cADPR stimulates SERCA activity in Xenopus oocytes

Abstract

The intracellular second messenger cyclic ADP-ribose (cADPR) induces Ca²⁺ release through the activation of ryanodine receptors (RyRs). Moreover, it has been suggested that cADPR may serve an additional role to modulate sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump activity, but studies have been complicated by concurrent actions on RyR. Here, we explore the actions of cADPR in Xenopus oocytes, which lack RyRs. We examined the effects of cADPR on the sequestration of cytosolic Ca²⁺ following Ca²⁺ transients evoked by photoreleased inositol 1,4,5-trisphosphate (InsP₃), and by Ca²⁺ influx through expressed nicotinic acetylcholine receptors (nAChR) in the oocytes membrane. In both cases the decay of the Ca²⁺ transients was accelerated by intracellular injection of a non-metabolizable analogue of cADPR, 3-Deaza-cADPR, and photorelease of cADPR from a caged precursor demonstrated that this action is rapid (a few s). The acceleration was abolished by pre-treatment with thapsigargin to block SERCA activity, and was inhibited by two specific antagonists of cADPR, 8-NH₂-cADPR and 8-br-cADPR. We conclude that cADPR serves to modulate Ca²⁺ sequestration by enhancing SERCA pump activity, in addition to its well established action on RyRs to liberate Ca²⁺.

1. Introduction

Diverse cellular functions are regulated by changes in cytosolic [Ca²⁺]. In addition to Ca²⁺ influx across the plasma membrane, intracellular organelles including the endoplasmic/sarcoplasmic reticulum (ER/SR), Golgi apparatus, mitochondria and lysosome-related acidic compartments serve as Ca²⁺ sources [1–5]. Liberation of Ca²⁺ from these stores is regulated by intracellular Ca²⁺ releasing messengers that act on Ca²⁺-permeable receptor/channel molecules in the organelle membranes. One major pathway involves inositol 1,4,5-trisphosphate (InsP₃), which binds to InsP₃ receptor/channels (InsP₃R) in the endoplasmic reticulum (ER) membrane [6, 7]. InsP₃-mediated Ca²⁺ signalling is a well-established system in many cells types, and has served as paradigm for discovery of other described Ca²⁺mobilizing messengers [8–10]. A second major pathway involves ryanodine receptors (RyRs), which are abundantly expressed in ER/SR membranes. Both InsP₃R and RyR channels are regulated by cytosolic Ca²⁺ itself, and are further modulated by enzymes such as PKA [11–13].

Following a Ca²⁺ signal, cytosolic [Ca²⁺] must ultimately be restored to its basal level. Canonical mechanisms involved in cytosolic Ca²⁺ removal are the plasma membrane Ca²⁺-ATPase and Na⁺/Ca²⁺-exchanger, the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA), and the mitochondria Ca²⁺ uniporter [14]. The relative contributions of those Ca²⁺ removal mechanisms vary widely among cell types and with developmental stage [15, 16].

The nucleotide, cADPR has been implicated as an additional Ca²⁺-mobilizing messenger [9, 17]. It is synthesized by ADPR-ribose cyclase enzymes, CD38 or CD157 in mammals [18, 19], which are widely distributed, pointing to a ubiquitous role of cADPR. The ability of cADPR to mobilize cytosolic Ca²⁺ is distinct from that of InsP₃, but instead is thought to involve an action on RyRs in the ER membrane. The interaction of cADPR with RyRs to enhance Ca²⁺ liberation was initially demonstrated in sea urchin egg homogenates by showing the sensitivity of cADPR-mediated Ca²⁺ release to pharmacological inhibitors of RyRs [20], and has subsequently been studied extensively in cardiac muscle. Most studies concur that the primary target of cADPR is the RyR [21–28]. However, a report by Lukyanenko showing that Ca²⁺ uptake by cardiac microsomes is accelerated by cADPR led to the suggestion that the enhanced Ca²⁺ liberation through RyR activity may also arise indirectly because increased SR Ca²⁺ uptake leads to an elevated SR Ca²⁺ content [29, 30]. This hypothesis consistent also with more recent studies [31] demonstrating a dual effect of cADPR in ventricular myocytes; that is, an initial, rapid effect to increase SR Ca²⁺ release by changing sensitivity of RyRs to cytosolic Ca²⁺ without changing SR Ca²⁺ content or affecting L-type Ca²⁺ channels, followed by a slower enhancement of SR Ca²⁺ levels consistent with an increased rate of Ca²⁺ uptake.

