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CARDIOVASCULAR

In Vitro Evidence That Carteolol Is a Nonconventional Partial Agonist of Guinea Pig Cardiac ₁-Adrenoceptors: A Comparison with Xamoterol

Department of Pharmacology and Anesthesiology, Pharmacology Section (M.F., G.F., L.Q., P.D.) and Eye Clinic (M.T.D.), University of Padova, Padova, Italy; and Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, Ferrara, Italy (K.V., P.A.B.)

Received for publication May 6, 2005
Accepted September 8, 2005.

	Abstract

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The present study was designed to verify our previous hypothesis that carteolol, a ₁/₂-adrenoceptor-blocking agent, is a nonconventional partial agonist of cardiac ₁-adrenoceptors. To this purpose, we characterized the effects of carteolol in guinea pig myocardial preparations and measured the affinities of carteolol for high- and low-affinity sites of ₁-adrenoceptors labeled by CGP12177 [(–)4-(3-t-butylamino-2-hydroxypropoxy)-2-benzimidazol-2-one]. All experiments were performed in comparison with xamoterol, a cardioselective ₁-adrenoceptor partial agonist. Both drugs caused cAMP-dependent positive inotropic and chronotropic effects, but carteolol was less effective and less potent than xamoterol, and its cardiac actions were not affected by conventional concentrations of the -blocker propranolol. Both carteolol and xamoterol antagonized the cardiac effects of isoprenaline, but although the antagonistic concentrations of xamoterol were almost equal to those producing cardiostimulation, the antagonistic concentrations of carteolol were 3 log units lower than those causing cardiostimulant effects. Both carteolol and xamoterol competed with (–)[³H]CGP12177 for a high-affinity site of ₁-adrenoceptors, but carteolol showed a higher affinity than xamoterol. Moreover, carteolol, unlike xamoterol, bound also to a low-affinity site of the receptors. The binding affinity constants of the drugs for the high-affinity site correlated well with the respective blocking potencies against isoprenaline, whereas the affinity constant of carteolol for the low-affinity site was well related to its agonist potency. In conclusion, our findings demonstrate that carteolol, unlike xamoterol, is a nonconventional partial agonist, which causes agonistic effects through interaction with the low-affinity propranolol-resistant site of ₁-adrenoceptors and antagonistic actions through the high-affinity site of the same receptors.

Within the last 20 years, much evidence has been accumulated that partial agonists of the -adrenoceptors have two modes of action. Although conventional partial agonists produce stimulant effects at concentrations equal to those causing blockade of -adrenoceptors, other compounds, namely the nonconventional partial agonists, produce cardiostimulation at concentrations greater than those blocking the effects of catecholamines (Kaumann and Blinks, 1980; Kaumann, 1989; Takayanagi et al., 1989), their agonistic effects being resistant to blockade by the classic -antagonist propranolol (Sarsero et al., 1999). Currently, the behavior of these agents is ascribed to the existence of two different active sites or conformations of the ₁-adrenoceptor: an H site through which the effects of catecholamines are blocked and an L site generally defined as the propranolol-resistant state of the ₁-adrenoceptor (Bundkirchen et al., 2002) through which the stimulant effects of these partial agonists are mediated (Konkar et al., 2000; Granneman, 2001; Baker et al., 2003; Arch, 2004; Baker, 2005). Various compounds have been recently classified as nonconventional partial agonists of the ₁-adrenoceptor (Sarsero et al., 1999; Bundkirchen et al., 2002; Lowe et al., 2002; Joseph et al., 2003). Among them, CGP12177 contains, like carteolol, an aryloxypropanolamine moiety (Fig. 1) (Sarsero et al., 1998, 1999; Konkar et al., 2000; Sarsero et al., 2003; Joseph et al., 2004). Therefore, we wondered whether carteolol also behaves as a nonconventional partial agonist of ₁-adrenoceptors. We recently showed that, in rat-isolated myocardial preparations, the drug antagonized the effects of isoprenaline at concentrations 2 log units lower than those causing positive inotropic and chronotropic effects (Floreani et al., 2004). Moreover, cardiostimulation by carteolol was resistant to a concentration of propranolol (1 µM) greater than that antagonizing the effects of catecholamines in the same preparations. Collectively, these findings indicate that carteolol behaves as a nonconventional partial agonist of rat ₁-adrenoceptors.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Chemical structures of carteolol and CGP12177 showing the common aryloxypropanolamine moiety.

Our results demonstrate, for the first time, that carteolol binds with different affinities to both the high- and low-affinity sites of ₁-adrenoceptors present in guinea pig heart membranes. Moreover, here we show that carteolol behaves like a nonconventional partial agonist, eliciting agonistic responses through its interaction with the low-affinity propranolol-resistant site of ₁-adrenoceptors while antagonizing the effects of catecholamines through its interaction with the high-affinity site. Therefore, carteolol markedly differs from xamoterol, which behaves as a conventional partial agonist.

