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RECEPTORS AND SIGNAL TRANSDUCTION
Centre National de la Recherche Scientifique UMR 6542, Physiologie des Cellules Cardiaques et Vasculaires, Faculté des Sciences, Université François-Rabelais, Tours, France
Submitted 1 June 2007 ; accepted in final form 16 October 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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420 nM, and was insensitive to SR59230A. In muscles from pertussis toxin-treated guinea pigs, the salbutamol-induced positive inotropic effect was sensitive to low concentrations of ICI-118551 in an unusual manner. Experiments in reserpinized animals revealed the importance of the phosphorylation-dephosphorylation processes. PKA inhibition reduced and suppressed the effects obtained at low and high concentrations, respectively, indicating that its activation was a prerequisite to the NIE. The effect occurring at nanomolar concentrations depended upon PKA/phosphatidylinositol 3-kinase/cytosolic phospholipase A2 (cPLA2) activations leading to nitric oxide (NO) release via the arachidonic acid/cyclooxygenase pathway. NO release via PKA-dependent phosphorylation of the receptor was responsible for the inotropic effect observed at submicromolar concentrations, which is negatively controlled by cPLA2. The possibility that these effects are due to an equilibrium between different affinity states of the receptor (Gs/Gi coupled and Gi independent with different signaling pathways) that can be displaced by ICI-118551 is discussed. We conclude that β2-adrenoceptors are functional in guinea pig heart and can modulate the inotropic state. salbutamol; β-adrenoceptor antagonists; cardiac contractility; Gs/-Gi coupling; active conformations
70–80%/30–20%; Ref. 6), are more tightly coupled to the Gs protein and thus induce a more pronounced activation of the adenylate cyclase (7). However, their inotropic effects are less than expected because of their localization in caveolae and the presence of a barrier of phosphodiesterases around them that limits the diffusion of cAMP resulting from their activation inside the cell (for review, see Ref. 18). Besides the Gs/cAMP-dependent PKA signaling pathway, β2-adrenoceptors can activate Gi protein-dependent pathways (22, 50, 58). For example, phosphatidylinositol 3-kinase (PI3K; Ref. 26) and cytosolic phospholipase A2 (cPLA2; Ref. 1) have been shown to functionally restrict in a pertussis toxin (PTX)-sensitive manner the β2-adrenoceptor-mediated cAMP/PKA signaling in the rat heart. Thus this coupling limits the Gs-mediated effect, especially the contractile response, an effect that may be of particular importance in pathological situations such as heart failure. Indeed, heart failure and aging are characterized by an increase in sympathetic activity leading to a decrease of functional responsiveness of cardiac β-adrenoceptors due to a diminution in β1-adrenoceptor density and uncoupling of β2-adrenoceptors from the Gs protein-adenylate cyclase pathway. Albeit controversial, an increased coupling of β2-adrenoceptors to the Gi protein has also been reported to occur (for review, see Refs. 7, 8), possibly leading to a more pronounced impairment of adrenergic responses. Although the existence of β2-adrenoceptors in the guinea pig heart has been demonstrated by radioligand binding studies, their functionality is still a matter of controversy. Several studies argue against a functional role of β2-adrenoceptors. In fact, the effects obtained at rather high concentrations of agonist are either blocked by the β1-adrenoceptor antagonist atenolol or sensitive to the specific β2-adrenoceptor antagonist ICI-118551 at concentrations known to also affect β1-adrenoceptors (23, 40, 54). However, other studies reported that β2-adrenoceptor activation induced an increase in heart rate (31, 52) and L-type calcium current of isolated ventricular cells (30, 36) and that these effects were sensitive to low concentrations of ICI-118551. The results of the former studies against a functional role of β2-adrenoceptors are surprising since their population in guinea pig hearts represents, as in the human heart, a nonnegligible proportion of total β-adrenoceptors, i.e., 15–36% in the ventricles (31, 52) and 15–25% in the atria (37, 52).
