feline heart. A beta 2-adrenoceptor-mediated positive inotropic effect possibly unrelated to cyclic AMP. Naunyn Schmiedebergs Arch Pharmacol 344: 56-69, 1991[Web of Science][Medline].

21.   Liggett, SB, Wagoner LE, Craft LL, Hornung RW, Hoit BD, and McIntosh TC. The Ile164 beta 2-adrenergic receptor polymorphism adversely affects the outcome of congestive heart failure. J Clin Invest 102: 1534-1539, 1998[Web of Science][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[Free Full Text].

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[Abstract/Free Full Text].

24.   Schubert, B, VanDongen AM, Kirsch GE, and Brown AM. beta -Adrenergic inhibition of cardiac sodium channels by dual G-protein pathways. Science 245: 516-519, 1989[Abstract/Free Full Text].

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[Web of Science][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[Web of Science][Medline].

27.   Wynne, DG, Poole-Wilson PA, and Harding SE. Incomplete reversal of beta -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[Web of Science][Medline].

28.   Xiao, RP, Avdonin P, Zhou YY, Cheng H, Akhter SA, Eschenhagen T, Lefkowitz RJ, Koch WJ, and Lakatta EG. Coupling of beta 2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes. Circ Res 84: 43-52, 1999[Abstract/Free Full Text].

29.   Xiao, RP, Ji X, and Lakatta EG. Functional coupling of the beta 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. beta 1-Adrenoceptor stimulation and beta 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[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 278(6):H1807-H1814
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
R. V. Iancu, G. Ramamurthy, S. Warrier, V. O. Nikolaev, M. J. Lohse, S. W. Jones, and R. D. Harvey
Cytoplasmic cAMP concentrations in intact cardiac myocytes
Am J Physiol Cell Physiol, August 1, 2008; 295(2): C414 - C422.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ranu, H. K.
Right arrow Articles by Harding, S. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ranu, H. K.
Right arrow Articles by Harding, S. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online