	Materials and Methods

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Animals
The procedures described below involving animals and their care were in conformity with institutional guidelines that comply with national and international laws and policies (National Institutes of Health Guide for the Care and Use of Laboratory Animals, National Institutes of Health Publication 85-23, 1985; European Economic Community Council Directive 86/609, OJ L 358, 1, Dec. 12, 1987).

Female guinea pigs (300–380 g), obtained from Harlan Italy (S. Pietro al Natisone, Udine, Italy), were kept in controlled environmental conditions (temperature, 23 ± 2°C; light/dark cycle, 7:00 AM to 7:00 PM). Animals had free access to a standard laboratory diet and to water. To obtain myocardial tissues depleted in endogenous catecholamines, the animals were treated daily for 2 days with reserpine (2 mg/kg i.p.) before sacrifice. At the time of sacrifice, animals were anesthetized by inhalation of methoxyflurane and then killed by cervical dislocation; the hearts were then rapidly removed.

Isolated Myocardial Preparations
Evaluation of Chronotropic and Inotropic Effects. The experiments were carried out on both spontaneously beating atria and electrically driven (2 Hz) left atria, as previously described (Floreani et al., 2003). Since myocardial preparations were isolated from reserpine-treated animals, the occurred depletion of catecholamines was confirmed by the lack of any effect following addition of 1.5 µM tyramine before the beginning of each experiment. Atria responding to tyramine, arrhythmic atria, as well as atria with basal frequency less than 100 and more than 160 beats/min, were discarded. In each atrial preparation, a cumulative concentration-response curve for isoprenaline was obtained before the addition of the drugs to determine the maximum effect (E_max) induced by the catecholamine. When three consecutive increasing concentrations of isoprenaline did not significantly modify the functional response of atria, the elicited effect was considered to be the E_max. Noncumulative concentration-effect curves were then obtained for increasing concentrations of both carteolol (from 10 nM to 0.1 mM) and xamoterol (from 1 nM to 10 µM). Three minutes after the maximum response to each drug concentration was reached, the atria were washed before the addition of a higher concentration. Chronotropic effects, determined in spontaneously beating atria, were defined as the difference between the heart rate (beats per minute) before and after drug additions and were expressed as percentages of the E_max induced by isoprenaline in the same preparation. Inotropic effects, determined both in spontaneously beating atria and in electrically driven (2 Hz) left atria, were defined as the difference between the force of contraction before and after drug addition and were expressed as percentages of the E_max induced by isoprenaline in the same preparation. The concentration-response data from each individual curve were evaluated by means of the Prism 3.03 software (GraphPad Software Inc., San Diego, CA) by sigmoidal curve fitting using eq. 1,

(1)

where E_min is the effect in the absence of the agonist, E_max represents the maximum agonist-induced effect, X is the molar concentration of the agonist, n_H is the Hill slope, and log EC₅₀ is the log of molar concentration of the agonist that produces a half-maximal response. In this way, we obtained the individual values of the negative log of EC₅₀; i.e., the pD₂ value [pD₂ = –log EC₅₀ (molar)], as well as the Hill slope value. Intrinsic activity (), defined as the fraction of the maximum effect of the drug compared with the maximum response elicited by isoprenaline in the same preparation, was calculated from individual concentration-effect curves. Thereafter, the individual values of each parameter were averaged and the obtained values were used to fit the curves from the averaged experimental data. Isometric contraction curves were analyzed for time to peak force (t₁), relaxation time (t₂), mean velocity of force development (S₁), and mean velocity of relaxation (S₂), according to Reiter (1972). In some sets of experiments, propranolol, butoxamine, prazosin, carbachol, or 3-isobutyl-1-methylxanthine (IBMX) was added to the bath medium 20 to 30 min before the addition of carteolol or xamoterol.

