A more recent version of this article appeared on September 28, 2001

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Article

Potentiation of Ca²⁺ Release by cADP-Ribose in the Heart Is Mediated by Enhanced SR Ca²⁺ Uptake Into the Sarcoplasmic Reticulum

Valeriy Lukyanenko, Inna Györke, Theodore F. Wiesner Sandor Györke

From the Department of Physiology (V.L., I.G., S.G.), Texas Tech University Health Sciences Center, and the Department of Chemical Engineering (T.F.W.), Texas Tech University, Lubbock.

Correspondence to Dr Sandor Györke, Texas Tech University HSC, 3601 4th St, Lubbock, TX 9430-6551. E-mail physg{at}ttuhsc.edu

	Abstract

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Abstract — cADP-Ribose (cADPR) is a novel endogenous messenger that is believed to mobilize Ca²⁺ from ryanodine-sensitive Ca²⁺ stores. Despite intense research, the precise mechanism of action of cADPR remains uncertain, and experimental findings are contradictory. To elucidate the mechanism of cADPR action, we performed confocal Ca²⁺ imaging in saponin-permeabilized rat ventricular myocytes. Exposure of the cells to cADPR resulted in a slow (>2 minutes) and steady increase in the frequency of Ca²⁺ sparks. These effects on local release events were accompanied by a significant increase in sarcoplasmic reticulum (SR) Ca²⁺ content. In comparison, sensitization of ryanodine receptors (RyRs) by caffeine, a true RyR agonist, caused a rapid (<1 second) and transient potentiation of Ca²⁺ sparks followed by a decrease in SR Ca²⁺ content. When the increase in the SR load was prevented by partial inhibition of the SR Ca²⁺ with thapsigargin, cADPR failed to produce any increase in sparking activity. cADPR had no significant impact on activity of single cardiac RyRs incorporated into lipid bilayers. However, it caused a significant increase in the rate of Ca²⁺ uptake by cardiac SR microsomes. Our results suggest that the primary target of cADPR is the SR Ca²⁺ uptake mechanism. Potentiation of Ca²⁺ release by cADPR is mediated by increased accumulation of Ca²⁺ in the SR and subsequent luminal Ca²⁺-dependent activation of RyRs.

Key Words: ventricular myocytes • ryanodine receptors • sarcoplasmic reticulum Ca²⁺ • Ca²⁺ sparks • cADP-ribose

	Introduction

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In mammalian cardiac myocytes, contractile activation occurs in response to Ca²⁺ influx from the extracellular space. This triggers Ca²⁺-induced Ca²⁺ release via Ca²⁺ release channels known as ryanodine receptors (RyRs), which reside in the membrane of the sarcoplasmic reticulum (SR).^1,2 The function of cardiac RyRs has been studied extensively by monitoring global and local intracellular Ca²⁺ release signals with fluorescent Ca²⁺ indicators, by measuring Ca²⁺ fluxes from isolated SR preparations, and by recording single-channel currents by using the planar lipid bilayer technique.³ These studies have shown that the activity of the RyR is controlled by a number of endogenous ligands, including Ca²⁺ in both the cytosol and the SR lumen, ATP, Mg²⁺, and calmodulin. Consequently, the RyR is thought to carry the corresponding binding sites.