We hypothesize that these discrepancies might be explained by the dual coupling of β2-adrenoceptors to Gs and Gi proteins leading to activation of different signaling pathways with opposing effects. Indeed, depending on the preponderant pathway, inotropic responses may be positive (Gs-dominant pathway), negative (Gi-dominant pathway), or even absent. Moreover, as shown for β3-adrenoceptors (for review, see Ref. 19), β2-adrenoceptor activation can induce nitric oxide (NO) release (15, 27) and thus possibly a direct negative inotropic effect (although not yet described). On that basis, we have reinvestigated the functionality of β2-adrenoceptor activation in guinea pig right papillary muscles by studying their potential inotropic effects. Surprisingly, we found that β2-adrenoceptor agonists can induce at low concentrations a significant ICI-118551-sensitive negative inotropic effect when β1-adrenoceptor activation is prevented by atenolol, an effect that cannot be ascribed to nonspecific activation of β3-adrenoceptors. We show here the dual Gs/Gi coupling of guinea pig β2-adrenoceptors and the predominant role of NO release in the biphasic negative inotropic effect of salbutamol and investigate the underlying signaling pathways.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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After heparinization (5 IU/g ip), guinea pigs were humanely killed by cervical dislocation to avoid any interaction with anesthetics. After quick removal of the heart and retrograde perfusion through the aorta, papillary muscles from the right ventricle were carefully dissected under a microscope in normal physiological saline at room temperature and mounted in a 5-ml thermostated and oxygenated glass experimental chamber maintained at 37°C. The base of the muscle was fixed in the bottom of the chamber coated with Sylgard (Dow Corning, Seneffe, Belgium) with minutien pins. The tendinous tip was connected via a small hook to a Pioden dynamometer UF1 force-displacement transducer (Pioden Controls, Canterbury, UK) mounted on a three-dimensional micromanipulator (Narishige International, London, UK). Muscles were stretched to 80–90% of length yielding maximum force and allowed to equilibrate before the start of the experiment until stable contraction amplitude was obtained. The contractions elicited by field stimulation (2 Hz, 1-ms suprathreshold rectangular pulse typically of 3.5–4 V), low-pass filtered at 40 Hz, were visualized on a digital oscilloscope (Tektronix TDS310, Tektronix, Les Ulis, France) and recorded both on a chart paper (GRASS 7400, Astro-Med, Trappes, France) and on magnetic tape (DAT DTR 1202, Bio-Logic Science Instruments, Claix, France) for further off-line analysis with IOX software (version 1.7.0; EMKA Technologies, Paris, France).
During the experiments, the muscle was continuously superfused at 37°C with Tyrode solution at a constant flow rate (1 ml/min) maintained by a peristaltic pump (Minipuls 2, Gilson International, Roissy en France, France), except for the establishment of the cumulative concentration-response curves, during which the perfusion was stopped. A stabilization period, usually of 10 min, was then necessary for the muscle to reach a new stable contraction amplitude before the first concentration of β2-agonist was added. When used, antagonists of β-adrenoceptors and inhibitors were directly added to the Tyrode solution at the final concentration and allowed to equilibrate for 10–15 min, except for N
-nitro-L-arginine methyl ester (L-NAME; 20 min). The Tyrode solution contained (in mM) 120 NaCl, 5.4 KCl, 1 MgCl2, 2 CaCl2, 0.6 NaH2PO4, 25 NaHCO3, 5 pyruvic acid, 0.05 ascorbic acid, and 11 glucose, pH adjusted to 7.35 (95% O2-5% CO2).