Evaluation of pA₂ and Constants of Dissociation (K_b) for Carteolol and Xamoterol. Some experiments evaluated the antagonistic effects of carteolol and xamoterol toward the positive chronotropic and inotropic effects of isoprenaline in spontaneously beating atria and in electrically driven left atria, respectively. To this end, a cumulative concentration-response curve for isoprenaline was first obtained in the presence of increasing concentrations of isoprenaline (from 1 nM to 3 µM). The cardiac tissue was then washed, and the bathing fluid was replaced two to three times until the developed tension was stable. Appropriate concentrations of carteolol or xamoterol were then added to the bathing medium, and the cardiac preparation was allowed to equilibrate for 30 min before the onset of the cumulative concentration-response curve for isoprenaline. To quantify the potency of carteolol and xamoterol as antagonists toward isoprenaline effects, experiments were carried out in the presence of three increasing concentrations of carteolol (from 5 to 50 nM) or xamoterol (0.1, 0.5, and 1 µM). Individual pA₂ values were determined from each experiment by linear regression analysis of log (DR-1) against log [B] plots (Arunlakshana and Schild, 1959), where [B] represents the molar concentration of the antagonist and DR, the dose ratio, is the ratio of the concentrations of isoprenaline required to produce an identical response in the presence and absence of the antagonist. Since the maximum response to isoprenaline was identical both in the absence and presence of the antagonist, DR was calculated as the ratio of EC₅₀ values in the presence and absence of the antagonist. In these experiments, the slope of the Schild plots appeared near to unit for both drugs (1.01 ± 0.03 and 1.18 ± 0.14, n = 3, for carteolol and xamoterol, respectively), indicating that pA₂ was independent from the concentrations of carteolol or xamoterol (MacKay, 1978). Therefore, it was possible to routinely estimate each pA₂ value using the relationship: pA₂ = log(DR-1) – log [B] (MacKay, 1978). Moreover, the dissociation constants K_b for carteolol and xamoterol were obtained using a single antagonist concentration, from the equation: K_b = [B]/(DR-1) (Besse and Furchgott, 1976).

Biochemical Assays
Preparation of Membranes from Guinea Pig Ventricular Tissue. Guinea pig cardiac ventricular tissue was homogenized in 10 volumes (w/v) of ice-cold 50 mM Tris-HCl, pH 7.4, and 10 mM MgCl₂. The homogenate was centrifuged at 48,000g for 10 min. The pellet was suspended in the same buffer, centrifuged again at 48,000g for 10 min, and used for binding assays and adenylate cyclase activity measurement. Protein content was determined according to a Bio-Rad method (Bredford, 1976) with bovine serum albumin as standard.

(–)[³H]CGP12177 Saturation Binding Experiments. In (–)[³H]CGP12177 receptor saturation binding assays, the membrane suspensions (100–120 µg of proteins/100 µl) were incubated for 120 min at 25°C in a medium (0.25 ml final volume) containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, and 18 to 20 increasing concentrations (from 0.1–100 nM) of (–)[³H]CGP12177. Nonspecific binding, which increased linearly with concentrations, was defined with 20 µM propranolol and represented 25 ± 3% of total radioactivity bound. Bound radioligand was isolated by filtration through Whatman GF/C glass fiber filters using a Brandel cell harvester (Brandel Inc., Gaithersburg, MD). Filters were washed three times with 50 mM Tris-HCl, pH 7.4, at 4°C and placed in liquid scintillation vials containing 5 ml of Aquassure (PerkinElmer Life and Analytical Sciences, Boston, MA). The radioactivity retained on filters was determined in a Beckman Liquid Scintillation Spectrometer at 55% efficiency. Dissociation equilibrium constants for saturation binding of (–)[³H]CGP12177 (K_d), as well as the maximum densities of specific binding sites (B_max) for (–)[³H]CGP12177, were calculated for a system of one or two binding site populations by nonlinear curve fitting using the program Ligand (Munson and Rodbard, 1980) purchased from Kell Biosoft (Ferguson, MO).

Competition Binding Studies. The affinities of carteolol and xamoterol for -adrenoceptor high- and low-affinity sites were determined by their ability to displace (–)[³H]CGP12177. Increasing concentrations (from 0.1 nM to 100 µM) of the tested compounds were assessed for displacement of both 1 nM [³H]CGP12177 bound to the high-affinity site and 30 nM [³H]CGP12177 bound to the low-affinity site. Nonspecific binding was determined in the presence of 20 µM (±)-propranolol and was about 25 to 30% of total binding. After 120 min of incubation at 25°C in a medium (0.25 ml of final volume) containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, and membrane suspensions (100–120 µg of proteins), bound and free radioactivity were separated by filtering the assay mixture through Whatman GF/C glass-fiber filters using a Brandel cell harvester. Filters that were washed three times with 50 mM Tris-HCl, pH 7.4, at 4°C were then placed in liquid scintillation vials containing 5 ml of Aquassure (PerkinElmer Life and Analytical Sciences), and the radioactivity retained on filters was determined in a Beckman Liquid Scintillation Spectrometer. IC₅₀ values for carteolol and xamoterol (where IC₅₀ is the concentration of the drug that displaces 50% of the labeled ligand) were obtained for the binding of (–)[³H]CGP12177 to both its high- and low-affinity sites and were calculated for a system of one or two binding site populations by nonlinear curve fitting using the program Ligand (Munson and Rodbard, 1980). pK_i values [=–log K_i (molar)], where K_i is the binding inhibition constant, were calculated from the Cheng-Prusoff equation (1973) with K_i = IC₅₀/(1 + C*/K_d*), where C* is the radioligand concentration used (1 and 30 nM for the high- and low-affinity sites, respectively) and K_d* is the obtained dissociation constant of radioligand from the high- and low-affinity sites (see Results).

