Cardiovascular Research

Cardiovascular Research 2002 53(2):326-333; doi:10.1016/S0008-6363(01)00471-0
© 2002 by European Society of Cardiology

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Copyright © 2001, European Society of Cardiology

Effect of cardiac A₁ adenosine receptor overexpression on sarcoplasmic reticulum function

Riccardo Zucchi^a,*, Rachael J Cerniway^b, Simonetta Ronca-Testoni^a, R.Ray Morrison^c, Giovanni Ronca^a and G.Paul Matherne^b

^aDepartimento di Scienze dell'Uomo e dell'Ambiente, University of Pisa, 56126 Pisa, Italy
^bDepartment of Pediatrics and the Cardiovascular Research Center, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
^cDepartment of Pediatrics, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA

^* Corresponding author r.zucchi{at}med.unipi.it

Received 18 June 2001; accepted 4 September 2001

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: We investigated the effect of A₁ adenosine receptor overexpression, which has been reported to increase myocardial tolerance to ischemia-reperfusion injury, on sarcoplasmic reticulum (SR) Ca²⁺ handling. Methods: Transgenic mouse hearts (300-fold A₁ adenosine receptor overexpression) and wild-type mouse hearts were perfused in the Langendorff mode and subjected either to 80 min of aerobic perfusion or to 30 min of aerobic perfusion, 20 min of global ischemia and 30 min of reperfusion. The hearts were then homogenized and used to assay SR oxalate-supported ⁴⁵Ca²⁺ uptake and [³H]–ryanodine binding. Results: Transgenic hearts showed increased resistance to ischemia-reperfusion, as shown by lower diastolic tension (1.5±0.2 vs. 2.6±0.1 g, P<0.05) and higher recovery of developed tension (45±3 vs. 30±4% of the baseline, P<0.05) following ischemia-reperfusion. Under baseline conditions, oxalate-supported ⁴⁵Ca²⁺ uptake was lower in transgenic hearts, owing to reduced V_max (10.6±2.0 vs. 17.8±2.7 nmol/min per mg of protein, P<0.05), and the difference was preserved after ischemia-reperfusion (10.0±1.0 vs. 15.7±2.5 nmol/min per mg of protein, P<0.05). No significant difference in [³H]–ryanodine binding was observed. Conclusions: A₁ adenosine receptor overexpression is associated with a decreased rate of active Ca²⁺ transport into the SR. We hypothesize that changes in SR function may cause a depletion of the SR Ca²⁺ pool, which might protect from ischemic injury by delaying the development of cytosolic Ca²⁺ overload during ischemia.

KEYWORDS Adenosine; Ca-pump; Ischemia; Receptors; Reperfusion; SR (function)

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This article is referred to in the Editorial by K. Mubagwa (pages 286–289) in this issue.

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Adenosine exerts a protective action in different models of myocardial injury [1,2]. Protection is usually mediated by the stimulation of A₁ receptors, although a role for other receptor types, particularly A₃ receptors, has been demonstrated in some species. A novel approach to study the cardioprotective action of adenosine is provided by the development of transgenic mice overexpressing A₁ adenosine receptors [3]. A₁ receptor overexpression affords increased resistance to ischemic injury and can mimic ischemic preconditioning [3,4]. The molecular mechanisms responsible for these effects are still largely unknown. A₁ receptor overexpression improves energy state after ischemia and reperfusion [5], which might be responsible, at least in part, for the protective effect. However, alternative possibilities need investigation and in particular, there is evidence that adenosine may influence Ca²⁺ homeostasis, acting on sarcolemmal or intracellular Ca²⁺ channels [6,7]. Cytosolic Ca²⁺ overload has major importance in the pathophysiology of ischemic injury, and changes in sarcoplasmic reticulum (SR) Ca²⁺ handling have profound effects on susceptibility to ischemia [8]. Therefore, we assessed SR function in transgenic hearts overexpressing A₁ adenosine receptors under basal conditions and after ischemia-reperfusion, by measuring oxalate-supported ⁴⁵Ca²⁺ uptake and [³H]–ryanodine binding.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1 Transgenic mouse model
This investigation conforms with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and the work was approved by the Institutional Animal Care and Use committee. Transgenic mice with the rat A₁ adenosine receptor cDNA under the control of the cardiac-specific -myosin heavy chain promoter were produced as described previously [3].