Drugs and reagents. Reserpine, salbutamol hemisulfate, terbutaline hemisulfate, procaterol hydrochloride, (±)isoproterenol hydrochloride, ICI-118551 hydrochloride, atenolol, H-89 dihydrochloride, wortmannin, indomethacin, nordihydroguaiaretic acid (NDGA) and L-NAME hydrochloride were purchased from Sigma (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France). SR59230A hydrochloride, okadaic acid sodium salt, and arachidonyl trifluoromethyl ketone (AACOCF3) were obtained from Tocris (Tocris Bioscience; Fisher-Bioblock, Illkirch, France). All drugs, prepared as stock solutions in distilled water kept at –20°C, were freshly diluted in Tyrode solution each day before use. When a vehicle (DMSO or ethanol) was used to prepare stock solutions, control experiments were carried out in the presence of equivalent final concentrations of the vehicle. No differences were observed in the concentration-response curves to salbutamol in the presence and absence of vehicle alone. All other chemicals were also purchased from Sigma.
Guinea pig treatments. To reduce the amount of circulating catecholamines, seven guinea pigs (mean wt 338.6 ± 10.4 g) received 24 h before death an intraperitoneal injection of freshly prepared reserpine (brought into solution in double-distilled water containing 2% glacial acetic acid) at a dose of 1 mg/kg. This procedure has been reported to cause an almost 95% depletion of endogenous catecholamines (53). All animals presented a significant weight loss at the time of the experiment (11.0 ± 1.2% of initial body wt).
To partially inactivate Gi proteins, nine guinea pigs (mean wt 296.6 ± 3.6 g) received an intraperitoneal injection of 100 µg/kg PTX (Calbiochem, Merck Chemicals, Nottingham, UK). The choice of the concentration used was based on the experiments of Hopwood et al. (25) on guinea pig papillary muscles. To reduce mortality and weakness (2 died after 72-h treatment, and 1 was very weak with complete loss of muscular tone), guinea pigs were killed between 71 and 72 h after injection and had a mean weight of 297.6 ± 5.8 g at that time. Four animals presented a significant weight loss, which represented 7.5 ± 1.3% of their initial body weight. Efficacy of the treatment was assessed by the reduction of the antiadrenergic effect of acetylcholine (see Fig. 3A): only muscles presenting a significant reduction were included in the study.
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Tables 2 and 3 as follows: the amplitude of the first component is given by (Amin,1 – Amax) and that of the second component by (Amin,2 – Amin,1) and (Amin,2 – Amax), when Eqs. 2 and 3, respectively, were used to adjust the data points. The choice of the model used to fit the experimental data with a single or double logistic equation was determined by least-squares fitting and statistical testing using the corrected Akaike's Information Criteria (AICc) and Fisher's test, as described by Motulsky and Christopoulos (39). Other statistical significances were assessed by paired Student's t-test and one-way or two-way ANOVA with Tukey's post hoc test. A P value <0.05 was considered as statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The shape of the mean concentration-response curve in the presence of 4 µM atenolol suggested that the negative inotropic effect of salbutamol might be biphasic. Adjusting the data with a simple logistic equation gave an EC50 of 19.9 ± 5.8 nM and a slope factor of 0.5 ± 0.05, far below unity. With a double logistic equation, the two EC50 values (3.0 ± 0.6 nM and 418.1 ± 157.9 nM; n = 19) were statistically different and the slope factor was 0.9 ± 0.1 (see MATERIALS AND METHODS), a value close to unity. As shown in Table 1, both fits appeared quite good when the R2 values were considered. However, comparison of the two models with Fisher's test and AICc indicated that the double logistic model has a highly significant (P < 0.002; Table 1) lower sum of squares from least-squares nonlinear regression (SSR) and a lower AICc. Calculation of the information ratio (ratio of the probabilities for each model) indicates that the model double logistic is 85 times more likely to be correct than the model simple logistic (Table 1). Therefore, two distinct mechanisms, each one having different threshold and sensitivity, might be involved in the biphasic negative inotropic effect of salbutamol.