Assay of Adenylate Cyclase Activity. Membrane preparations (100 µg/100 µl) obtained from guinea pig heart were preincubated for 10 min in a shaking bath at 30°C in a medium (final volume 0.5 ml) containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 1 mM EDTA, pH 7.4, 5 µM GTP, and 0.5 mM IBMX, as a nonspecific phosphodiesterase inhibitor. At the end of the preincubation period, 0.5 mM ATP and increasing concentrations (1 nM to 10 µM) of carteolol or xamoterol were added. The reaction was stopped after 10 min by transferring the tubes to a boiling water bath for 2 min. The samples were then cooled to room temperature and centrifuged at 2000g for 10 min at 4°C, and the supernatants were analyzed for cAMP content by a competition protein binding assay as previously reported (Varani et al., 1998). In brief, [³H]cAMP was incubated with binding protein, previously prepared from beef adrenals, added to the samples, and incubated at 4°C for 150 min. At the end of the incubation time and after the addition of charcoal, the samples were centrifuged at 2000g for 10 min. The clear supernatant was mixed with Atomlight and counted in a LS-1800 Beckman scintillation counter. In our experimental conditions, the basal value of adenylate cyclase activity was 140 ± 15 pmol cAMP/10 min/mg protein. EC₅₀ values were calculated by nonlinear least-squares curve fitting using the Prism 3.03 software.

Drugs. (±)-Carteolol hydrochloride was provided by Società Industria Farmaceutica Italiana S.p.A. (Catania, Italy). Xamoterol hemifumarate, (±)-propranolol hydrochloride, butoxamine hydrochloride, (±)CGP12177 hydrochloride, reserpine, tyramine hydrochloride, (±)-isoprenaline hydrochloride, carbamylcholine chloride (carbachol), prazosin hydrochloride, IBMX, and ATP were from Sigma-Aldrich (St. Louis, MO). (–)[³H]CGP12177 was purchased from PerkinElmer Life and Analytical Sciences, and [³H]cAMP was obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). All other reagents were of analytical grade and obtained from commercial sources. Reserpine was dissolved in distilled water containing 10% ascorbic acid. Ascorbic acid (0.1 mM) was also added to isoprenaline solution to prevent auto-oxidation of the catecholamine. Carteolol and xamoterol were dissolved in saline.

Analysis of the Data. All data are expressed as arithmetic means ± S.E.M. Statistical analyses were performed using Prism 3.03 software. The evaluated variables were tested for normality with the Kolmogorov-Smirnov test, and all were found to be parametric. Therefore, statistical comparisons of data were performed by one-way analysis of variance followed by Newman-Keuls post hoc test. Bonferroni test was used for multiple comparisons of data sets. Critical values of P < 0.05 were judged statistically significant.

	Results

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Positive Chronotropic and Inotropic Effects of Carteolol: Comparison with Xamoterol. The effect of carteolol on heart frequency was determined in spontaneously beating atria and was compared with that caused by xamoterol. The positive chronotropic effects of the drugs were expressed as percentages of the maximum effect (E_max) induced by isoprenaline (3 µM) in the same myocardial preparations. Results are shown in Fig. 2 (top panel). Both carteolol and xamoterol elicited a concentration-dependent positive chronotropic effect. Their effects were very rapid in onset and reached their peak within 3 to 4 min after drug additions to the bathing medium. The effect of carteolol peaked at 10 µM (27.24 ± 4.92% of E_max) and decreased at higher concentrations. Xamoterol was more effective and more potent than carteolol, its effect being more marked (56.64 ± 7.70% of E_max) than that of carteolol and peaking at 1 µM. As for carteolol, the effect of xamoterol tended to decrease at higher concentrations. The values of intrinsic activity () and potency (pD₂) of both drugs are summarized in Table 1.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Mean concentration-effect curves for the chronotropic and inotropic effects of carteolol and xamoterol in guinea pig spontaneously beating atria. The effects of the drugs on the rate of contraction (top) and on the force of contraction (bottom) are expressed as percentages of the maximum effects (Emax) caused by 3 µM isoprenaline in the same preparation. Predrug frequency of atria was 134.4 ± 3.7 beats/min (n = 31), and predrug force of contraction of atria was 7.46 ± 0.29 mN (n = 31). Isoprenaline-induced maximum increases in heart rate and force of contraction were 65.12 ± 4.88 and 115.71 ± 13.98% over the basal values, respectively. The curves have been obtained as explained in detail under Materials and Methods. The experimental data in brackets were excluded in the fitting. Results are means ± S.E.M. from seven assays carried out using seven different guinea pig hearts for each drug.