Studies to confirm and elucidate how cADPR may modulate SR Ca²⁺ uptake are obviously complicated in cell types that display RyR-mediated Ca²⁺ liberation. In the present study, we thus address whether cADPR exerts a specific action on SERCA by using Xenopus laevis oocytes, which express InsP₃Rs but not RyRs [32]. Consistent with this, and with earlier reports [33], photorelease of cADPR failed to evoke detectable Ca²⁺ signals in oocytes. We then examined the effects of cADPR on the clearance of cytosolic Ca²⁺ transients evoked by photoreleased InsP₃ and by influx through expressed plasmalemmal nicotinic acetylcholine receptors (nAChR) in response to hyperpolarizing voltage-clamped pulses. Both photoreleased cADPR and intracellular loading of a non-metabolizable cADPR analogue, 3-Deaza-cADPR [34], accelerated the decay of these cytosolic Ca²⁺ transients. Moreover, this acceleration was abolished by the specific SERCA inhibitor thapsigargin [35], and was antagonized by the cADPR inhibitors 8-NH₂-cADPR and 8-Br-cADPR [36]. We thus conclude that cADPR may play a physiological role in promoting SERCA activity.

2. Methods

2.1 Oocyte preparation

Xenopus laevis (purchased from Nasco International, Fort Atkinson, WI, USA) were sacrificed according to the protocols approved by the UC Irvine Institutional Animal Care and Use committee, and stage V–VI oocytes were isolated after removing ovaries. Oocytes were treated with collagenase (1 mg/ml of collagenase type A1 for 30 min) to remove enveloping cell layers and were stored in modified Barth’s solution (mM: NaCl, 88; KCl, 1; NaHCO₃ 2.4; MgSO₄, 0.82; Ca(NO₃)2, 0.33; CaCl₂, 0.41; Hepes, 5; gentamicin, 1mg/ml; pH 7.4) for 1 – 5 days before use.

2.2. Expression of nAChRs

Plasmids containing cDNA clones coding for the muscle nicotinic receptor α, β, γ, and δ subunits were linearized and transcribed in vitro, and corresponding cRNAs (molar ratio 2 : 1 : 1 : 1) were mixed to a final concentration of 0.1–1 mg/ml and injected (50 nl) into oocytes. Oocytes were maintained at 16 °C for 1–3 days to express nACh-Rs in the plasma membrane. Expression level was monitored using a voltage clamp to measure current in response to 100 – 500 nM ACh: oocytes showing currents >1 μA at −80 mV were selected for experiments.

2.3. Microinjection of oocytes

Intracellular microinjections were performed using a Drummond microinjector in Ca²⁺ -free Barth’s solution. Final intracellular concentrations given below were calculated assuming a cytosolic volume of 1 μl. Oocytes were loaded with combinations of following: fluo 4 dextran, high affinity, 40 μM; Oregon Green 488 BAPTA-5N (OGBTA-5N), 40 μM; P₄₍₅₎-(1-(2-nitrophenyl)ethyl)ester, tris (triethylammonium) salt (caged-InsP₃), 8μM; cyclic adenosine 5′-diphosphate ribose, 1-(1-(2-nitrophenyl)ethyl ester (caged-cADPR), 16 μM; cyclic 3-deaza-adenosine 5′-diphosphate ribose (3-Deaza-cADPR), 0.8 μM; 8-amino-cyclic adenosine 5′-diphosphate ribose (8-NH₂-cADPR), 16 μM; 8-bromo-cyclic adenosine diphosphate ribose (8-Br-cADPR), 80 μM. Oocytes were left for 15 – 30 min after injections to allow agents to diffuse throughout the cell before use.