In recent years, cADP-ribose (cADPR) has emerged as a potential endogenous regulator of RyR activity.⁴ Despite intense research, the precise mechanism of action of cADPR remains uncertain, and experimental findings are contradictory. Early in vitro studies reported that cADPR can directly activate RyRs reconstituted into lipid bilayers.⁵ Subsequent studies, however, indicated no effects or detected changes that would be expected to be abolished in the presence of physiological concentrations of ATP.^6,7 On the other hand, most experiments performed in cardiac cells yielded results that are consistent with a role for cADPR in the modulation of SR Ca²⁺ release. In guinea pig ventricular cells, the cADPR antagonists 8-amino-cADPR and 8-Br-cADPR have inhibited both depolarization-induced Ca²⁺ transients and spontaneous Ca²⁺ waves.^8,9 It has been further demonstrated that cADPR applied to the cytosol increases cell-average Ca²⁺ transients and contractions^10–13 and enhances the frequency of local Ca²⁺ release events (ie, sparks) in both intact and permeabilized cardiac myocytes.^11,14 The effects of cADPR on Ca²⁺ release are highly temperature dependent and relatively slow kinetically.^10,11 These observations have been interpreted as cADPR sensitizing RyRs to cytosolic Ca²⁺, thereby enhancing Ca²⁺-induced Ca²⁺ release. The marked temperature dependence and complex kinetics of these effects suggest that the action of cADPR on Ca²⁺ release might involve intermediate signaling steps, such as interactions of cADPR with other proteins of the RyR complex or with calmodulin.¹¹ However, precisely how cADPR influences the function of Ca²⁺ release channels has not been elucidated. To explore the mechanism of action of cADPR on SR Ca²⁺ release, we investigated the effects of cADPR on Ca²⁺ release and on accumulation by the SR in permeabilized rat ventricular myocytes. Our experiments showed that the primary target of cADPR is the SR Ca²⁺ uptake mechanism. Potentiation of Ca²⁺ release by cADPR is mediated indirectly by increased accumulation of Ca²⁺ in the SR and subsequent luminal Ca²⁺-dependent activation of RyRs.

	Materials and Methods

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Confocal Microscopy
Single ventricular myocytes were obtained from adult male Sprague-Dawley rat hearts (n=33) by enzymatic dissociation.¹⁵ The standard Tyrode’s solution contained (in mmol/L) NaCl 140, KCl 5.4, MgCl₂ 0.5, CaCl₂ 1, HEPES 10, NaH₂PO₄ 0.25, and glucose 5.6, pH 7.3. The cells were permeabilized by exposure to saponin (0.01% for 45 to 60 seconds).¹⁴ The permeabilization solution contained (in mmol/L) K aspartate 100, KCl 20, MgATP 3, MgCl₂ 0.81 ([Mg²⁺]_free=1 mmol/L), EGTA 0.5, phosphocreatine 10, HEPES 20, and 5 U/mL creatine phosphokinase, pH 7.2. The control experimental solution contained (in mmol/L) K aspartate 100, KCl 20, MgATP 3, EGTA 0.5, CaCl₂ 0.114 ([Ca²⁺]_free=100 nmol/L), MgCl₂ 0.81 ([Mg²⁺]_free=1 mmol/L), phosphocreatine 10, HEPES 20, Fluo-3 K-salt 0.03 (TefLabs), and 5 U/mL creatine phosphokinase, pH 7.2. The free [Ca²⁺] and [Mg²⁺] at given total Ca²⁺, Mg²⁺, ATP, and EGTA concentrations were calculated by using (WinMAXC 1.80). All chemicals were from Sigma unless otherwise specified. All experiments were performed at room temperature (21°C to 23°C). Changes in [Ca²⁺] were recorded with a Bio-Rad Laser Scanning Confocal system (MRC-1024ES, Bio-Rad Laboratories) with an Olympus 60x 1.4 N.A. objective.¹⁶ Fluo-3 was excited by light at 488 nm (25-mW argon laser, intensity attenuated to 0.3%), and the fluorescence was acquired at wavelengths of >515 nm in the line scan mode at rate of 6.0 ms per scan. Ca²⁺ sparks were quantified by using a detection computer algorithm, and their statistics were corrected for missed events and amplitude distortions introduced by instrumental noise.^16,17 Data were expressed as means±SE. Comparisons were performed by using the paired t-test, and significance was defined at P<0.05.

Preparation of SR Membrane Vesicles
Heavy SR microsomes were isolated by differential centrifugation from the ventricles of dog heart as described previously.¹⁵