2.2 Langendorff perfused heart model
Hearts were isolated from 25-week-old male and female wild-type (body weight 28±2 g, n=10) and transgenic mice (body weight 29±2 g, n=10) overexpressing A₁ adenosine receptors. Mice were anesthetized with 50 mg/kg sodium pentobarbital administered intraperitoneally, a thoracotomy performed and hearts rapidly excised into heparinized ice-cold perfusion buffer. The aorta was cannulated (20 gauge stainless steel blunt needle) and the hearts retrogradely perfused at a constant pressure of 80 mm Hg with modified Krebs buffer containing: 120 mM NaCl, 25 mM NaHCO₃, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 1.2 mM Mg₂SO₄, 15 mM glucose, and 0.05 mM EDTA (free Ca²⁺ 2.41 mM, free Mg²⁺ 2.36 mM). Buffer was equilibrated with 95% O₂, 5% CO₂ at 37°C, giving a pH of 7.4. Hearts were immersed in perfusate within a water-jacketed bath maintained at 37°C. The left ventricle was vented with a small polyethylene apical drain. Coronary flow was continuously monitored via an ultrasonic Doppler flow-probe (Transonic Systems Inc., Ithaca, NY, USA) located in the aortic perfusion line. Developed tension was used as an indicator of contractile function, and this was assessed using a small stainless steel hook attached to the cardiac apex and connected to a tension transducer (Model FT03C, Grass). Transducer position was adjusted to yield a diastolic tension of 1.0 g. Developed tension and aortic pressure were recorded with a MacLab data acquisition system (ADInstruments, Castle Hill, Australia) connected to an Apple 7300/180 computer. The tension signal was digitally processed on-line (using MacLab Chart 3.5.6, ADInstruments, Castle Hill, Australia) to yield heart rate and developed tension. Hearts were allowed to equilibrate for 20 min at intrinsic heart rate. All hearts were then paced at a constant rate of 380 bpm (ventricular pacing with 5 ms square waves, 30% above threshold, typically 4–5 V) for a further 10 min of equilibration. The electrodes used for pacing were placed within the electrolyte-containing bath in proximity to the heart, without direct contact with either atria or ventricles. In the ischemia-reperfusion groups, ventricular pacing was then stopped and the aortic inflow line occluded for 20 min of normothermic ischemia. Following ischemia, hearts were reperfused for 30 min with pacing at 360 bpm resumed after 1 min. In the perfusion only (control) groups, pacing was continued after the equilibration period at 360 bpm for 50 min, to allow for the same duration as the ischemia-reperfusion experiments.

2.3 Preparation of cellular fractions
Hearts were initially flash frozen in liquid nitrogen at the end of each experiment. They were then finely minced and homogenized in five volumes of 300 mM sucrose and 10 mM imidazole (pH 7.0 at 4°C) by 15+15 passes in a Potter–Elvejheim homogenizer set at 800 rpm and kept in a cold room at 4°C. The homogenate was filtered through one layer of cheesecloth and used to assay ⁴⁵Ca²⁺ uptake and [³H]–ryanodine binding.

2.4 Assay of SR Ca²⁺ uptake
Oxalate-supported Ca²⁺ uptake was determined in the crude homogenate in the presence of a concentration of ryanodine able to block the Ca²⁺ release channel, as described previously [9]. The homogenate was preincubated at 37°C for 5 min in the presence or absence of 900 µM ryanodine. The uptake medium contained (final concentration): 40 mM imidazole (pH 7.0 at 37°C), 100 mM KCl, 60 mM sucrose, 8 mM NaN₃, 3 mM MgCl₂, 0.145 mM EGTA, various amounts of ⁴⁵CaCl₂ (0.3–0.4 mCi/ml), required to produce the desired free Ca²⁺ concentration, 10 mM potassium oxalate, 5 mM K₃ATP, 0.1–0.2 mg/ml of homogenate protein, and 810 µM ryanodine where appropriate.