2.4. Confocal laser scanning microscopy

Confocal linescan Ca²⁺ images were obtained as described previously [37], employing a custom-built confocal scanner interfaced to an Olympus IX70 inverted microscope [38]. Fluorescence excitation was provided by the 488 nm line of an argon ion laser, with the laser spot focused by a 40 × oil immersion objective (NA 1.35) and scanned along a 50 μm line. Emitted fluorescence was detected at wavelengths > 510 nm through a confocal pinhole, providing lateral and axial resolutions of about 0.3 and 0.8 μm, respectively. Images were collected using custom-written image acquisition software (Labview). Recordings were made at 16–18 °C, imaging at the level of the pigment granules in the animal hemisphere of oocytes bathed in normal Ringer solution (mM: NaCl₂, 120; KCl, 2; CaCl₂, 1.8; HEPES, 5;pH 7.4). InsP₃ was uniformly photoreleased throughout a 100 μm spot surrounding the image scan line [39], and the relative concentration of InsP₃ was controlled by using an electronic shutter to vary flash duration and/or neutral density filters (N.D.). Intervals of 2m in were allowed between recordings. Fluorescence signals are expressed as ratios (ΔF/F₀) of the fluorescence (F) at each pixel relative to the mean resting fluorescence (F₀) at that pixel prior to stimulation. Custom routines written in the IDL programming environment (Research Systems, Boulder, CO, USA) were used for image processing and measurements were exported to Microcal Origin version 6.0 (OriginLab, Northamptom, MA, USA) for analysis and graphing.

2.5. Reagents

Fluo 4 dextran, high affinity (K_d:~350 nM), OGBAPTA-5N (Kd:~20 μM), caged-InsP₃, caged-cADPR and 8-NH₂-cADPRwere purchased from Invitrogen (Carlsbad, CA, U.S.A.). 3-Deaza-cADPR and collagenase type A were from Sigma-Aldrich (St. Louis, MO, U.S.A.), and 8-Br-cADPR (IC₅₀: ~1 μM) was from BIOMOL International, L.P. (Plymouth Meeting, PA, U.S.A.).

3. Results

3.1. cADPR does not mobilize Ca²⁺ in oocytes

We first confirmed previous findings that cADPR fails to evoke Ca²⁺ liberation in Xenopus oocytes [33]. Oocytes were loaded with the high affinity (K_d= 345 nM) Ca²⁺ indicator fluo-4 dextran (40 μM final intracellular concentration) and caged-cADPR (16 μM final intracellular concentration). No responses were detected upon even very strong stimuli (repeated sequences of 200 ms flashes at maximal UV intensity; 3 regions tested in each of 3 oocytes); whereas weaker photolysis flashes evoked large responses in oocytes loaded with caged InsP₃ (8 μM final intracellular concentration) (e.g. Fig. 1A). Moreover, no change in basal Ca²⁺-dependent fluorescence was seen after injecting oocytes with the non-metabolizable analogue 3-Deaza-cADPR to a final intracellular concentration of 0.8 μM (control: 4.3 ± 1.0 fluorescence unit, 3-Deaza-cADPR: 4.3 ± 0.4 fluorescence unit, n = 10, p > 0.05).

Fig. 1.

3-Deaza-cADPR accelerates the decay of global Ca²⁺ signals generated by photoreleased InsP₃. (A). Representative confocal line scan images illustrating fluo-4 dextran fluorescence signals evoked by photoreleased InsP₃ in a control oocyte (upper panel) and in an oocyte pre-loaded with 3-Deaza-cADPR to a final cytosolic concentration of ~0.8 μM (lower panel). In both cases the durations of the photolysis flashes was about double the threshold required to evoke a detectable Ca²⁺ signal. (B) Fluorescence profiles from (A) averaged across the scan line, after normalizing peak amplitudes to 100%. (C) Mean decay times (75 % recovery from peak to baseline) of fluorescence signals in control oocytes (black; 10.0 ± 0.8 s n = 6 oocytes), and 3-Deaza-cADPR-loaded oocytes (red; 4.6 ± 0.5 s n = 10; p = 0.01). (D) Mean peak fluorescence signals are not significantly different (p > 0.05) between control (ΔF/F₀ = 27.8 ± 3.0, n = 6) and 3-Deaza-cADPR-loaded oocytes (ΔF/F₀ = 26.6 ± 2.8, n = 10).

3.2. cADPR accelerates the decay of InsP₃-evoked Ca²⁺ transients

We then explored possible actions of cADPR independent of Ca²⁺ mobilization through RyRs by examining the effects of 3-Deaza-cADPR on global and local Ca²⁺ signals generated by photorelease of InsP₃ from a caged precursor. Fig. 1A shows representative confocal linescan images of global Ca²⁺ transients evoked by relatively strong photorelease of InsP₃ in a control oocyte (upper panel) and in an oocyte previously loaded with 3-Deaza-cADPR (lower). To compare kinetics in the face of cell-to-cell variability we normalized responses to individual peak amplitudes (Fig. 1B), and measured times to 75 % recovery from peak to baseline. The mean recovery time was accelerated about two-fold by 3-Deaza-cADPR (Fig. 1C: control, 10.0 ± 0.8 s, n= 6 oocytes; 3-Deaza-cADPR, 4.6 ± 0.5 s, n= 10, p < 0.01), whereas there was no significant effect of 3-Deaza-cADPR on peak amplitudes (Fig. 1D).