Lipid Bilayer Experiments
Single RyRs were reconstituted by fusing SR microsomes into planar lipid bilayers as described previously.¹⁸ Channel incorporation was performed in solutions containing (in mmol/L) CsCH₃SO₃ 350, CaCl₂0.02, HEPES 20 (pH 7.4) on the cytosolic (cis) side of the bilayer, and CsCH₃SO₃ 20, CaCl₂ 0.02, HEPES 20 (pH 7.4) on the lumenal (trans) side of the bilayer. The experimental solutions contained (in mmol/L) CsCH₃SO₃ 350, CaCl₂ 0.02 ([Ca²⁺]_free=5 µmol/L), MgATP 3, MgCl₂ 0.6 ([Mg²⁺]_free=0.9 mmol/L), HEPES 20 (pH 7.4) (cis), and CsCH₃SO₃ 20, CaCl₂ 0.02, HEPES 20 (pH 7.4) (trans). Single channels were monitored at +30 mV in symmetrical Cs⁺ (350 mmol/L). Single-channel currents were recorded with an Axopatch 200A (Axon Instruments) patch-clamp amplifier. Data were filtered at 2 kHz and digitized at 5 to 10 kHz. Acquisition and analysis of data were performed by using pClamp 6.01 software (Axon Instruments).

SR Ca²⁺ Uptake Measurements
Ca²⁺ uptake measurements were performed with spectrofluorometer D-Scan (PTI). We monitored [Ca²⁺] outside the membrane vesicles using the ratiometric Ca²⁺ indicator Fura 2FF (1 mmol/L, K⁺ salt). The medium in the cuvette consisted of (in mmol/L): K aspartate 100, KCl 20, MgCl₂ 0.81, phosphocreatine 10, MgATP 3, and 5 U/mL creatine phosphokinase, pH 7.2. To inhibit the Ca²⁺ release through RyRs, the experimental medium was supplemented with 10 µmol/L ruthenium red. Membranes (0.5 to 1.0 mg of protein per milliliter) were added to the cuvette, and active Ca²⁺ uptake was initiated by the administration of 500 nmol/L Ca²⁺.

	Results

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Effects of cADPR on Ca²⁺ Sparks
In accordance with our previous studies, cADPR caused an increase in the frequency of Ca²⁺ sparks in permeabilized myocytes.¹⁴ Representative line scan images of a cell acquired before and at different times after addition of 5 µmol/L cADPR are shown in Figure 1A. The changes in frequency and amplitude of events along with the values of basal fluorescence in the same experiment are documented in panel B. After a fast (0.1-second) application of the agent, the frequency of events gradually (ie, within 2 to 5 minutes) increased from approximately 4 to 8 events/s/100 µm and, in experiments without washout, remained elevated at this level for the duration of the experiment (10 minutes, n=5, not shown). The basal fluorescence was relatively constant (gray bars), suggesting that laser illumination did not cause significant dye bleaching or photo-damage to the cell in the course of the experiment. The effect of cADPR on Ca²⁺ sparks was reversible, as the frequency of events returned to levels close to control within 2 to 3 minutes after the removal of cADPR. The results of the effects of cADPR on Ca²⁺ sparks are summarized in Figure 1C. As can be seen, cADPR caused approximately a 50% increase (47±9%, P<0.001) in the frequency of detected events. At the same time, although there was no significant change in the average amplitude of events, the magnitude of the 5% of the brightest sparks (most likely to be situated at the center of the line scan) significantly increased in each of the consecutive images (22±10%, P<0.05, n=5). Consistent with our previous observations,¹⁴ cADPR had no significant effects on the half-maximal duration (21.0±1.1 versus 20.8±1.0 ms in control and presence of 5 µmol/L cADPR, respectively) and half-maximal width (1.9±0.1 and 1.6±0.2 µm in control and presence of 5 µmol/L cADPR, respectively) of the events. The potentiation of Ca²⁺ release by cADPR was prevented by the specific antagonist of cADPR 8-Br-cADPR,¹⁹ confirming that the effects of cADPR were on specific cADPR binding sites (Figure 2). In this series of experiments, the cardiomyocytes were pretreated with 5 µmol/L 8-Br-cADPR for 5 minutes before the exposure to cADPR. Under these conditions, cADPR failed to produce any changes in the frequency and amplitude of events (Figure 2B). Taken together, these results are consistent with the ability of cADPR to produce a slow enhancement in the functional activity of Ca²⁺ release sites by activating a cADPR-specific signaling pathway.