In spontaneously beating atria, both carteolol and xamoterol caused also a concentration-dependent increase in the force of contraction (Fig. 2, bottom panel). The pattern of this effect was similar to that of their chronotropic action, peaking at 10 and 1 µM for carteolol and xamoterol, respectively, and decreasing at higher concentrations. The pD₂ values, calculated for the inotropic effect of carteolol and xamoterol (6.30 ± 0.11 and 7.18 ± 0.13, respectively) were very similar to those reported for their respective chronotropic effects (Table 1).

Because of the strict relationship existing between heart frequency and developed cardiac tension, the effects of carteolol and xamoterol on the cardiac force of contraction were also studied in preparations of the left atria electrically driven at 2 Hz. We chose this frequency of stimulation, because it was rather similar to the spontaneous rate of our guinea pig heart preparations (134.4 ± 3.7 beats/min, n = 31). In these experimental conditions, the extent of the positive inotropic effect of xamoterol was rather similar to that observed in spontaneously beating atria (Fig. 3). Moreover, its pD₂ value (7.41 ± 0.14) was comparable with that calculated from data obtained from spontaneously beating atria. In contrast, the positive inotropic response to carteolol in electrically driven left atria (Fig. 3) was smaller (13.88 ± 2.47% of E_max induced by isoprenaline) than that observed in spontaneously beating atria. Furthermore, pD₂ value for carteolol in the left atria (5.91 ± 0.03) was slightly lower than that calculated in spontaneously beating atria (for a comparison, see Table 1).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Mean concentration-effect curves for the inotropic effect of carteolol and xamoterol on guinea pig left atria electrically driven at 2 Hz. The positive inotropic effects of increasing concentrations of carteolol and xamoterol were expressed as percentage of the maximum positive inotropic effect (Emax) caused by 3 µM isoprenaline in the same preparation. Predrug force of contraction of atria was 5.84 ± 0.41 mN (n = 11). Isoprenaline-induced maximum increases in force of contraction were 114.92 ± 13.72% over the basal value (n = 11). The curves have been obtained as explained in detail under Materials and Methods. The experimental data in brackets were excluded in the fitting. Results are means ± S.E.M. from four assays carried out using four different guinea pig hearts for each drug.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Concentration-effect curves for the chronotropic effects of carteolol, xamoterol, and isoprenaline in the absence and presence of propranolol. The effects of the drugs on the rate of contraction of spontaneously beating atria were expressed as percentage of the maximum positive inotropic effect (Emax) caused by 3 µM isoprenaline in the same preparation. Propranolol (1 µM) was added to the bathing medium 30 min before the addition of the tested drugs. For each drug, the results are means ± S.E.M. from three assays carried out using three different guinea pig hearts. *, P < 0.05; **, P < 0.01; ***, P < 0.001; a, no significant differences were detected (P > 0.05).

Similarly, to exclude any contribution of ₁-adrenoceptors to the cardiac actions of carteolol, additional experiments evaluated the effects of the drug in the presence of the ₁-antagonist prazosin. The concentration-effect curves of carteolol were not affected at all by the presence of prazosin (10 nM) in the bathing medium (data not shown).

Functional Evidence of the Involvement of cAMP in the Cardiac Effects of Carteolol. To ascertain the involvement of cAMP in the cardiac actions of carteolol, three different evaluations were performed. First, the isometric contraction curves obtained in spontaneously beating atria in the presence of increasing concentrations of carteolol and xamoterol were analyzed for time to peak force (t₁), relaxation time (t₂), mean velocity of force development (S₁), and mean velocity of relaxation (S₂). There is general agreement that modifications of such parameters are indicative of variations in cardiac cAMP concentrations (Reiter, 1972). As shown in Table 2, although t₁ and t₂ values were not significantly modified by carteolol, a clear trend of decrease of these parameters was observed at 1 and 10 µM carteolol. In contrast, S₁ values showed a statistically significant increase, indicating an increased velocity of force development. In the presence of xamoterol, a significant shortening of t₁ value occurred and increases in S₁ and S₂ values were observed, indicating increased mean velocities of both force development and relaxation. For comparison, Table 2 also shows the values of the same parameters obtained in the presence of isoprenaline.

