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1 Faculté de Médecine3 Faculté de Sciences et de Génie Informatique, Université Saint Esprit de Kaslik, BP 446 Jounieh, Lebanon 2 Université de Nantes, CNRS, UMR 6204, Biotechnologie, Biocatalyse et Biorégulation, Faculté des Sciences et des Techniques, 2 rue de la Houssinière, BP 92208, F-44322 Nantes, France
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Abstract |
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(Received 6 January 2006;
accepted after revision 29 March 2006; first published online 31 March 2006)
Corresponding author W. Hleihel: Faculté Médecine, Université Saint Esprit de Kaslik, BP 446 Jounieh, Lebanon. Email: walidhleihel{at}usek.edu.lb
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Introduction |
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In fact, endogenous adenosine can cross the cell membrane via a nucleotide transporter and then enter the interstitial space, where it produces its physiological effects via specific cell surface receptors. Four subtypes of adenosine receptors (A1, A2A, A2B and A3 receptors) are known to exist (Palmer & Stiles, 1995). Adenosine has a protective effect against various types of myocardial injury (Ely & Berne, 1992), and this protection is usually mediated by stimulation of the A1 receptors (Shryock & Belardinelli, 1997). By inhibiting adenylyl cyclase, the A1 receptors attenuate the positive inotropic effect of ß-adrenergic receptor stimulation. Other adenosine receptor subtypes also exist in ventricular myocytes and may have important physiological functions. It was demonstrated that A2A receptors are expressed in rat ventricular cardiomyocytes and that, in isolated rat hearts and isolated adult rat ventricular cardiomyocytes, the antiadrenergic effects of adenosine are potentiated in the presence of selective A2A antagonists. Experiments conducted on isolated rat ventricular myocytes have suggested that adenosine A2A receptors on the sarcolemma are involved in the increase in inotropism via cAMP-dependent and -independent mechanisms (Dobson & Fenton, 1997). Adenosine A2B receptors have also been suggested to be expressed in ventricular cardiomyocytes based on studies in isolated embryonic cells. Like A2A receptors, A2B receptors are positively coupled to adenylyl cyclase and appear to antagonize the antiadrenergic effects of A1 adenosine receptors. In addition to the A2 receptors, there is also evidence that A3 receptors, the most recently identified subtype, may be expressed in ventricular cardiomyocytes. The A3 receptor, like the A1 receptor, is negatively coupled to adenylyl cyclase. Using embryonic chick cardiomyocytes, it has been shown that A3 receptor agonists inhibit isoproterenol-induced increases in cAMP and that activation of A3 receptors provides tolerance against hypoxic injury (Strickler et al. 1996). Another study, however, indicates that although rat ventricular myocytes appear to express adenosine A2a receptors, stimulation with an A2a agonist exerts no functional effects, possibly because of the subcellular localization of the A2a receptor (Kilpatrick et al. 2002). Interestingly, ‘cytosolic’ adenosine A2a receptors have been observed in human skeletal muscle and PC12 cells (Lynge & Hellsten, 2000; Arslan et al. 2002). Moreover, adenosine has been shown to produce direct activation of the sarcoplasmic reticulum ryanodine receptors of cardiac channels (RyR2) incorporated into phosphatidylethanolamine bilayers (McGarry & Williams, 1994). Furthermore, it has recently been proposed that adenosine may have an indirect effect on the ryanodine Ca2+ channels (RyR1) in mammalian skeletal muscle. It has been demonstrated that adenosine inhibits the caffeine-induced release of Ca2+ from saponin-skinned fibres via the RyR1 and/or A1 receptors present in the sarcoplasmic reticulum (Hleihel et al. 2001). It was therefore of interest to see whether adenosine affects the release of Ca2+ from the sarcoplasmic reticulum in mammalian cardiac muscle by a similar mechanism.