To determine whether the observed negative inotropic effect of salbutamol was specific to this molecule or a more common feature of short-acting β2-adrenoceptor agonists, concentration-response curves to other agonists were made in the presence of 200 nM atenolol. Terbutaline and procaterol also induced a biphasic negative inotropic effect (Fig. 1C) of –17.5 ± 3.4% (at 2 µM) and –14.9 ± 5.2% (maximum at 0.6 µM), respectively, with EC50 values of 1.9 ± 1.2 and 110.9 ± 91.5 nM (n = 7) and of 3.1 ± 1.5 and 45.1 ± 11.3 nM (n = 5). Therefore, under our experimental conditions, we can conclude that short-acting β2-adrenoceptor partial agonists induce a biphasic negative inotropic effect.
To test an eventual implication of β3-adrenoceptors in the biphasic negative inotropic effect of salbutamol, concentration-response curves were determined in the additional presence of SR59230A, a selective β3-adrenoceptor antagonist. At a concentration of 200 nM, which should block almost 98% of β3-adrenoceptors with minimal effects on β2-adrenoceptors (33), the concentration-response curves to salbutamol determined in nine muscles (Fig. 1D) were not significantly affected (Table 3). Therefore, β3-adrenoceptors do not appear to be involved in the negative inotropic effect of salbutamol.
Effect of ICI-118551 on negative inotropic effect. The effect of ICI-118551, a selective β2-adrenoceptor antagonist, was examined in the presence of 4 µM atenolol. Figure 2 shows that, in the presence of 40 nM ICI-118551, salbutamol induced a positive inotropic effect with an EC50 of 1.9 ± 0.4 nM (n = 5) similar to that of the first negative component in control conditions (Table 2). It was followed at higher concentrations by a negative inotropic effect, as in control conditions (EC50 of 316.9 ± 61.4 nM). These results indicate that β2-adrenoceptor activation is involved in the negative component induced by nanomolar concentrations of salbutamol.
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27 times from 224.8 ± 50.4 nM to 6.2 ± 1.5 µM; n = 8; apparent pA2 of 7.82). Although at this concentration ICI-118551 should also bind onto β1-adrenoceptors, we found that these receptors were functionally not significantly affected since the EC50 of the concentration-response curve to isoproterenol was not changed (not shown). Moreover, the concentration of atenolol used during these experiments (4 µM) is sufficient to already induce >95% block of β1-adrenoceptors. Thus an additional effect of ICI-118551 on these receptors is probably irrelevant. Therefore, these results strongly suggest that β2-adrenoceptors are likely involved in both components of the negative inotropic effect of salbutamol. Role of Gi protein coupling and antagonism by ICI-118551. Since Gs-coupled β2-adrenoceptors can simultaneously activate a pathway that leads to functional inhibition via a PTX-sensitive Gi protein (55), we hypothesized that the negative inotropic effect of salbutamol resulted from the coupling of the receptors to Gi proteins. The effect of salbutamol was therefore tested in papillary muscles from PTX-treated guinea pigs. PTX pretreatment did not affect the Gs-mediated positive inotropic effect induced by 50 nM isoproterenol alone [control: +266.7 ± 66.9% (n = 7); PTX-treated: +244.0 ± 49.8% (n = 8); not shown] but markedly reduced the antiadrenergic effect of acetylcholine as illustrated in Fig. 3A. Only half of the papillary muscles presenting a marked reduction of the antiadrenergic effect of acetylcholine were stable enough in the presence of salbutamol to perform reliable measurements. Figure 3B shows that when Gi proteins were inactivated, salbutamol alone induced only a concentration-dependent positive inotropic effect (+5.8 ± 0.6%, n = 4) with an EC50 of 0.101 ± 0.007 nM, 10 times lower than in papillary muscles from nontreated guinea pigs, as expected for a dual Gs/Gi coupling of the β2-adrenoceptor in nontreated animals. The results obtained in the presence of 40 nM ICI-118551 were unexpected since the salbutamol concentration-response curve was biphasic: the maximum positive inotropic effect was significantly reduced by almost 50% (Table 2), with no changes in its EC50, and was followed by a negative inotropic effect (EC50 of 25.1 ± 6.6 nM, 10 times lower than that observed in papillary muscles from control guinea pigs). Increasing ICI-118551 concentration to 400 nM further reduced the positive inotropic effect and increased the amplitude of the negative component of the concentration-response curve (Table 2). Thus ICI-118551 has a dual action: it exerts a noncompetitive-like antagonism on the Gs-mediated positive inotropic effect occurring at low concentrations and allows salbutamol to activate at higher concentrations a second signaling pathway leading to a negative inotropic effect.