Second, we determined the effect of carteolol on the force of contraction of electrically driven left atria in the presence of the phosphodiesterase inhibitor IBMX. The slight positive inotropic effect of carteolol was concentration-dependently increased by IBMX (Fig. 5, top panel), indicating involvement of a cAMP-dependent pathway in the positive inotropic action of the drug. As expected, IBMX addition increased also the positive inotropic effect of xamoterol (Fig. 5, top panel).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Functional evidences of cAMP involvement in the cardiostimulant effects of carteolol and xamoterol. Top, positive inotropic effects of carteolol (10 µM) and xamoterol (10 nM) were evaluated in electrically driven left atria in the absence and presence of increasing concentrations of IBMX (2 and 10 µM) and were expressed as percentage of increase over the basal values. IBMX was added 20 min before the addition of carteolol or xamoterol. The force of contraction developed in the presence of IBMX was considered the basal force of contraction of atria. Bottom, positive inotropic effects caused by carteolol (10 µM) and xamoterol (1 µM) in guinea pig spontaneously beating atria were assessed in the absence and presence of 50 nM carbachol. The effects of the drugs were expressed as percentage of maximum positive inotropic effect (Emax) caused by 3 µM isoprenaline in the same preparations. Carbachol was added to the bathing fluid 20 min before the addition of carteolol or xamoterol. Basal force of contraction was decreased to 10 to 20% by this concentration of carbachol. Results are means ± S.E.M. from four assays carried out using four different guinea pig hearts. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus values obtained in the absence of IBMX or carbachol.

Carteolol and Xamoterol Differ in Their Antagonistic Action toward Isoprenaline Effects. In guinea pig myocardial preparations, carteolol, at concentrations (from 5 to 50 nM) not affecting per se the heart rate and the force of contraction, significantly antagonized the chronotropic and inotropic effects of increasing concentrations of the full -agonist isoprenaline. Results of representative experiments are shown in Fig. 6. Carteolol parallel rightwardly shifted the concentration-response curves of isoprenaline without modifying the maximum responses to isoprenaline. Higher concentrations of carteolol (0.1 µM) completely prevented the effects of conventional concentrations of isoprenaline (data not shown). From this set of data, we calculated both the blocking potency (pA₂) of carteolol toward isoprenaline-induced chronotropic and inotropic effects (9.04 ± 0.05 and 9.23 ± 0.18, respectively) (Table 3) and its dissociation constants (K_b values of 0.94 ± 0.04 and 0.68 ± 0.03 nM, n = 6, in spontaneously beating and in left atria, respectively).

View larger version (27K): [in this window] [in a new window]   Fig. 6. Representative concentration-effect curves for the positive chronotropic and inotropic effects of isoprenaline in the presence of increasing antagonistic concentrations of carteolol and xamoterol. The positive chronotropic effect of isoprenaline was determined in spontaneously beating atria, whereas the positive inotropic effect was assessed in left atria electrically driven at 2 Hz. The effects of xamoterol on the basal rate and force of contraction, obtained in the absence of isoprenaline, were expressed as percentage of the maximum chronotropic or inotropic effect (Emax) caused by isoprenaline.

An analysis of the effects of xamoterol against the chronotropic and inotropic effects of isoprenaline showed that the antagonistic activity of xamoterol became evident at a concentration (0.1 µM) affecting per se the rate and the force of cardiac contraction. Both the frequency of spontaneously beating atria and the force developed by electrically driven left atrium were increased by xamoterol. This compound shifted to the right the concentration-response curves of isoprenaline, showing a behavior typical of a partial agonist against a full agonist endowed with higher affinity. Results of representative experiments are shown in Fig. 6. The pA₂ values for xamoterol against isoprenaline-induced positive chronotropic and inotropic effects were 7.92 ± 0.28 and 7.99 ± 0.39, respectively (Table 3). The K_b values were 11.38 ± 0.77 and 10.41 ± 0.48 nM (n = 6) in spontaneously beating and in left atria, respectively.

Carteolol Binds to Both High- and Low-Affinity Sites in Cardiac Membranes. To determine the affinities of carteolol and xamoterol for ₁-adrenoceptors of guinea pig cardiac membranes, we measured their ability to specifically displace (–)[³H]CGP12177 from its binding sites. Because (–)CGP12177 is a nonconventional partial agonist that binds to both the high- and low-affinity sites of ₁-adrenoceptors in rat (Sarsero et al., 1998), ferret (Lowe et al., 2002), and human (Sarsero et al., 2003) cardiac tissues, as well as in recombinant human ₁-adrenoceptors (Joseph et al., 2004), we first assayed the binding of increasing concentrations (from 0.1 to 100 nM) of (–)[³H]CGP12177 in guinea pig cardiac membranes. The data collected from saturation binding experiments are shown in Fig. 7; computer analysis of these data showed a significantly better fit to a two-site than to a one-site binding model. By means of this analysis, we resolved the curve into two sites: H site (pK_d, 9.88 ± 0.01; B_max, 28 ± 3 fmol/mg protein; n = 3) and L site (pK_d, 7.52 ± 0.7; B_max, 36 ± 4 fmol/mg protein; n = 3).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Saturation of [3H]CGP12177-specific binding to guinea pig cardiac membranes. Experimental details are reported under Materials and Methods. Each data point is the mean ± S.E.M. of three independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 8. Mean competition curves between [3H]CGP12177 and carteolol or xamoterol for specific binding on guinea pig cardiac membranes. The experiments were carried out using 1 nM (A) and 30 nM [3H]CGP12177 (B) to evaluate the binding inhibition constants of the two drugs for the H and L sites of 1-adrenoceptors. pKi values were calculated using the Cheng-Prusoff (1973) equation, as reported under Materials and Methods. Each data point is the mean ± S.E.M. of three independent experiments.