During excitation–contraction coupling, the Ca2+ that enters the cytosol via plasmalemmal voltage-dependent Ca2+ channels binds to and activates RyRs at high-affinity cytosolic Ca2+ activation sites (Meissner et al. 1997). When RyRs open, a much larger amount of Ca2+ is released to the myoplasm, leading to activation of contractile proteins. This mechanism is known as Ca2+-induced Ca2+ release (Bers, 2002). The RyRs are activated by Ca2+ and by chemical substances, such as caffeine (Lai et al. 1988; Rousseau et al. 1988; Rousseau & Meissner, 1989; Sitsapesan et al. 1995). Besides the high-affinity cytosolic activation sites, the RyR is positively controlled by low-affinity Ca2+ sensing sites accessible from the luminal side of the channel (Ching et al. 2000). Dissociation of Ca2+ from these sites leads to RyR deactivation and robust termination of Ca2+-induced Ca2+ release. Abnormal regulation of RyRs by luminal Ca2+ has been implicated in certain pathological states, including Ca2+ overload, arrhythmias and heart failure (Györke et al. 2002). One possibility to explain these effects is that the luminal Ca2+ sensor is a part of the RyR protein itself. Alternatively, auxiliary proteins in the heart with luminal localization, such as calsequestrin, triadin 1 and junctin, could mediate the effects of luminal Ca2+ on RyRs. The effect of caffeine has been tested in planar lipid bilayers (Rousseau et al. 1988), and it has been shown that this substance, which acts on the sarcoplasmic reticulum RyRs, also increases the probability and duration of open events without changing the conductance of the channel. Caffeine elicited a large transient contracture in isolated trabeculae from the ferret heart by releasing Ca2+ from intracellular stores (Chapman & Leoty, 1976). Moreover, caffeine is commonly used to induce contractile responses in both intact and saponin-skinned cardiac fibres.
In the present experiments, which were performed on saponin-skinned fibres isolated from the papillary muscles of the adult ferret heart, the modifications in caffeine contractures were used to indicate changes in Ca2+ release from the sarcoplasmic reticulum via RyR2. Our findings show that adenosine and the specific adenosine receptor agonist CGS 21680 both induced a dose-dependent potentiation of the caffeine-induced Ca2+ release, and that this potentiation was reduced by ZM 241385, a specific A2A receptor antagonist. However, in Triton X-100-skinned fibres, neither adenosine nor CGS 21680 produced any significant effects on the properties of the contractile apparatus. It is therefore proposed that adenosine may potentiate the release of Ca2+ from the sarcoplasmic reticulum by caffeine by directly activating the RyR2 Ca2+ release channel and/or by acting on A2A receptors present in the sarcoplasmic reticulum.
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Methods |
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Experimental protocol for skinned fibre experiments
Chemical skinning was carried out immediately after dissection, using either Triton X-100 or saponin. The skinned fibres were then transferred to a chamber and mounted between two stainless-steel tubes that supported a metal target facing the sensor of a transducer (model 0.5 SU, Displacement Measuring System KD 2300, Kaman Instrumentation, Colorado Springs, CO, USA) according to the protocol previously described by Huchet & Leoty (1993). At the beginning of each experiment, the fibre was adjusted to its slack length and then stretched progressively until the tension developed at pCa 4.5, maximal tension (Tmax) was generally reached once length was increased by 20%. All experiments were performed at 22°C.