Since the slope of the modified Schild's equation for noncompetitive antagonism (2) was far below unity (0.31; pD2' value of 7.31), the ICI-118551 effect (decrease of the positive inotropic effect of salbutamol) is not truly noncompetitive.
These unexpected results strongly suggest the existence of an equilibrium between two active forms of the β2-adrenoceptor [as proposed by Gether and Kobilka (20) and Hopkinson et al. (24)] with different ligand sensitivities, which can be induced and displaced by ICI-118551.
Role of endogenous catecholamines. Because we noted that the biphasic inotropic effect in response to β2-adrenoceptor agonists was more consistently obtained during periods in which guinea pigs presented a higher L-type calcium current density than usual (which was also less sensitive to isoproterenol; unpublished observations), we hypothesized that the basal phosphorylation level due to circulating endogenous catecholamines was of importance. To assess this point, responses to salbutamol of right papillary muscles dissected from the heart of control and catecholamine-depleted guinea pigs were compared. In catecholamine-depleted guinea pigs, the response to increasing concentrations of salbutamol in the presence of 200 nM atenolol was overall a positive inotropic effect that appeared to be triphasic (Fig. 4): contraction amplitude first significantly increased (+9.9 ± 2.9% at 2 nM; n = 7), then decreased (+4.9 ± 2.9% at 60 nM) and increased again to a maximum of +22.8 ± 9.8% at 6 µM. Adjusting a double logistic equation to the data (concentrations up to 60 nM) gave an EC50 of 0.47 ± 0.17 nM for the positive inotropic effect (+15.0 ± 2.4%) and an EC50 of 5 nM for the decreasing limb of the curve, a value that was similar to that obtained in control animals (4.9 nM, Fig. 4). Thus our results strongly suggest that the ability of salbutamol to induce a positive or a negative inotropic effect depends on the basal phosphorylation state of papillary muscles and that phosphorylation is a prerequisite to the negative inotropic effect.
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50% of that observed in control conditions (Table 3), with no significant change in the EC50 (from 11.0 ± 4.5 nM to 14.0 ± 3.7 nM; n = 5). Thus PKA activation appeared to play a central role in the salbutamol-induced negative inotropic effect.
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Involvement of PI3K/cPLA2/arachidonic acid pathway. In rat ventricular myocytes, PI3K blockade enhances the contractile responses to zinterol, a β2-adrenoceptor agonist, in a PTX-sensitive manner, showing that PI3K functionally restricts the β2-adrenoceptor-mediated cAMP/PKA signaling (26). We therefore tested the implication of PI3K in the negative inotropic effect of salbutamol, which we showed to depend on Gi. In the presence of 100 nM wortmannin, the inotropic effect occurring at low concentrations was statistically reduced by almost 50% with no changes in the EC50 (see Table 3 and Fig. 6 Aa). In the presence of a higher concentration of wortmannin (400 nM; Fig. 6Ab), it was reversed to a positive one with an EC50 identical to that of the negative inotropic effect obtained under control conditions (Table 3). The negative inotropic effect occurring at higher concentrations remained unaffected. Thus, PI3K activation is also implicated in the negative inotropic effect occurring at low concentrations.