	Discussion

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In the present study, we demonstrate for the first time that, also in guinea pig cardiac membranes as previously observed in preparations of cardiac tissue from rat (Sarsero et al., 1998), ferret (Lowe et al., 2002), and humans (Sarsero et al., 2003), as well as in recombinant human ₁-adrenoceptors (Joseph et al., 2004), two different sites or conformations of ₁-adrenoceptors exist and bind the aryloxypropanolamine (–)[³H]CGP12177. The first one is H site (pK_H = 9.88 ± 0.01, B_max = 28 ± 3 fmol/mg protein), and the other one is L site (pK_L = 7.52 ± 0.7, B_max = 36 ± 4 fmol/mg protein), these values being almost equal to those reported by Sarsero et al. (1998) for the rat atrium. Here, we show for the first time that carteolol, whose molecule contains an aryloxypropanolamine moiety identical to that of CGP12177, is able to compete with (–)[³H]CGP12177 for both sites. When bound to the H site, carteolol behaves as an antagonist of catecholamine effects while behaving as a partial agonist when bound to the L site. Therefore, carteolol is a nonconventional partial agonist. Moreover, by comparing the cardiac effects of carteolol with those of xamoterol, a well known cardioselective ₁-adrenoceptor partial agonist, this study gives new insights into the differences existing among the -blockers provided with ISA. In fact, in guinea pig myocardial preparations, both carteolol and xamoterol were able to antagonize the positive chronotropic and inotropic effects of isoprenaline, shifting to the right, in a parallel manner, the concentration-effect curves for isoprenaline. However, a marked difference exists in the behavior of the two drugs. Interestingly, carteolol antagonized the effects of isoprenaline at concentrations not affecting per se the heart rate and/or the force of contraction. In contrast, xamoterol behaved as an antagonist toward isoprenaline at the same concentrations that increased per se the heart rate and the force of contraction, thus showing the typical effect of a conventional partial agonist on the action of a full agonist. The fact that the binding inhibition constant of carteolol for the high-affinity site of cardiac membranes (pK_iH = 9.37 ± 0.92) was very similar to its blocking potency against isoprenaline (pA₂ values of 9.04 ± 0.05 and 9.23 ± 0.18 toward isoprenaline-induced chronotropic and inotropic effects, respectively) is consistent with the hypothesis that carteolol prevents the effect of catecholamine through interaction with an H site of ₁-adrenoceptors labeled with 1 nM (–)[³H]CGP12177. However, the binding of carteolol to this H site is not able to trigger biological responses, as indicated by lack of any cardiostimulant effect by its antagonistic concentrations. On the contrary, evaluating the potency of xamoterol as an antagonist toward isoprenaline, we obtained pA₂ values (7.92 ± 0.28 and 7.99 ± 0.39 against chronotropic and inotropic effects of isoprenaline, respectively) rather similar to its binding inhibition constant for the high-affinity site (pK_iH = 7.34 ± 0.80) and in the same range of concentrations causing cardiostimulant effects. Although at present we cannot exclude a priori that neuronal and/or extraneuronal uptake of isoprenaline influenced the above-mentioned results, this seems to be unlikely because the neuronal uptake limits the synaptic concentrations of physiological catecholamines but not that of isoprenaline (Leenen et al., 2005 and references therein); moreover, in guinea pig atria, the extraneuronal uptake of isoprenaline has been found to be poor (Trendelenburg, 1988, and references therein). In this regard, guinea pig heart seems to be markedly different from rat heart in which Brotto (2003) recently demonstrated a significant uptake of isoprenaline.