The rationale for using ryanodine is the following: In vitro the SR is fragmented and forms a heterogeneous set of vesicles [10]. Some vesicles contain both Ca²⁺ channels and Ca²⁺ pump (SR Ca²⁺-ATPase, SERCA), so that Ca²⁺ uptake actually represents the difference between active (ATP-driven) Ca²⁺ uptake and passive Ca²⁺ efflux through release channels [11]. In order to get an appropriate estimate of the former, Ca²⁺ uptake must be measured after channel blockade, which was achieved in our experiments by pre-incubation with 900 µM ryanodine [9]. The stimulation of SR Ca²⁺ uptake produced by ryanodine has often been considered as an indirect index of SR Ca²⁺ efflux [9,11].

The uptake reaction was started by adding ATP and potassium oxalate to the other reagents. Aliquots of the reaction mixture were taken at 15 s intervals and filtered by suction under vacuum through cellulose nitrate filters with pores of 0.45 µm (Sartorious, Göttingen, Germany), which were quickly washed with 2x2 ml aliquots of washing buffer (40 mM imidazole, 100 mM KCl, 8 mM NaN₃, 3 mM MgCl₂, 0.2 mM EGTA). Radioactivity was measured by scintillation counting at 88% efficiency (LKB Wallac 1214, Finland) and the uptake rate was calculated by linear regression analysis of the first 60 s. Ca²⁺ uptake was determined in duplicate immediately after tissue homogenization.

Some assays were performed at different free Ca²⁺ concentrations to determine the Ca²⁺-dependence of the uptake reaction. The apparent dissociation constant for Ca²⁺ (K_Ca) and the Hill coefficient (n) were calculated by non-linear least square fitting of experimental data to the following equation:

where v is the rate of Ca uptake, V_max is the maximum rate of Ca²⁺ uptake, [Ca²⁺] is free Ca²⁺ concentration [12,13].

2.5 Assay of [³H]–ryanodine binding
High affinity ryanodine binding was assayed at different free Ca²⁺ concentrations, as described previously [9,14]. Briefly, vesicles were incubated at 37°C in a buffer containing 25 mM imidazole (pH 7.4 at 37°C), 1 M KCl, 0.2 to 50 nM [³H]–ryanodine (6 Ci/mmol), 0.950 mM EGTA, and variable amounts of CaCl₂, in order to obtain the desired free Ca²⁺ concentration. After 60 min, the binding reaction was stopped by filtration through cellulose nitrate filters with pores of 0.45 µm (Sartorius), presoaked in 25 mM imidazole and 1 M KCl (washing buffer). The filters were then washed with 2x5 ml aliquots of washing buffer and shaken overnight in 8 ml of scintillation fluid (Optiphase II, LKB). Radioactivity was counted at 50% efficiency in an LKB Wallac 1214 scintillation counter. Incubations were performed in duplicate, and non-specific binding was measured in the presence of 10 µM unlabeled ryanodine. The difference between the counts of duplicate samples was <10% in all cases.

2.6 Chemicals and radionucleotides
Ryanodine was purchased from Calbiochem (San Diego, CA). EGTA was obtained from Sigma Chemicals (St Louis, MO). All other reagents were of analytical grade. Free Ca²⁺ concentration ([Ca²⁺]) was calculated according to Fabiato and Fabiato [15]. [Ca²⁺] was also measured with the antipyrylazo III technique [16], and the results of the assay were in accordance with the theoretical values. [³H]–ryanodine and ⁴⁵CaCl₂ were obtained from New England Nuclear – DuPont (Milan, Italy).