3.3. Thapsigargin inhibits the action of cADPR to accelerate the decay of InsP₃-evoked Ca²⁺ signals

To address whether cADPR affects Ca²⁺ sequestration by targeting SERCA activity we used thapsigargin as a specific inhibitor of the SERCA pump [35]. Global Ca²⁺ signals were evoked by flash photorelease of InsP₃ as before, and paired measurements of decay rates were obtained in the same oocytes before and 15 min after addition of 20 μM thapsigargin to the bathing solution. Surprisingly, thapsigargin caused only a slight slowing of Ca²⁺ decay in control oocytes (Fig. 2A: 75 % recovery before thapsigargin 10.1 ± 0.8 s; after thapsigargin 11.3 ± 1.3 s, n = 6, p > 0.05), suggesting that SERCA activity plays only a minor role in the decay of InsP₃-evoked Ca²⁺ signals under basal conditions. On the other hand, oocytes loaded with 3-Deaza-cADPR showed a more rapid Ca²⁺ decay (4.7 ± 0.5 s, n = 10, vs. 10.1 ± 0.8 s, n = 6 in control oocytes; p = 0.01). Importantly, this was markedly slowed following thapsigargin treatment, returning close to the value in control cells without cADPR (Fig. 2C, 75% recovery after thapsigargin 10.2 ± 0.5 s, n = 10; p = 0.01).

Fig. 2.

Thapsigargin antagonises the effect of 3-Deaza-cADPR on accelerating the decay of Ca²⁺ signals evoked by photoreleased InsP₃. (A) Corresponding decay times in control oocytes (without 3-Deaza-cADPR) before (solid black, 10.1 ± 0.8 s) and 15 min after application of 20 μM thapsigargin (broken black, 11.3 ± 1.3 s, n = 6; p > 0.05). (B)Thapsigargin slowed the decay of InsP₃-evoked Ca²⁺ signals in oocytes preloaded with 3-Deaza-cADPR (solid grey, before thapsigargin, 4.7 ± 0.5 s; broken grey, 15m in after thapsigargin, 10.2 ± 0.5 s, n = 10; p = 0.01).

3.4. cADPR accelerates clearance of Ca²⁺ entering through expressed plasmalemmal nAChRs

The results in Figure 2 suggest that cADPR likely accelerates cytosolic Ca²⁺ clearance by promoting SERCA pump activity. However, interpretation of the decay rate of InsP₃-evoked Ca²⁺ signals is complicated because this reflects both the rates of sequestration mechanisms that remove Ca²⁺ ions from the cytosol, and the rate at which Ca²⁺ flux into the cytosol through InsP₃R terminates [40]. To address whether the action of cADPR on Ca²⁺ sequestration is independent of activity of InsP₃Rs, we expressed Ca²⁺-permeable nicotinic acetylcholine receptors (nAChRs) in the oocyte membrane to serve as a ‘Ca²⁺ switch’ so as to precisely control the kinetics of cytosolic Ca²⁺ elevations [41, 42]. Acetylcholine was maintained at a low, non-desensitizing concentration in the bathing solution. Oocytes were voltage clamped at 0 mV to minimize the electrochemical gradient for Ca²⁺ influx at rest and pulsed to −120 mV to evoke Ca²⁺ transients. Figure 3A shows representative line scan images in response to hyperpolarizing steps with durations of 1.0 and 3.0 s, and corresponding measurements of fluorescence ratio changes averaged across the scan line are shown in Fig. 3B. The decay of the fluorescence signal following termination of Ca²⁺ influx upon stepping back to 0 mV was appreciably slower with the 3 s pulse than with the 1 s pulse. A likely explanation for this difference is that decay of [Ca²⁺] after a brief influx is dominated by passive diffusion of Ca²⁺ ions into the enormous interior volume of the oocyte, whereas this process is slowed by accumulation of Ca²⁺ during longer pulses so that other active sequestration mechanisms assume greater prominence.

Fig. 3.