View larger version (50K): [in this window] [in a new window]   Figure 1. Effects of cADPR on properties of Ca2+ sparks in permeabilized rat ventricular myocytes. A, Representative line scan fluorescence images under control conditions (a), and at different times (b through d) after exposure of the cell to 5 µmol/L cADPR (the experimental protocol is presented schematically at the top). B, Ca2+ spark frequency (dark red) and amplitude (blue), and F0 from corresponding images as a function of time before and after the addition of cADPR (arrow) into the bathing solution in the same experiment. The basal fluorescence is indicated by the gray portion at the bottom of the amplitude bars. Stages a through d correspond to images depicted by panel A. C, Averaged spark frequency (red) and amplitude (blue) in control solution 5 minutes after application of cADPR and 3 minutes after washout of the drug. *P<0.01, n=10.

View larger version (92K): [in this window] [in a new window]   Figure 2. Prevention of cADPR-induced potentiation of Ca2+ sparks by 8-Br-cADPR. A, Representative line scan fluorescence images under control conditions (a), 1 minute after exposure of the cell to 5 µmol/L 8-Br-cADPR (b), and 5 minutes after 5 µmol/L of cADPR was added into the bathing solution (c). The experimental protocol is presented schematically at the top. B, Averaged spark frequency (red) and amplitude (blue) in control solution and in 5 minutes after application of 5 µmol/L cADPR (n=5).

Effects of Direct Activation of RyRs on Ca²⁺ Sparks
To better understand the mechanism of action of cADPR on Ca²⁺ release, we contrasted the impact of cADPR with that of the well-known RyR agonist caffeine on spontaneous Ca²⁺ sparks in permeabilized myocytes. We used small concentrations of caffeine (0.1 to 0.3 mmol/L) to produce potentiating effects of similar scale as observed with cADPR. In contrast to the slow and maintained effects observed with cADPR, caffeine produced rapid and transient enhancement in the frequency of events (by 60%). These were followed by a decrease in sparking activity below the control level. The effects of 0.1 mmol/L caffeine on frequency and amplitude of Ca²⁺ sparks are illustrated in Figure 3. Similar rapid and transient changes in spark frequency on the addition of small doses of caffeine were observed in 10 cells used in this series of experiments. The rapid action of caffeine is consistent with the direct interaction of this agent with the RyRs. The secondary decline in frequency of events could be caused by reductions in the SR Ca²⁺ content as a result of enhanced Ca²⁺ release through RyRs. The profound differences in the action of cADPR and caffeine on Ca²⁺ sparks suggest that cADPR acts through a different mechanism, specifically one that does not involve a direct activation of the RyR.

View larger version (68K): [in this window] [in a new window]   Figure 3. Effects of low doses of caffeine on properties of Ca2+ sparks. A, Representative line scan fluorescence images under control conditions (a), as well as 1 ssecond and 2.5 minutes after exposure of the cell to 0.1 mmol/L caffeine (b and c, respectively). The experimental protocol is presented schematically at the top. B, Ca2+ spark frequency (dark red) and amplitude (gray) as a function of time before and after the addition of caffeine (arrow) into the bathing solution in the same experiment.

Effects of cADPR on SR Ca²⁺ Content
To further explore the mechanisms of action of cADPR, we performed measurements of the SR Ca²⁺ content using large concentrations of caffeine (20 mmol/L) to liberate Ca²⁺ from the SR. Exposure of the cells to 5 µmol/L cADPR for 5 minutes caused approximately a 50% increase in the peak amplitude of caffeine-induced Ca²⁺ transients (from 3.2±0.5 to 4.7±0.4 F/F₀, P<0.01; Figure 4A and 4C), implying that the SR Ca²⁺ load was enhanced by cADPR. Under similar conditions, exposure of the cells to 0.1 mmol/L caffeine, the amplitude of Ca²⁺ transients decreased by 46% (from 3.1±0.4 to 1.6±0.4 F/F₀, P<0.05; Figure 4B and 4D), indicating a reduction in the SR Ca²⁺ load. This effect of caffeine is consistent with a direct and selective activation of RyRs, which leads to a net loss in the SR Ca²⁺ content. On the other hand, the ability of cADPR to increase the SR Ca²⁺ content suggests that its effects on SR Ca²⁺ release might be mediated by potentiating the uptake of Ca²⁺ into the SR. Elevation of SR Ca²⁺ content has been reported to increase the frequencies of Ca²⁺ sparks in both intact and permeabilized cardiac myocytes.^17,20,21 Thus, the enhanced sparking activity illustrated in Figure 1 could be secondary to elevation of luminal Ca²⁺ in the presence of cADPR.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effects of cADPR and caffeine on SR Ca2+ content. A and B, Representative Ca2+ transients induced by application of 20 mmol/L caffeine to the whole bath under control conditions and in the presence of 5 µmol/L cADPR (A) or 0.1 mmol/L caffeine (B). C and D, Averaged amplitude of caffeine-induced Ca2+ transients in the control solution and 5 minutes after application of cADPR (C, n=5) or 0.1 mmol/L caffeine (D, n=5).