2.7 Statistical analysis
Results are expressed as mean±S.E.M. Data analysis was performed using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego, CA), using the models mentioned in the description of the specific experiments. Differences between groups were evaluated as follows. One-way analysis of variance was used as a global test for differences between means. If between-groups variance was significantly (P<0.05) higher than within-groups variance, individual groups were compared by Student–Newman–Keuls test. Functional data from the isolated heart experiments were evaluated by two way repeated measures ANOVA (one factor repetition) and Student–Newman–Keuls post-hoc test.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 Contractile function and ischemic injury
No differences in baseline functional parameters for wild-type (wet heart weight 149±11 mg, n=10) and transgenic (wet heart weight 159±13 mg, n=10) isolated hearts were observed (Table 1). Global normothermic ischemia immediately reduced heart rate and contractile function, with full arrest within 5 min, causing a rapid rise in diastolic tension (see Fig. 1 for typical tracings and the upper panel of Fig. 2 for cumulative results). The maximum degree of diastolic tension achieved during ischemia was 2.64±0.10 g in wild-type hearts versus only 1.46±0.14 g in transgenic hearts (P<0.05). The time course of myocardial function, as indicated by developed tension, during ischemia-reperfusion is shown in the lower panel of Fig. 2. With the onset of reperfusion, hearts resumed spontaneous contraction within 30 s and exhibited an immediate recovery of developed tension. After an initial increased recovery, there was a decline in function followed by a progressive recovery of developed tension for the remainder of reperfusion. Transgenic hearts demonstrated a significantly improved recovery of function compared with wild-type hearts at all times during reperfusion. This difference was particularly marked at 2 min of reperfusion, when transgenic hearts recovered to 68±8% of pre-ischemia compared to only 25±5% in wild-type hearts (P<0.05). Similarly, final recovery of developed tension in transgenic hearts was also markedly improved compared to wild-type hearts (45±3 vs. 30±4%, P<0.05).

View this table: [in this window] [in a new window]   Table 1 Baseline functional data

 

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Representative tracings of left ventricular tension and coronary flow for wild-type (A) and transgenic (B) isolated hearts. At the end of 20 min of ischemia, wild-type hearts had higher diastolic tension than transgenic hearts. Recovery of developed tension during reperfusion was greater in transgenic hearts compared to wild-type hearts. See Fig. 2 for average values and S.E.M. at different time points.

 

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Time course of diastolic tension (upper panel), and of left ventricular developed tension (lower panel), in wild-type (, n=5) and transgenic (, n=5) hearts subjected to 20 min ischemia and 30 min reperfusion. Diastolic tension was adjusted to 1 g before the onset of ischemia. Actual baseline values averaged 0.98±0.01 and 0.99±0.02 g in wild-type and transgenic hearts, respectively. Developed tension is expressed as percent of baseline values, which averaged 2.2±0.3 and 2.1±0.1 g in wild-type and transgenic hearts, respectively. * P<0.05 vs. wild type, by multivariate ANOVA for repeated measures and Student–Newman–Kuels test.

 
3.2 SR Ca²⁺ uptake
In all samples Ca²⁺ uptake was measured at [Ca²⁺]=1.5 µM (pCa=5.82). This concentration was chosen because it is close to peak cytosolic [Ca²⁺] in systole and to time-averaged cytosolic [Ca²⁺] after brief ischemia [17–20]. Results obtained in the presence of ryanodine are shown in the upper panel of Fig. 3. Under control conditions (perfusion only groups) the uptake rate averaged 10.2±1.2 nmol/min per mg of protein in wild-type hearts, and it was significantly lower in transgenic hearts (7.1±0.5 nmol/min per mg of protein, P<0.05). After ischemia and reperfusion the difference between wild-type and transgenic hearts was preserved, although the rate of Ca²⁺ uptake was slightly reduced in both groups (9.1±0.7 nmol/min per mg of protein in the wild-type group vs. 6.9±0.5 nmol/min per mg of protein in the transgenic group, P<0.05).

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 The upper panel shows the results of the assay of oxalate-supported Ca2+ uptake performed at [Ca2+]=1.5 µM, in the presence of 900 µM ryanodine. The lower panel shows the effect of ryanodine on oxalate-supported Ca2+ uptake, i.e. the difference between the uptake rate measured in the presence and in the absence of 900 µM ryanodine. CTRL, control (perfusion only); I/R, ischemia-reperfusion; WT, wild-type; Trans, transgenic. Bars represent mean±S.E.M. of seven to eight hearts in each group. * P<0.05, by ANOVA and Student–Newman–Keuls test.