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85%) from –4.8 ± 0.3% to –9.0 ± 0.7%, with no significant modification in the EC50 (Table 3). This suggests that cPLA2 might likely induce a negative feedback control on the signaling pathway involved for that component. Thus, besides PI3K, cPLA2 activation is also necessary for salbutamol to induce a negative inotropic effect at low concentrations (in the nanomolar range). cPLA2 hydrolyzes glycerophospholipids of the membrane to liberate arachidonic acid (AA), which is then metabolized by three major pathways: cyclooxygenases (COX), lipoxygenases, and cytochrome P-450. In the presence of 10 µM indomethacin, an inhibitor of COX-1 and COX-2, only the negative inotropic effect of salbutamol occurring at low concentrations was strongly reduced by >70% (from –7.2 ± 0.4% to –2.1 ± 0.2%; n = 9; Fig. 6C and Table 3). This suggests that at least one of the COX-generated AA metabolites might be involved in the negative inotropic effect of salbutamol. In contrast, the lipoxygenase pathway does not seem to be involved in the negative inotropic effect of salbutamol, because experiments carried out in the presence of 30 µM NDGA, an inhibitor of lipoxygenases, showed no significant alterations in the concentration-response curves to salbutamol (data not shown).
Central role of nitric oxide synthase activation. NO is a ubiquitous signaling molecule that is well known to attenuate the chronotropic and inotropic response to β-adrenoceptors agonists in the heart (see, e.g., Refs. 4, 5; for review, see Ref. 10). We therefore investigated its potential role by comparing responses to salbutamol in the presence and absence of L-NAME, a nonselective inhibitor of nitric oxide synthase (NOS). In the presence of 100 µM L-NAME (Fig. 7), the negative inotropic effects occurring at low and high concentrations of salbutamol were considerably reduced and no more significant compared with the basal contraction amplitude. Thus our results clearly demonstrate that salbutamol-induced negative inotropic effect depends on NO synthesis. It should be noted that 100 µM L-NAME alone induced a positive inotropic effect of +10.6 ± 3.0% (n = 10), indicating a constitutive activity of NOS in guinea pig papillary muscles.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Negative inotropic effect of β2-adrenoceptor activation. Concentration-response curves to salbutamol, terbutaline, and procaterol built in the presence of atenolol have revealed that the activation of β2-adrenoceptors leads to a negative inotropic effect that is masked by the concomitant positive inotropic effect induced by β1-adrenoceptor activation. A nonspecific activation of β3-adrenoceptors that are known to induce a negative inotropic effect (for review, see Ref. 19) was ruled out since a β3-adrenoceptor antagonist had no effect. SR59230A was used at a concentration of 200 nM since at a higher concentration it lacks specificity toward β3-adrenoceptors (IC50 of 408 and 648 nM for β1- and β2-adrenoceptors, respectively; Ref. 33). On the other hand, involvement of β2-adrenoceptors was confirmed by the effect of a low concentration (40 nM) of ICI-118551 (see below), a specific antagonist of these receptors.
This negative inotropic effect induced by β2-adrenoceptor activation is surprising. As shown in several studies, the expected inotropic effect is a positive one, resulting from the classical coupling of β2-adrenoceptors to the Gs protein, leading to activation of the adenylate cyclase/PKA pathway. However, Xiao et al. (55) have demonstrated that β2-adrenoceptors from rat ventricular myocytes are simultaneously coupled to Gs and Gi proteins. The PKA-mediated coupling to Gi protein (12) activates signaling pathways shown to confine the β2-adrenoceptor/PKA signaling to restricted subsarcolemmal spaces (see, e.g., Refs. 11, 26, 56) and to limit the positive inotropic effect that should normally result from their activation. Our results are in agreement with such a dual coupling to Gs and Gi proteins since in papillary muscles from PTX-pretreated guinea pigs salbutamol induced only a positive inotropic effect. Thus the negative inotropic effect observed in muscles from control animals suggests a preponderant activation of Gi-dependent signaling pathways, consistent with our working hypothesis. Moreover, the biphasic nature of the negative inotropic effect indicates that β2-agonists can activate two signaling pathways with different threshold and sensitivity.