Preventing Effects of Thapsigargin
To test whether changes in the SR Ca²⁺ content in fact are necessary and sufficient to produce the cADPR-induced increase in sparking activity, we used a specific inhibitor of the SR Ca²⁺ pump (SERCA), thapsigargin, in concert with cADPR. If enhanced accumulation of Ca²⁺ were required for potentiation of Ca²⁺ sparks by cADPR, one would expect little potentiation of Ca²⁺ sparks when the resequestration mechanism is suppressed by thapsigargin. This hypothesis was supported by the results of our experiments (Figure 5). In this series of experiments, the cardiomyocytes were pretreated with 0.1 mmol/L thapsigargin for 1 minute before addition of cADPR. As shown previously,²² thapsigargin at this concentration reduces SR Ca²⁺ accumulation without altering the probability of generation of Ca²⁺ sparks at resting cytosolic [Ca²⁺] (100 nmol/L). With this protocol, the SR Ca²⁺ content of cells exposed to thapsigargin and cADPR was not significantly different from that of control cells (-5±4.3%, n=6; Figure 5D). As illustrated by panels A through C of Figure 5, when the SR Ca²⁺ load of cells exposed to cADPR was the same as it was in control cells, cADPR failed to induce any changes in Ca²⁺ sparks. In several cells, we also examined the effects of thapsigargin alone on Ca²⁺ sparks. In accordance with Song et al,²² 100 nmol/L thapsigargin had no significant effects on spontaneous activity of release sites (the frequency of sparks was 4.8±0.8 and 4.1±7 events/s/100 µm in control and 5 minutes after addition of the drug, respectively, n=5). This apparent lack of influence of reduced SR Ca²⁺ content on release sites can be attributed to the relatively low affinity and steep dependence of the RyR on luminal Ca²⁺ (K_D2.5 mmol/L; Hill Coefficient2).¹⁸ Shannon and Bers²³ have estimated that the resting [Ca²⁺] inside the SR approaches only about 0.7 mmol/L at cytosolic [Ca²⁺] of 100 nmol/L. Therefore, reducing SR Ca²⁺ accumulation may simply decrease [Ca²⁺]_SR below the range where it can effectively influence RyR gating. Taken together, these results suggest that the increase in frequency of Ca²⁺ sparks by cADPR is caused not by direct sensitization of the RyR, but rather it is mediated by elevation in the SR Ca²⁺ content.

View larger version (62K): [in this window] [in a new window]   Figure 5. Preventing cADPR-induced potentiation of Ca2+ sparks by thapsigargin. A, Representative line scan fluorescence images under control conditions (a), 1 minute after addition of 0.1 µmol/L thapsigargin (b) and at different times (c and d) after administration of 5 µmol/L cADPR (the experimental protocol is presented schematically at the top panel). B, Ca2+ spark frequency (dark red) and amplitude (blue) as a function of time before and after the addition of thapsigargin and cADPR into the bathing solution in the same experiment. Stages a through d correspond to images represented in the panel A. C, averaged spark frequency (red) and amplitude (blue) in control and 5 minutes after application of cADPR in the presence of 0.1 µmol/L thapsigargin (n=8). D, representative Ca2+ transients induced by application of 20 mmol/L caffeine to the whole bath under control conditions and in the presence of cADPR and thapsigargin (5 minutes).