 
When ryanodine was omitted from pre-incubation and reaction buffers, the rate of Ca²⁺ uptake decreased by 10–30% in all groups, due to Ca²⁺ efflux through ryanodine-sensitive channels. As discussed below, the response to ryanodine has often been considered as an indirect index of SR Ca²⁺ efflux. Such responses are shown in the lower panel of Fig. 3: the stimulation of Ca²⁺ uptake was slightly higher in wild-type hearts perfused under control conditions, but the difference between groups did not reach the threshold of statistical significance.

In some cases it was possible to assay oxalate-supported Ca²⁺ uptake at six different free Ca²⁺ concentrations, ranging from 0.1 to 40 µM. Results are shown in Fig. 4. As expected [13], the relationship was bell-shaped, since the rate of Ca²⁺ uptake increased at increasing [Ca²⁺], reaching a maximum at [Ca²⁺]10 µM, and decreasing slightly at higher concentrations. The ascending limb of each curve was analyzed as described in the Methods section: differences between wild-type and transgenic hearts were due to changes in the maximum uptake rate (17.8±2.7 vs. 10.6±2.0 nmol/min per mg of protein under control conditions; 15.7±2.5 vs. 10.0±1.0 nmol/min per mg of protein after ischemia and reperfusion; P<0.05 in both cases), while the affinity for Ca²⁺ was unchanged (K_Ca averaged 0.65 µM, without significant differences between groups). No significant difference was observed with regard to the Hill coefficient, that was close to 2 in all groups (average value 1.83).

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Ca2+-dependence of oxalate-supported Ca2+ uptake. Data points represent mean of three hearts per group. =wild type, control; =transgenic, control; =wild type, ischemia-reperfusion; =transgenic, ischemia-reperfusion. Each curve was analyzed by GraphPad Prism 3.02 software, using a sigmoidal dose-response model. In the wild-type control group EC50 averaged 0.63 µM (pEC50=6.20±0.12) and maximum uptake rate averaged 17.84±2.67 nmol/min per mg of protein. In the transgenic control group EC50 was not significantly modified (pEC50=6.17±0.17), whereas maximum uptake rate decreased to 10.63±2.00 nmol/min per mg of protein (P<0.05). Differences between control and transgenic hearts were preserved after ischemia and reperfusion (maximum uptake rate 15.71±2.51 vs. 10.03±0.98 nmol/min per mg of protein, P<0.05; pEC50=6.12±0.15 vs. 6.25±0.08, P=NS). The Hill coefficient was close to 2 in all groups (average value: 1.83).

 
3.3 [³H]–Ryanodine binding
Saturation binding curves were determined at [Ca²⁺]=10 µM. Each curve was well-fitted by a single binding site model, and the Hill coefficient was close to one. Estimated values for binding site number (B_max) and dissociation constant (K_d) are shown in Table 2. Although B_max was slightly higher in wild-type hearts, perfused under control conditions, the difference did not reach the threshold of statistical significance. K_d was also unchanged. Ryanodine binding is strongly Ca²⁺-dependent. To evaluate this issue, some binding experiments were performed with [Ca²⁺] ranging from 0.1 to 100 µM at fixed [³H]–ryanodine concentration (2 nM). We observed that EC₅₀ for Ca²⁺ was similar in all groups, and it averaged 5.1 µM (data not shown).

View this table: [in this window] [in a new window]   Table 2 [3H]–Ryanodine binding

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Overexpression of A₁ adenosine receptors increases cardiac resistance to ischemia [3], but the mechanism of this protection is not fully understood. Metabolic effects or activation of ATP-dependent K⁺ channels have been proposed as mediators of the protection [5,21]. While these mechanisms might contribute to reduced ischemic injury, another possibility, which has not been investigated so far, is represented by modulation of intracellular Ca²⁺ homeostasis. Evidence that acute administration of adenosine may affect SR Ca²⁺ release has recently been reported [7]. SR Ca²⁺ release is also known to play a major role in the pathophysiology of ischemia-reperfusion [8]. Therefore, we investigated whether overexpression of A₁ adenosine receptors influenced SR Ca²⁺ handling. Due to the limited amount of tissue available, we used simple techniques, which have been extensively validated in crude preparations, i.e. the assays of oxalate-supported ⁴⁵Ca²⁺ uptake and of [³H]–ryanodine binding.