Tension–pCa relationships in Triton X-100-skinned fibres
Preparations were incubated for 1 h in relaxing solution (pCa 9.0) containing 1% Triton X-100 (v/v) to solubilize the sarcolemma and the sarcoplasmic reticulum membranes, and subsequently washed several times in relaxing solution without detergent. Following skinning, the fibres were stored at –20°C in relaxing solution containing 50% glycerol (v/v). Tension–pCa relationships were obtained by exposing Triton X-100-skinned fibres sequentially to solutions with decreasing pCa values until the maximal Ca2+-activated tension (Tmax) was reached (at pCa 4.5); the fibres were then returned to a low-[Ca2+] solution (pCa 9.0). A full set of solutions containing different Ca2+ concentrations was prepared, and each solution then divided into aliquots, one solution serving as the control and adenosine (100 nM) or CGS 21680 (50 nM) plus caffeine (2.5 mM) being added to the others. The isometric tension was recorded continuously using a paper chart recorder (model 1200, Linear, Reno, NV, USA), and baseline tension was measured at the steady state in a relaxing solution. To obtain the Ca2+ sensitivity curve, data for the relative tension (T/Tmax) were fitted using a modified Hill equation (Huchet & Leoty, 1993):
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Sarcoplasmic reticulum Ca2+ uptake and release in saponin-skinned fibres. The saponin-skinned fibre technique, used to assess the properties of the sarcoplasmic reticulum, was performed by incubating the preparations for 30 min in relaxing solution at pCa 9.0 containing saponin at 50 µg ml–1. The skinned fibres were then washed several times in relaxing solution without detergent. Saponin is an agent that binds to cholesterol and forms relatively large pores in the membrane and, at this low concentration, saponin permeabilizes the sarcolemma without affecting the ability of the sarcoplasmic reticulum to accumulate and release Ca2+ (Endo & Kitazawa, 1978). Controls experiments were carried out to confirm the integrity of the sarcoplasmic reticulum (see below). The preparation was immersed sequentially in five different solutions (Table 1): the first solution to deplete the sarcoplasmic reticulum of Ca2+; the second to wash out caffeine; the third to load the sarcoplasmic reticulum with Ca2+; the fourth as another wash-out solution to prepare the fibre for the last solution, which was used to release Ca2+ using caffeine, an activator of RyRs (Herrmann-Frank et al. 1999). The ionic composition of these solutions was the same as that of the relaxing and activating solutions (pCa 9.0 and 4.5, respectively). However, the concentrations of EGTA, Mg2+ and Ca2+ were varied (Table 1). For these experiments, sarcoplasmic reticulum Ca2+ uptake was performed in a solution containing 1 mM Mg2+, a physiological concentration. In contrast, Ca2+ was released in a solution containing caffeine at a low [Mg2+] (0.1 mM) that favours the release of Ca2+ by caffeine (Kabbara & Stephenson, 1994; Fryer & Stephenson, 1996). The application of 10 mM caffeine generates a transient contracture. At the beginning of each experiment, two or three 10 mM caffeine contractures were then generated to check the integrity of the sarcoplasmic reticulum after the saponin skinning treatment, and control caffeine-induced contractures were recorded at regular intervals. The amplitude of the control caffeine contractures did not change significantly, suggesting that the sarcoplasmic reticulum remained in a functional state. After successive control caffeine applications, the fibres in which the decrease in the contracture amplitude was more than 5% were discarded. To investigate the effects of adenosine on the caffeine-induced Ca2+ release, various adenosine concentrations (1–100 nM) were added to the release solution that contained 2.5 mM of caffeine (Table 1, solution 5). The incubation time in solutions 1–4 was 2 min, as described in Table 1. Furthermore, to investigate possible effects of adenosine on the Ca2+ loading cycle of the sarcoplasmic reticulum Ca2+ pump, 10, 50 and 100 nM of adenosine were then added to the uptake medium (Table 1, solution 3). The sarcoplasmic reticulum of saponin-skinned fibres was then loaded with Ca2+ solution (pCa 6.5) in the presence of adenosine for various periods of time, and the sarcoplasmic reticulum Ca2+ content was then estimated by the application of 2.5 mM caffeine.
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Solutions and chemicals
The composition of the skinned fibre solutions was calculated according to Godt & Nosek(1989). The relaxing (pCa 9.0 with pCa = –log10[Ca2+]; [Ca2+] = 10–9M) and activating (pCa 4.5 with pCa = –log10[Ca2+]; [Ca2+] = 10–4.5M) solutions consisted of (mM): EGTA, 10; imidazole, 30; Na+, 30.6; Mg2+, 1; MgATP, 3.16; and phosphocreatine, 12. The ionic strength was adjusted to 160 mM by adding KCl, and the pH was adjusted to 7.1 with HCl or KOH. In saponin-skinned fibre experiments, the solutions also contained phosphocreatine kinase (17.5 IU ml–1) and sodium azide (1 mM). For both Triton X-100- and saponin-skinned fibre experiments, solutions with intermediate concentrations of Ca2+ were obtained by mixing the pCa 9.0 and 4.5 solutions in appropriate proportions. EGTA, 1,3-dipropyl-8-cyclopentylxanthine(DPCPX), 2-chloro-N6-cyclopentyladenosine(CCPA), adenosine and phosphocreatine were obtained from Sigma Chemical Co. (St Louis, MO, USA). DPCPX and CCPA were dissolved in ethanol and methanol, respectively, with a maximal final concentration of 0.8%. ZM 241385 and CGS 21680, obtained from Tocris Bioblock Scientific (Illkirch, France), were dissolved in dimethyl sulphoxide (DMSO), with a maximal final concentration of 0.5%. The final concentrations of the different solvents used had no effect on the Ca2+ sensitivity of contractile proteins.