Lipid Bilayer Experiments
To evaluate the ability of cADPR to influence the functional activity of the RyR more directly, we performed experiments with single RyRs reconstituted into lipid bilayers. Heavy cardiac SR microsomes were fused into planar lipid bilayers, and single RyR channels were monitored by using Cs⁺ as the charge carrier. The solutions on the cytosolic (cis) side of the channel contained 3 mmol/L ATP and 3.6 mmol/L Mg²⁺ ([Mg²⁺]_free=0.9 mmol/L) to match the concentration of these ions in the internal solutions in our permeabilized myocyte experiments. Addition of cADPR (5 µmol/L) to the cytosolic side of the channel had no significant effect on the open probability (P_o) of the channel (Figure 6). Our results are in agreement with previous studies reporting no significant effects of cADPR on the RyR in the presence of physiological [ATP].⁷ They are also consistent with the results of our experiments in permeabilized myocytes (see above), suggesting that the effects of cADPR on RyR involve indirect mechanisms, such as activation by elevated luminal Ca²⁺.

View larger version (33K): [in this window] [in a new window]   Figure 6. Effect of cADPR on characteristics of single ryanodine receptor channels. A, Representative single channel recordings performed in symmetrical Cs+ and Ca2+ under control conditions and after addition (4 minutes) of 5 µmol/L cADPR. The cis chamber contained 3 mmol/L MgATP and 0.6 mmol/L MgCl2 ([Mg2+]free=0.9 mmol/L). Channel openings are upward. Holding potential was +30 mV. B, Averaged (means±SEM) Po in the control and presence of cADPR (P>0.5; n=4).

SR Ca²⁺ Uptake Measurements
To examine the effects of cADPR on Ca²⁺ uptake by the SERCA, we performed Ca²⁺ uptake measurements fluorometrically in isolated cardiac microsomes using Fura 2FF. To inhibit the Ca²⁺ leak through the RyRs, the medium was supplemented with 10 µmol/L ruthenium red, a known RyR channel blocker. Active Ca²⁺ uptake was initiated by administration to the cuvette solution of 500 nmol/L Ca²⁺. Thus, the preparation featured simple uptake to the SR by SERCA without concurrent release via RyRs. In the representative experiment shown in Figure 7A, free calcium concentration outside of the microsomes declined much more rapidly in the presence of cADPR. The decay constant in the presence of the modulator was approximately half that of the control. Based on results of 17 independent measurements like that in 7A, we found the mean decay constant in the presence of cADPR to be approximately 20 seconds. This compares to an average of 40 seconds for control conditions. This confirms the results of our experiments in permeabilized cells, suggesting that cADPR enhances the rate of Ca²⁺ uptake by the SR and establishes the SR Ca²⁺ uptake mechanism as a likely target of cADPR in the cardiac muscle.

View larger version (17K): [in this window] [in a new window]   Figure 7. Effects of cADPR on Ca2+ uptake by cardiac SR vesicles. A, Cardiac microsomes (0.6 mg/mL) were administered 500 nmol/L CaCl2 under control conditions () and in the presence of 5 µmol/L cADPR (, both in the presence of 10 µmol/L ruthenium red. The time courses of uptake were best fit by exponential functions (solid lines). B, Averaged (means±SEM) time constants of Ca2+ uptake in the control and presence of cADPR (P<0.05; 10 and 8 independent measurements, respectively).

	Discussion

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cADPR is commonly viewed as a specific agonist of RyR channels that acts by sensitizing the RyR to cytosolic Ca²⁺. Our results show for the first time that the primary target of cADPR in the heart is SERCA-dependent Ca²⁺ uptake. Potentiation of Ca²⁺ release by cADPR is mediated by increased SR Ca²⁺ load and subsequent luminal Ca²⁺–dependent activation of RyRs (Figure 8). Luminal Ca²⁺ is increasingly recognized as a modulator of RyR activity in cardiac muscle. It has been recently shown that increases in [Ca²⁺]_SR are detected at specific sites on the luminal side of the RyR or associated regulatory protein. Elevated luminal calcium enhances the open probability of the channel.^18,24 The luminal sensor mechanism, which links the functional activity of the RyR to the loading state of the SR, appears to be a part of a dynamic control system that allows the cells to autoregulate the size and functional state of their SR Ca²⁺ pool.^17,18,25 In the present study, we show that activation of RyRs by Ca²⁺ within the SR also represents the final downstream step in a cADPR-dependent signaling cascade that operates to produce maintained positive inotropic effects on the heart.