3-Deaza-cADPR accelerates the decay of Ca²⁺ signals evoked by Ca²⁺ influx through nAChRs expressed in the plasma membrane. (A) Linescan images illustrating Ca²⁺ transients evoked by stepping the membrane potential from 0 mV to −120 mV for 1.0 s (left) or 3.0 s (right) to increase the electrochemical driving force for influx of extracellular Ca²⁺ through nAChR activated by 100 – 500 nM ACh in the bathing solution. (B) Corresponding fluorescence profiles averaged across the scan line. (C) Comparison of averaged, normalized fluorescence profiles from control oocytes (black) and oocytes pre-loaded with 3-Deaza-cADPR (grey) for hyperpolarizing pulses of 1.0 s (left) and 3.0 s (right) duration. Inset bar graphs show mean values of 75% decay times.

We next compared the decay of normalized signals in control oocytes (Fig. 3C, black traces) with that in oocytes pre-loaded with 0.8 μM 3-Deaza-cADPR (grey traces). An acceleration was evident following both 1.0 s and 3.0 s hyperpolarizing pulses, and mean data are summarized by the inset bar graphs in Figure 3C (1.0 s hyperpolarizing pulse: control, 1.7 ± 0.2 s, n= 4, and 3-Deaza-cADPR, 0.9 ± 0.3 s, n = 3, p = 0.03: 3.0 s hyperpolarizing pulse: control, 3.7 ± 0.2, n = 25, and 3-Deaza-cADPR, 2.9 ± 0.2 s, n = 36, p< 0.01).

3.5. Rapid action of cADPR on accelerating Ca²⁺ uptake

The experiments described above utilized a non-metabolizable cADPR analogue preloaded into the oocyte several minutes before recording. To then determine how rapidly cADPR may act on Ca²⁺ sequestration we used photolysis of caged cADPR to elevate cytosolic [cADPR] shortly before inducing Ca²⁺ influx through nAChRs (Fig. 4A). Control experiments showed that exposure to UV light in oocytes not loaded with caged cADPR produced no artifactual change in Ca²⁺ decay kinetics following a hyperpolarizing pulse to induce Ca²⁺ influx (Fig. 4B: 3.6 ± 0.4 s without UV in solid black; 3.5 ± 0.3s with UV in broken black; p > 0.05). In contrast, sustained exposure to photolysis light beginning 5.0 s before the 3.0 s hyperpolarizing pulse resulted in a small but statistically significant acceleration of decay in oocytes loaded with 16 μM caged cADPR (Fig. 4C: 75% recovery times 3.2 ± 0.1 s without UV exposure grey trace, 2.7 ± 0.1 s with exposure, broken trace; n = 10, p < 0.05).

Fig. 4.

Photorelease of cADPR rapidly induces an acceleration of Ca²⁺ decay. (A) Diagram illustrating the experimental protocol. (B) Control experiment, comparing Ca²⁺ decay kinetics with and without UV exposure in oocytes not loaded with 3-Deaza-cADPR. Traces show averaged, normalized fluorescence profiles, beginning 1.0 s before the end of the hyperpolarizing pulse. Inset bar graphs show paired measurements of mean decay times to fall to 75% of peak in 4 oocytes (without UV in solid black and with UV in broken black). (C) Photorelease of cADPR accelerates Ca²⁺ decay. Traces and bar graphs compare decay kinetics in oocytes not loaded with caged cADPR and not exposed to UV (black), loaded with caged cADPR but not exposed to UV (solid grey) and loaded with caged cADPR and exposed to UV (broken grey).

3.6. Thapsigargin blocks the cADPR-mediated acceleration of Ca²⁺ decay following influx through nAChRs

To confirm whether the effect of cADPR on speeding the clearance of Ca²⁺ that had entered the cytosol through nAChR is mediated via actions on SERCA, we again applied 20 μM thapsigargin to block SERCA activity. In control oocytes (without 3-Deaza-cADPR), incubation with thapsigargin for 30 min resulted in almost no change in decay of the Ca²⁺ transient following influx evoked by 3.0 s hyperpolarizing pulses (Fig. 5A, 75% decay time before thapsigargin in solid black, 3.7 ± 0.2 s, n = 25 oocytes; with thapsigargin in grey 3.7 ± 0.4, n = 5 oocytes, p >0.05); concordant with the similar lack of effect on decay of InsP₃-evoked Ca²⁺ transients (see Fig. 2A). On the other hand, the accelerated decay seen in oocytes loaded with 3-Deaza-cADPR was substantially slowed by thapsigargin (Fig. 5B; 75 % recovery time before thapsigargin 2.9 ± 0.1 s, n = 36; with thapsigargin 4.4 ± 0.3 s, n = 10, p < 0.01).