Fitting of inhibition curves and statistical analysis
The accumulation of Ca2+ by the sarcoplasmic reticulum during the loading period was estimated by calculating the Ca2+ concentration at the myofilaments during caffeine application. The sarcoplasmic reticulum Ca2+ loading capacity can be estimated by using contractile proteins as an internal sensor. Conventionally, the Ca2+ released by caffeine of saponin skinned fibre preparations was then estimated via the isometric force transient, which results from the Ca2+-evoked activation of the troponin–tropomyosin complex. It is well known that the Ca2+ sensitivity of the contractile proteins is different between skeletal and cardiac muscles. Furthermore, caffeine used to release Ca2+ from the sarcoplasmic reticulum and various pharmacological tools also affects the properties of the contractile proteins. Thus, the same amount of Ca2+ released from the sarcoplasmic reticulum could cause different force transients. Consequently, to compare all the results presently obtained on ferret cardiac muscle and to discard the effects of the drugs on the contractile proteins, the amplitude of each caffeine contracture was converted into a Ca2+ transient according to the method developed by Makabe et al. (1996) and Uttenweiler et al. (1998). The caffeine contracture amplitude was expressed as a percentage of the maximal Ca2+-activated tension, which has been recorded at pCa 4.5 for each fibre. This was achieved by using the individual pCa–force relationship, which was established for each saponin-skinned fibre in the presence of caffeine and/or adenosine and/or an adenosine receptor agonist and antagonist. The percentage change in the caffeine-induced Ca2+ release was then estimated. The results obtained at various concentrations of adenosine or CGS 21680 were fitted using a sigmoid equation (Hill equation). All values are expressed as the means ± S.E.M. ANOVA followed by Dunnett's post hoc test was used to compare the various parameters in the different groups. Statistical significance was reached when P < 0.05.
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Results |
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In cardiac saponin-skinned fibres, the application of caffeine (2.5 or 10 mM) after a Ca2+ loading step in a solution that contains 10–6.5 M of Ca2+ during 2 min induced a transient contracture. The amplitude of the response (13.2 ± 2.6 mN mm–2, n = 16) induced by 2.5 mM caffeine was about 50% of the amplitude of the contracture (24.9 ± 3.4 mN mm–2, n = 16) induced by 10 mM of caffeine, which had been demonstrated to be nearly the maximal response produced by caffeine application. In this study, caffeine was used at a concentration of 2.5 mM, because this provided a better estimate of any change in the caffeine response. Under similar loading conditions, in the absence (Fig. 1A) or presence (Fig. 1B and C) of adenosine (10 or 100 nM), the release of Ca2+ by caffeine resulted in a similar transient contracture. In the presence of adenosine, a concentration-dependent increase in the amplitude of the 2.5 mM caffeine responses was observed without significant modification for the time to peak and the time of half-relaxation (Fig. 1). The Ca2+ released by caffeine of saponin skinned-fibre preparations was estimated via the isometric force transient, which results from the Ca2+-evoked activation of the troponin–tropomyosin complex. The amplitude of the contractile response is mostly due to the sarcoplasmic reticulum Ca2+ content but is also dependent on the Ca2+ sensitivity of the contractile proteins. The potentiation of the amplitude of caffeine contractures observed in the presence of adenosine suggests that adenosine could act on the Ca2+ uptake or release and/or on the contractile proteins. To find out whether the apparent Ca2+ sensitivity and the maximal Ca2+-activated tension were modified by adding adenosine, we then conducted some experiments using Triton X-100-skinned fibres.