View larger version (44K): [in this window] [in a new window]   Figure 8. Schematic presentation of the mechanism of action of cADPR on SR Ca2+ release in cardiac myocytes. cADPR enhances SR Ca2+ uptake either through direct activation of the SERCA pump or through relieving the inhibition of the pump by phospholamban (PLB). This results in elevation of [Ca2+] in the lumen of the SR. The increased [Ca2+]SR enhances the functional activity of the RyR through luminal Ca2+ sensing sites.

Our finding that cADPR acts by enhancing the SR Ca²⁺ uptake reconciles a body of apparently contradictory results. First, it helps to explain why cADPR has no significant effect on isolated RyR channels reconstituted into lipid bilayers^6,7 (and the present study), although it enhances SR Ca²⁺ release from ryanodine-sensitive stores in most in situ studies.^10–12,14 Several previous studies did report increases in RyR P_o under simple ionic conditions (ie, absence of ATP).^5,26 Those effects, however, can be attributed to the action of cADPR on adenine nucleotide binding sites.⁷ This explains why cADPR appears to be ineffective in the presence of millimolar ATP. Second, our results could also account for the slow time course and high temperature dependence of the effects of cADPR described in the literature.^10,11 The slow time course can be ascribed to the dynamic lag associated with accumulation of Ca²⁺ in the SR, a lag which would not be present if cADPR acted directly on the RyR. The high temperature dependence is consistent with the fact that the SR Ca²⁺ATPase is more temperature dependent than the RyR (the former has a Q₁₀=2.2, whereas the latter has a Q₁₀=1.5).²⁷ Based on the reported temperature dependence of the effects of cADPR on whole-cell Ca²⁺ transients,¹⁰ we would expect the effects observed in this study at room temperature to be even more pronounced at body temperature. Finally, our findings explain how cADPR produces maintained stimulation of RyR activity without causing a compensatory decrease in the loading of the SR by Ca²⁺. It has been shown previously and confirmed here (Figure 3) that specific modulators of RyR activity, such as the agonist caffeine and the inhibitor tetracaine produce only transient effects on Ca²⁺ sparks in resting cardiac myocytes.^15,17 The transient nature of the effects arises as a result of SR Ca²⁺ load compensating for the primary changes in availability of the RyR with subsequent luminal Ca²⁺–dependent changes in release site activity. Similar biphasic effects of tetracaine and caffeine on SR Ca²⁺ release have been observed with globally measured systolic Ca²⁺ transients in rat ventricular myocytes.^25,28 One logical consequence of this dynamic regulation process is that maintained potentiation of SR Ca²⁺ release can be attained only by enhancing Ca²⁺ uptake or when the Ca²⁺ uptake and the release mechanisms are stimulated simultaneously.²⁵ Adrenergic stimulation of the SERCA and the RyR can be regarded as a well-established example of the second scenario. The effect of cADPR appears to present a case of a more specific modulatory influence, in which the release is enhanced solely by increasing the uptake, with no direct effects on the RyRs.

Although affecting the frequency of sparks, cADPR did not significantly influence their spatio-temporal characteristics. If the SERCA pump function is altered by cADPR, one might also expect changes in these parameters.²⁹ The decline of [Ca²⁺] during the Ca²⁺ sparks is determined primarily by Ca²⁺ diffusion from the source of Ca²⁺.³⁰ Indeed, complete inhibition of the SERCA pump by 5 µmol/L thapsigargin has been shown to cause only a relatively small prolongation of the [Ca²⁺] decay (by 30%) during Ca²⁺ sparks.³⁰ Thus, the cADPR-mediated change in the pump rate might not be sufficiently large to manifest itself in altered spark decay kinetics. In support of this explanation, 100 nmol/L thapsigargin selected to prevent the enhancement of the SR Ca²⁺ load by cADPR produced no significant changes in the spatio-temporal properties of sparks (Figure 3A). Consistent with our results, Santana et al³¹ found no significant difference in the decay time constants of sparks measured in ventricular myocytes from wild type and phospholamban-deficient transgenic mice, although the SR Ca²⁺ load and the frequency of events were significantly increased in transgenic cells.