Effect of ICI-118551. Unusual also are the effects of the selective β2-adrenoceptor antagonist ICI-118551, both in control and in PTX-treated muscles. Indeed, in PTX-treated muscles, the maximum amplitude of the salbutamol-induced positive inotropic effect decreased with increasing concentrations of ICI-118551, with no changes in its EC50 (noncompetitive-like antagonism). This reduction in amplitude was concomitant with the apparition of a negative inotropic effect of salbutamol for concentrations above 2 nM, whose amplitude increased with ICI-118551 concentration. A noncompetitive-like effect of ICI-118551 has already been reported to occur in two cell lines (24) with a low level of expression of β2-adrenoceptors: BC3H1 cells (endogenous constitutively active β2-adrenoceptors, 77.9 fmol/mg protein) and a stable Chinese hamster ovary (CHO) cell line (transfected with the human β2-adrenoceptor, 50 fmol/mg protein). These authors have suggested that the noncompetitive-like effect of ICI-118551 might be due to its interaction with different affinity states of the receptor. Although the control of the contractile state is a multistep process resulting from the activation of numerous signaling pathways, our results might suggest, as in these studies, the possible existence of an equilibrium between active conformations of the receptor that could be induced and displaced by low concentrations of ICI-118551. The observation of the inotropic consequences of such an hypothetical equilibrium is probably facilitated by the low reserve of β2-adrenoceptors in the guinea pig heart (38), as in BC3H1 cells (24).
Although experiments at the molecular level are needed to clearly demonstrate the existence of different active conformations of the β2-adrenoceptor in equilibrium, our hypothesis would also explain the paradoxical effect of low and high concentrations of ICI-118551 on the first negative component of the inotropic effect of salbutamol in control muscles. This component has an EC50 in the nanomolar range, as expected for activation of β2-adrenoceptors (i.e., Refs. 9, 57) and results from the activation of a dominant Gi-dependent signaling pathway, as can be concluded from the experiments in muscles from PTX-treated guinea pigs. Experiments carried out in the presence of 40 nM ICI-118551 strongly suggest that this first component results from the activation of β2-adrenoceptors. However, depending on the concentration of antagonist used, nanomolar concentrations of salbutamol can induce either a positive (Gs mediated) or a negative (Gi mediated) inotropic effect, a result consistent with an equilibrium between Gs-coupled and Gi-coupled conformations of the receptor (28, 29).
Our results strongly suggest that the second component of the negative inotropic effect might result from salbutamol acting on a specific conformation of β2-adrenoceptors. First, it can be observed in muscles from PTX-treated guinea pigs once the receptor has acquired a particular conformation in the presence of 40 nM ICI-118551, a concentration specific for the β2-adrenoceptors. Second, a concentration of 400 nM ICI-118551, sufficient to block 99% of the receptors, induced a 27 times increase in its EC50 in control muscles (leading to an apparent pA2 of ICI-118551 of 7.82). As already stated in RESULTS, an additional effect of ICI-118551 on β1-adrenoceptors is probably irrelevant since the concentration of atenolol used (4 µM) is already sufficient to induce >95% block of these receptors.
Signaling pathways involved. Our results show a key role of phosphorylation processes in mediating the negative inotropic effect of salbutamol: 1) in catecholamine-depleted animals, the inotropic effect is on the overall a positive one; 2) blockade of PKA with H-89 resulted in a strong reduction of the first component (EC50 in the nanomolar range) and the suppression of the second component (EC50 in the submicromolar range); and 3) when dephosphorylation processes are prevented by okadaic acid, the sensitivity to high concentrations of salbutamol is increased. The high concentration of H-89 used (20 µM) was required to block most of the inotropic response to isoproterenol in our experimental conditions. KT-5720, another PKA inhibitor, was not used since it was inefficient at concentrations up to 50 µM. Although we cannot exclude that H-89 might also have affected other protein kinases besides PKA (14, 32), we can nevertheless assume that PKA activation is likely underlying both negative inotropic effects.