In transgenic hearts we observed a significant reduction of the rate of SR Ca²⁺ uptake, which was accounted for by decreased V_max, while the Ca²⁺ affinity of the pump was unchanged. After ischemia-reperfusion, the difference between wild-type and transgenic hearts was preserved, although the rate of SR Ca²⁺ uptake slightly decreased in wild-type hearts, consistent with previous observations (reviewed in Ref. [22]). The effect of ischemia-reperfusion appeared to be blunted in transgenic hearts, and a similar phenomenon has been reported to occur after ischemic preconditioning [23].

Interestingly, treatment with SERCA inhibitors, such as thapsigargin or cyclopiazonic acid, has been found to be cardioprotective in several experimental models. In particular, pre-treatment with thapsigargin or cyclopiazonic acid improved functional recovery and reduced creatine kinase leakage in isolated hearts and in ventricular muscle preparations subjected to ischemia and reperfusion [24–27]. Arrhythmias induced by ischemia or ischemia-reperfusion were also decreased by either cyclopiazonic acid or thapsigargin [28].

The usual interpretation of these findings is that SERCA inhibition determines depletion of the SR Ca²⁺ pool. In fact, during diastole cytosolic Ca²⁺ is transported either into the SR, or outside the sarcolemma (by the sarcolemmal Ca²⁺-ATPase and Na⁺-Ca²⁺ exchanger, the latter playing a predominant role). SERCA inhibition causes more Ca²⁺ to be extruded from the cell, leading to a reduced SR Ca²⁺ pool. If less Ca²⁺ is available in the SR, ischemic injury should be reduced, since Ca²⁺ release from the SR plays a major role in the development of cytosolic Ca²⁺ overload in the early phase of ischemia [8].

Reduced SR Ca²⁺ uptake activity, in as much as it causes depletion of the SR Ca²⁺ pool, might be expected to reduce contractile performance. To this end, developed tension under basal conditions was lower in transgenic than in wild-type hearts [3], although this difference decreased, and was no longer statistically significant, when the hearts were paced at the same rate. It is likely that moderate reduction of SR Ca²⁺ content has minor effects on contractile protein activation, under baseline conditions. However, under conditions of increased Ca²⁺ cycling, e.g. during ischemia, even slight changes in SR Ca²⁺ uptake activity may become critical [7,8].

Beside SERCA, the other major SR structure involved in Ca²⁺ homeostasis is the Ca²⁺ release channel, also known as the ryanodine receptor. Stimulation of Ca²⁺ uptake by ryanodine has been proposed as an indirect index of channel function [9,11]. [³H]–Ryanodine binding experiments provide direct data on channel expression and function, since increased ryanodine binding is usually associated with increased duration of channel opening [29]. In transgenic hearts, we observed a slight reduction of the B_max for [³H]–ryanodine, and of ryanodine-induced stimulation of Ca²⁺ uptake, but the effect did not reach the threshold of statistical significance. In unpublished experiments, we observed that the decrease in [³H]–ryanodine binding was more prominent in selected strains of transgenic mice. On the whole, the relevance of ryanodine receptor changes in mice overexpressing A₁ receptors appears to be limited. However, such changes might contribute to reduce SR Ca²⁺ cycling.

In summary, A₁ adenosine receptor overexpression is associated with significant changes in SR Ca²⁺ handling, particularly with reduced active Ca²⁺ uptake, which might contribute to increase myocardial resistance to ischemia. The underlying molecular mechanisms remain to be determined. Either changes in gene expression or post-translational modifications of SERCA and/or of associated molecules (e.g. phospholamban) might be involved. Further studies based on the combination of biochemical and molecular biology techniques will be necessary to unravel the relevant signaling pathways and effectors. In general, the present results and previous observations [7] suggest that SR structures are important targets of the regulatory action of adenosine.

Time for primary review 24 days.

	Acknowledgements

 
This work was supported by MURST (COFIN 2000).

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