It has been reported that the decrease of the depolarization-induced Ca²⁺ transients and contractions produced by the cADPR antagonist 8-amino-cADPR is not accompanied by any decrease of SR Ca²⁺ content assessed by caffeine-induced contractions.⁸ This would imply that the effects of cADPR and its antagonists are not mediated by changes in the SR Ca²⁺ content. The reasons for these discrepancies are not known, but they may involve differences in experimental approaches. Myofilament Ca²⁺ sensitivity is subject to modulation by various factors, which might complicate the interpretation of results obtained with contraction measurements. For example, methylxanthines, including caffeine, are potent myofilament Ca² sensitizers that can influence contractile performance either through direct interaction with contractile proteins or via their ability to inhibit cardiac phosphodiesterases and thereby elevate cAMP.¹ Thus, it is possible that the estimates of SR Ca²⁺ content changes using contraction measurements were influenced by effects of caffeine on myofilaments. Interestingly, in a more recent study, Rakovic et al⁹ showed that 8-amino-cADPR reduced the frequency of spontaneous Ca²⁺ waves, although cADPR itself enhanced the incidents of waves in cardiac myocytes exposed to ouabain. Spontaneous Ca²⁺ waves are generally associated with increased SR Ca²⁺ content. RyR modulators such as the inhibitor tetracaine and the potentiator caffeine have been shown to cause only transient effects on Ca²⁺ waves because the alterations of RyR activity are compensated by changes in load.^15,25,28 Therefore, the sustained effects of cADPR and its antagonists on Ca²⁺ waves are more likely to be the result of SR pump-mediated changes in SR Ca²⁺ load rather than to modulation of the RyRs.

At the present time, we do not know the specific biochemical mechanisms underlying the enhancement of SR Ca²⁺ uptake by cADPR. Theoretically, increased SR Ca²⁺ uptake could be the result of either inhibition of Ca²⁺ leak or stimulation of Ca²⁺ uptake by the SERCA. It has been shown previously that RyRs represent the single most prominent pathway of Ca²⁺ leak from the SR in cardiac myocytes.^32,33 In our SR Ca²⁺ uptake measurements, the RyRs were inhibited by ruthenium red. In addition, cADPR has been reported to have no inhibitory effects on the RyR channels in vitro. Therefore it is unlikely that the increased rate of net Ca²⁺ uptake in our experiments is caused by reduced SR Ca²⁺ leak. Stimulation of the SERCA by cADPR could be produced either by direct potentiation of the SERCA or through the relieving of the inhibition of dephosphorylated phospholamban. Indeed, several agents have been reported either to stimulate the SERCA, such as gingerol and 1-(3,4-dimethoxyphenyl)-3-dodeconone,^34,35 or to inhibit the phospholamban interactions as described with tannin, ellagic acid and polyanionic compounds.^35,36 Phospholamban is expressed primarily in the cardiac, smooth, and slow-twitch skeletal muscles, and it has been found only in mammalian and avian species.³⁷ On the other hand, the intracellular Ca²⁺ mobilizing effects of cADPR have been described in various cell types across different phyla. This lack of correlation between the effects of cADPR and the presence of phospholamban would seem to imply that stimulation of Ca²⁺ uptake occurs through a mechanism that does not require the involvement of phospholamban. It is possible however that in other cellular systems, the role of phospholamban is played by homologous regulatory proteins. Regardless of the specific biochemical mechanisms, stimulation of SR Ca²⁺ uptake plays an important role in the enhancement of Ca²⁺ release through RyRs in cardiac muscle. Future studies will have to determine precisely how cADPR causes stimulation of SR Ca²⁺ uptake in the heart and whether these mechanisms apply to other cell types.

	Acknowledgments

 
This work was supported by grants from the American Heart Association, Texas Affiliate (V.L.) and the National Institutes of Health (HL 52620 and HL 03739 to S.G.).

Received May 7, 2001; accepted August 21, 2001.

	References

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