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The relative tension–pCa curves established in the presence of 2.5 mM of caffeine were shown to be shifted to the right by adenosine (100 nM; Table 2). However, the values indicate that in the presence of caffeine (2.5 mM), adenosine (100 nM) did not significantly change the pCa50 (obtained under control conditions) or the amount of Ca2+ required to produce 50% of the maximal Ca2+-activated tension (Table 2). Furthermore, nH and the maximal Ca2+-activated tension were not significantly affected by adenosine (Table 2). These findings suggest that the observed potentiation of caffeine responses in saponin-skinned fibres was not attributable to changes in the Ca2+ sensitivity of the contractile apparatus, but rather to an effect of adenosine on the Ca2+ pump and/or on the RyR2 receptors of the sarcoplasmic reticulum.
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Effects of adenosine on Ca2+ uptake by the sarcoplasmic reticulum
The caffeine response (2.5 mM) was not significantly affected by the presence of adenosine (10, 50 or 100 nM) at any time during the Ca2+ loading step. The values found were not significantly different (n = 5). For example, the maximal amount of Ca2+ released after 2 min of loading in the absence or in the presence of adenosine (50 nM) was 0.48 ± 0.07 (n = 5) and 0.46 ± 0.01 (n = 5), respectively. It had previously been demonstrated that adenosine activates the release of Ca2+ from the RyR2 in sheep cardiac sarcoplasmic reticulum incorporated into planar bilayers (McGarry & Williams, 1994). In the absence of any effect on contractile protein and on the sarcoplasmic reticulum Ca2+ uptake, the increase in the Ca2+ released by caffeine would suggest that adenosine was acting directly on the Ca2+ release channel of the sarcoplasmic reticulum.
Effects of adenosine on the caffeine-induced release of Ca2+ from the sarcoplasmic reticulum
On saponin-skinned fibres, the effects of adenosine (1–100 nM), added to the release solution, were tested on the 2.5 mM caffeine contracture. Caffeine amplitudes recorded in the absence (Fig. 1A) or presence of adenosine (Fig. 1B and C) were expressed as a percentage of the maximal tension, which was obtained at pCa 4.5 for each fibre. The data plotted versus adenosine concentrations displayed a sigmoid (Hill) relationship (Fig. 2A), with a maximal potentiation of the caffeine contracture amplitude by 36.1 ± 4.9%, a Hill coefficient of 1.8 ± 0.4, and a Km of 38.8 ± 3.9 nM. Therefore, the contracture amplitude values were converted into Ca2+ transients using the individual pCa–force relationship for each fibre. The calculated values plotted versus adenosine concentrations displayed a sigmoid (Hill) relationship (Fig. 2B), with maximal potentiation of the caffeine-induced release of Ca2+ by 22.2 ± 1.6%, a Hill coefficient of 2.1 ± 0.3 and a Km of 35.9 ± 3.6 nM. The increase in the caffeine-induced Ca2+ released by adenosine could be explained by a direct effect on the sarcoplasmic reticulum RyR2. However, an indirect effect, related to the presence of adenosine receptors, should also be taken into account, particularly since it was recently demonstrated that adenosine affects the Ca2+ release in skeletal muscle by acting on A1 receptors present at the sarcoplasmic reticulum membrane level (Hleihel et al. 2001). This possibility was investigated by testing the effects of A1 and A2A receptor agonists (CCPA and CGS 21680, respectively) and antagonists (DPCPX and ZM 241385, respectively) on saponin-skinned cardiac fibres.