In spontaneously beating atria, both carteolol and xamoterol were provided with positive chronotropic and inotropic effects, although they differed in potency and intrinsic activity. The pD₂ values of carteolol for the positive chronotropic and inotropic effects (6.14 ± 0.12 and 6.30 ± 0.11, respectively) were very similar to its pD₂ value (6.19 ± 0.65) for stimulation of adenylate cyclase activity and were also not significantly different from the binding inhibition constant (pK_iL = 7.14 ± 0.79) calculated for an L site of cardiac ₁-adrenoceptors labeled with 30 nM (–)[³H]CGP12177. In contrast, pD₂ values of xamoterol for chronotropic and inotropic effects (7.41 ± 0.20 and 7.18 ± 0.14, respectively) were quite similar to the above-reported binding inhibition constant (pK_iH = 7.34 ± 0.80) for the H site of cardiac membrane preparations, the above-mentioned pA₂ values against isoprenaline, and the pD₂ value for stimulation of adenylate cyclase activity (6.72 ± 0.79). Xamoterol, unlike carteolol, did not exhibit affinity for the L site of cardiac ₁-receptors labeled with 30 nM (–)[³H]CGP12177. Therefore, our results demonstrate the substantial difference existing between carteolol and xamoterol. Whereas the partial agonist activity of carteolol appears at concentrations 3 log units higher than those causing the blockade of ₁-receptors and is related to the interaction of the drug with the low-affinity site of receptors, xamoterol, as a typical partial agonist, exerts both its agonist and antagonist activity at the same concentrations through the interaction with the H site. Both in spontaneously beating atria and in electrically driven left atria, the intrinsic activity of xamoterol was significantly higher than that of carteolol. An explanation for this difference is certainly provided by the different extents of increase in adenylate cyclase activity triggered by the two drugs in guinea pig cardiac membranes (96.4 ± 8.7 and 40.7 ± 8.7% for xamoterol and carteolol, respectively).

Biochemical and functional data confirm the involvement of cAMP in the cardiostimulant effects of carteolol. First, we found a good relationship between the pD₂ values for activation of the adenylate cyclase activity in cardiac membranes by carteolol and those for its cardiostimulant effects. Second, in the left atrium, the addition of IBMX, a well established phosphodiesterase inhibitor, increased significantly and concentration-dependently the positive inotropic effect of carteolol. Furthermore, the effect of carteolol was significantly decreased by carbachol, which is known to impair the biological effects of compounds whose activity is mediated through a rise in cAMP levels (Endoh, 1979; Floreani et al., 2003). Third, at concentrations exerting positive inotropic effects (1–10 µM), carteolol caused decreasing trends in the t₁ and t₂ values and produced a statistically significant increase in S₁ values; this pattern of behavior is consistent with a cAMP-mediated effect (Tsien, 1977; Brixius et al., 1997; Floreani et al., 2003). The impact of carteolol on these parameters was less marked than that of xamoterol, probably because of the differences in the intrinsic activity and in the extent of cAMP increases induced by the drugs.

Moreover, the relative resistance of carteolol effects to propranolol further supports the conclusion that it is a nonconventional partial agonist, whose ISA is mediated through its interaction with the L site (namely the propranolol-insensitive site) of ₁-adrenoceptors, as previously shown for other -blockers including CGP12177 (Konkar et al., 2000; Joseph et al., 2004).

The results obtained in this study do exclude that carteolol causes its effects on guinea pig heart tissues through its interaction with ₂-adrenoceptors, because its actions were not affected at all by butoxamine, a selective ₂ antagonist. A recent study carried out by Bruck et al. (2004) in healthy volunteers suggests that the ISA of carteolol may be due to its interaction with ₂-adrenoceptors. However, the same authors, referring to our previous paper (Floreani et al., 2004), do not exclude but rather consider the possibility that their results could be explained also by the interaction of carteolol with the propranolol-resistant state of ₁-adrenoceptors (Bruck et al., 2004).

In conclusion, our results suggest that remarkable differences may exist among the -blockers endowed with ISA; therefore, the cardiac effects of the -blockers endowed with ISA cannot be generalized because there does not exist a paradigm of behavior for all partial agonists. These findings may impact the clinical usefulness of nonconventional partial agonists, such as carteolol. Due to the distance existing between their antagonistic and agonistic concentrations, they may be very safe drugs among the -blocking agents endowed with ISA.

	Acknowledgements

 
We thank Anna Scuderi (Società Industria Farmaceutica Italiana S.p.A., Catania, Italy) for the kind gift of carteolol, Robert Strobl for assistance in preparing the manuscript, Roberto Padrini for helpful discussions, and Enrico Secchi and Paolo Favero for excellent technical assistance.

	Footnotes

 
This work was supported by grants from the Ministry of Instruction, University, and Research (Rome, Italy), and by Società Industria Farmaceutica Italiana S.p.A. (Catania, Italy).

doi:10.1124/jpet.105.088963.

ABBREVIATIONS: ISA, intrinsic sympathomimetic activity; H, high affinity; L, low affinity; (–)CGP12177, (–)4-(3-t-butylamino-2-hydroxypropoxy)-2-benzimidazol-2-one; IBMX, 3-isobutyl-1-methylxanthine.

Address correspondence to: Prof. Paola Dorigo, Department of Pharmacology and Anesthesiology, Pharmacology Section, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy. E-mail: paola.dorigo{at}unipd.it

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