Our results also support a key role of NO in the two components of the negative inotropic effect, since they are almost suppressed in the presence of L-NAME. As previously reported, β-adrenoceptors activation can induce NO release (e.g., Refs. 15, 27). Moreover, NOS activity is regulated by phosphorylation-dephosphorylation processes (for review, see Ref. 34), like the inotropic effect we observed. Although specific NOS inhibitors were not used, a possible candidate is endothelial NOS (NOS3), which has been shown to be colocalized in caveolae with L-type calcium channels, β2-adrenoceptors, caveolin 3, and adenylate cyclase (see, e.g., Ref. 41; for review, see Ref. 34). Because of the multicellular nature of papillary muscles, the exact origin of the NO released (endothelial cells, fibroblasts, or myocardium) is uncertain and will require further investigation.
As shown and discussed above, the two NO-dependent components of the negative inotropic effects are differentially affected by the inhibitors used. These results strongly support activation of at least two different pathways leading to NO release. Figure 8 summarizes the signaling pathways that are likely involved in the β2-adrenoceptor activation induced by salbutamol in guinea pig right papillary muscles, as deduced from the results presented in this study and from data available in the literature. At low concentrations of salbutamol, β2-adrenoceptor coupling to Gs leads to activation of PKA and to an increase of intracellular calcium. The positive inotropic effect that would normally occur is overridden by simultaneous activation of cPLA2 by PI3K and Cai, leading to NO synthesis via AA metabolites produced by COX (resulting in the negative inotropic effect with an EC50 in the nanomolar range). Simultaneously, PKA also phosphorylates the receptor and induces its coupling to Gi. This coupling contributes also to the activation of cPLA2 [as described by Aït-Mamar et al. (1)] and to its positive control by PI3K. Because we found that the negative inotropic effect occurring at higher salbutamol concentrations (EC50 in the submicromolar range) was sensitive only to H-89, L-NAME, and AACOCF3, we hypothesize that PKA-dependent phosphorylation of the receptor leads to activation of NOS independently of the Gi-dependent PI3K/cPLA2 pathway and that cPLA2 exerts a negative control on this second pathway leading to NOS activation. Whether NO directly decreases Cai or acts via activation of the guanylate cyclase/cGMP/PKG pathway awaits further studies. It would be also of interest to investigate the possible cross talk between cPLA2, COX, and NOS.
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s protein-coupled (activation of MAPK pathway, EC50 
1 nM isoproterenol) to a G protein-independent signaling (tyrosine kinase Src-dependent pathway, EC50 1 µM isoproterenol). It is noteworthy that the EC50 values reported in this study are very similar to those determined for the β2-agonists-induced negative inotropic effect we found. Whether or not such a pathway exists in the guinea pig heart and is responsible for the second component of the negative inotropic effect remains to be determined. Because the inotropic effects of β2-agonists we found are small and unusual (negative ones), require β1-adrenoceptors to be blocked by atenolol, and show a peculiar unexpected sensitivity to ICI-118551, this might explain why β2-adrenoceptors have mostly been considered in the literature so far as nonfunctional in the guinea pig heart (see, e.g., Refs. 16, 23, 40). However, other studies have given evidence of a functionality of these receptors in the guinea pig heart: Lemoine et al. (31) and Voss et al. (52) have respectively reported a positive chronotropic effect of fenoterol and TA2005 in atrium, and antibodies raised against the second extracellular loop of the human β2-adrenoceptor have been shown to increase the L-type calcium current in ventricular heart cells (30, 36).
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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. Section 1734 solely to indicate this fact.
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, n = 4) or presence of 40 nM (
, n = 4) or 400 nM (
, n = 4) ICI-118551. Smooth lines represent nonlinear adjustment to the mean data points of 
, n = 5) in the presence of 200 nM atenolol. Terbutaline: Amax = 100%, Amin,1 = 90.36%, Amin,2 = 83%, EC50,1 = 1.93 nM, EC50,2 = 110.9 nM, p = 1.5; procaterol: Amax = 100%, Amin,1 = 95.77%, Amin,2 = 84.65%, EC50,1 = 3.05 nM, EC50,2 = 45.06 nM, p = 1.30. Smooth lines represent nonlinear adjustment to the data points of 