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On saponin-skinned fibres, CGS 21680 (1–50 nM) potentiated the 2.5 mM caffeine-induced release of Ca2+ in a concentration-dependent manner (Fig. 3A). A plot of the relationship between CGS 21680 concentrations and the calculated Ca2+ release potentiation displayed a sigmoid curve (Fig. 3B), with an apparent CGS 21680 Km value of 4.0 ± 0.4 nM (n = 6). The Hill coefficient was 1.9 ± 0.3 (n = 6), and the maximal potentiation of Ca2+ release was 10.9 ± 0.4% (n = 6). In Triton X-100-skinned fibres, in the presence of caffeine (2.5 mM), CGS 21680 (50 nM) did not significantly affect the pCa50 obtained under control conditions or the amount of Ca2+ required to produce a Ca2+-activated tension equal to 50% the maximal value. Furthermore, nH and the maximal Ca2+-activated tension were not significantly affected by CGS 21680 (Table 2). These findings suggest that the potentiation of caffeine responses by CGS 21680 was not related to changes in the sensitivity of the contractile apparatus. By comparison to the data obtained on the effects of adenosine on the sarcoplasmic reticulum Ca2+ release, the selective A2A receptor agonist CGS 21680 induced a maximal potentiation that represented 50% of that observed with adenosine. It could be then proposed that A2A receptors are implicated in the adenosine effects observed in cardiac saponin-skinned fibres. The effects of ZM 241385, a selective A2A receptor antagonist (Poucher et al. 1995), were then tested on saponin-skinned fibres, especially because ZM 241385 offers the best A2A/A1 selectivity ratio (Shryock & Belardinelli, 1997).
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The potentiation of caffeine-induced (2.5 mM) Ca2+ release by adenosine (50 nM; 15.3 ± 1.0%, n = 6, Fig. 4A) and by CGS 21680 (50 nM; 11.2 ± 0.4%, n = 6, Fig. 5A) were reduced by the selective A2A adenosine receptor antagonist ZM 241385 (50 nM) to 8.0 ± 1.4% (n = 4, Fig. 4B) and to 5.4 ± 1.2% (n = 4, Fig. 5B), respectively. According with these data, it could then be suggested that A2A receptors may be present and functional on the ferret cardiac sarcoplasmic reticulum, and implicated in the adenosine effect. However, it has been shown on the hippocampal membrane that ZM 241385 can also antagonize A1 receptors (Lopes et al. 1999). To find out whether A1 receptors were involved in the change observed here, the effects of a specific A1 receptor agonist and antagonist were investigated.
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The characteristics of the caffeine responses were not significantly modified by CCPA (1 and 50 nM), and the potentiation of the caffeine-induced release of Ca2+ by adenosine (50 nM) and CGS (50 nM) was not modified by DPCPX (10–50 nM). In the light of these findings, we propose that the contribution of A1 receptors to adenosine-induced effects was reduced in ferret cardiac saponin-skinned fibres.
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Discussion |
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It is recognized that adenosine produces its physiological effects by interacting with membrane-bound receptors located on the plasma membrane (Collis & Hourani, 1993; Fredholm et al. 1997). In our study, the results indicate that CGS 21680 (a selective A2A receptor agonist) increased caffeine-induced Ca2+ release in a dose-dependent manner, and that the potentiation of caffeine-induced Ca2+ release by adenosine and by CGS 21680 was reduced by the specific A2A receptor antagonist, ZM 241385. The fact that a selective A2A adenosine receptor antagonist induced a reduction in the effects of both adenosine and the agonist on the caffeine contractures induced in saponin-skinned fibres suggests that A2A receptors were present in the sarcoplasmic membrane. This proposal is supported by the range of concentrations of CGS 21680 and of ZM 241385 used, which had previously been reported to be specific for the A2A receptors (Palmer et al. 1995; Poucher et al. 1995, 1996; Keddie et al. 1996; Shryock & Belardinelli, 1997). It can therefore be proposed that the observed potentiation of Ca2+ release resulted in part from the effect of adenosine on A2A receptors present in the sarcoplasmic reticulum. However, the absence of any significant effect on caffeine responses of CCPA and DPCPX (selective A1 receptor agonist and antagonist, respectively) suggests that the participation of A1 receptors in the effect of adenosine on the release of Ca2+ from the sarcoplasmic reticulum was reduced. In cardiac muscle, major changes in the ionic medium and metabolites that occur during myocardial ischaemia affect Ca2+ release channel activity. These include depletion of the high-energy adenine nucleotide pool and increases in cytosolic free Ca2+ (Koretsune & Marban, 1990; Mohabir et al. 1991; Lee & Allen, 1992; Murphy et al. 1993). Measurements on sarcoplasmic reticulum vesicle Ca2+ efflux single channel and radioligand binding assays using [3H] ryanodine have indicated that cardiac release channel activity is dependent on cytosolic Ca2+ concentration modulated by Mg2+ and ATP (Coronado et al. 1994; Meissner, 1994). Our findings clearly show that the application of adenosine to saponin-skinned fibres induced an increase in the amplitude of caffeine contracture, thereby suggesting that adenosine, which can be derived from the catabolism of adenosine triphosphate (ATP) in cardiac muscle, also modulates the RyR2 by increasing Ca2+ release from the sarcoplasmic reticulum. This allows us to propose that adenosine affects cardiac cells by activating RyR2, leading to Ca2+ release from the sarcoplasmic reticulum. Our results show that the sarcoplasmic reticulum sensitivity to adenosine was observed at very low concentrations (10–50 nM), and the solutions may already contain a significant contamination level of adenosine. Our data then imply that the contamination levels cannot be much more than perhaps 10–50 nM. Furthermore, it would be very interesting to see the effects of very high concentrations of adenosine. In isolated, perfused rat hearts, it has been demonstrated that 10–4 M adenosine increases contractile performance via activation of A2A receptors (Monahan et al. 2000). By contrast, in rat skeletal muscle, using similar approaches, it was shown that adenosine reduced the caffeine-induced Ca2+ release (Hleihel et al. 2001). It was then proposed that, in skeletal muscle, adenosine modulates the sarcoplasmic reticulum Ca2+ release by a direct effect on the RyR1 or by an indirect effect owing to the activation of A1 receptors. The different effects of adenosine on the sarcoplasmic reticulum Ca2+ release between cardiac and skeletal muscles could be explained by the expression of two isoforms of RyR, type 2 and 1, respectively, but also by the different mechanisms involved in excitation–contraction coupling and in intracellular Ca2+ regulation in both types of muscles.
Adenosine has a number of activities that make it a possible cardioprotective and therapeutic agent for use in chronic heart failure (Kitakaze & Hori, 2000). It is likely that the formation of adenosine may be one of the mechanisms by which ischaemic preconditioning protects the heart against Ca2+ paradox injury. At the cardiac sarcolemmal level, the A1 receptors present are involved in ischaemic preconditioning and induce cardioprotection against Ca2+-paradox injury (Kawabata et al. 2000). In fact, an increase in the release of Ca2+ by the sarcoplasmic reticulum plays a key role in cardiac excitation–contraction coupling, and therefore the increased Ca2+ release facilitated by adenosine might increase myocardial O2 consumption and counteract the effects of adenosine A1 receptors. However, it has already been shown that adenosine stimulates the Na+–Ca2+ exchanger, extruding Ca2+, in ewe ventricular sarcolemma, by acting via A1 receptors (Brechler et al. 1990). Furthermore, in rat heart, cardiac protection has been demonstrated to be mainly due to A3 receptor stimulation that modulates the sarcoplasmic reticulum Ca2+ channel (Zucchi et al. 2001). In cultured newborn rat cardiac myocytes, after A3 receptor activation, an increase in the sarcoplasmic reticulum Ca2+ uptake and Na+–Ca2+ efflux of Ca2+ were sufficient not only for compensation of Ca2+ release, but also for effective prevention of extensive increase in intracellular Ca2+, and may provide a protective mechanism against intracellular Ca2+ overload (Shneyvays et al. 2004). Thus, coupled to the effects of adenosine previously described at the sarcolemmal membrane level, our findings suggest that adenosine is involved in another mechanism for the regulation of Ca2+ via A2A receptors present in the sarcoplasmic reticulum. This means that adenosine may play an important modulating role in the cardiac cell by preserving Ca2+ homeostasis, especially during ischaemia and hypoxia, by acting on sarcolemmal membrane receptors, and this study supplies the novel finding that adenosine A2A receptors may also be found on the sarcoplasmic reticulum membrane. The importance of the modulating influence of A2A receptor-mediated actions in the ischaemic, hypoxic or failing myocardium, where endogenous levels of adenosine are elevated, requires further investigation.
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