Fig. 5.

Thapsigargin antagonizes the effect of 3-Deaza-cADPR on accelerating the decay of Ca²⁺ following influx through nAChRs. Data were obtained following the protocol in Fig. 3, using 3 s hyperpolarizing pulses. (A) Representative examples of Ca²⁺ decay in a control oocytes (without 3-Deaza-cADPR) before (black trace) and after (broken trace) treatment with 20 μM thapsigargin for 30 min. Inset histogram shows mean values of 75% decay time (before thapsigargin, 3.7 ± 0.2 s, n = 25: after thapsigargin 3.7 ± 0.4 s, n = 5, p > 0.05). (B) Corresponding traces and measurements showing that thapsigargin reversed the acceleration of Ca²⁺ decay in oocytes injected with 3-Deaza-cADPR (before thapsigargin, 2.9 ± 0.1, n = 36: after thapsigargin, 4.4 ± 0.3; n = 10; p < 0.01).

3.6. cADPR inhibitors antagonise the action of 3-Deaza-cADPR on Ca²⁺ clearance

8-NH₂-cADPR and 8-br-cADPR are competitive antagonists of cADPR modulation of RyR-mediated Ca²⁺ signalling in many cell types, with respective IC₅₀ values of 9 nM and 1 μM in inhibiting Ca²⁺ signals induced by cADPR in sea urchin egg homogenate [36]. We thus examined whether they would similarly antagonize the action of cADPR on accelerating cytosolic Ca²⁺ clearance following influx through nAChRs. Comparison of decay rates in oocytes injected with 3-Deaza-cADPR alone (75 % recovery time 2.9 ± 0.2 s, n = 36) with those in oocytes loaded also with 8-NH₂-cADPR (16 μM final intracellular concentration) showed a strong antagonism (Fig. 6A; 75% recovery time; 3.6 ± 0.5 s, n = 10, p = 0.02). A lesser slowing was also apparent in oocytes loaded with 80 μM 8-br-cADPR (Fig. 6B; 75% recovery time 3.3 ± 0.3 s, n = 10, p = 0.03).

Fig. 6.

Competitive inhibitors of cADPR action antagonize the effect of 3-Deaza-cADPR on accelerating Ca²⁺ clearance. (A) Traces and bar graphs compare the kinetics of Ca²⁺ decay measured as in Fig. 3 following influx through nAChR in oocytes injected with 3-Deaza-cADPR alone (solid grey trace, mean time to decay to 75% from peak 2.9 ± 0.2 s, n =36) and together with 8-NH₂-cADPR (broken grey trace, 3.6 ± 0.5 s, n = 10, p < 0.05). (B) Corresponding action of 8-NH₂-cADPR (decay to 75 % of peak 3.3 ± 0.3 s, n = 10, p < 0.05).

4. Discussion

This study provides evidence for a distinct role of cADPR in the control of intracellular Ca²⁺ homeostasis via sequestration by SERCA pumps. Our main findings are that; (i) cytosolic calcium clearance is accelerated by cADPR, an action that is inhibited by the specific antagonists 8-NH₂-cADPR and 8-Br-cADPR, (ii) photorelease of cADPR from a caged precursor promotes acceleration of calcium clearance, and (iii) the effect of cADPR is abolished by thapsigargin, a specific blocker for SERCA pumps. Although previous reports had suggested that cADPR may modulate Ca²⁺ sequestration by SERCA [29, 30], definitive studies were hampered by its well-established action to sensitize Ca²⁺ liberation through RyR. We thus investigated the actions of cADPR in Xenopus oocytes, which lack RyRs [32] and do not show Ca²⁺ signals in response to cADPR [33].

The most direct evidence comes from experiments utilizing Ca²⁺-permeable nACh-R channels expressed in the oocyte membrane as a Ca^{2+ ‘}switch’, whereby Ca²⁺ influx could be abruptly attenuated by stepping the membrane potential to more positive voltages such that the subsequent decay of intracellular Ca²⁺ fluorescence signal exclusively reflects Ca²⁺ removal from the cytosol. Oocytes pre-loaded with the non-metabolizable cADPR analogue 3-Deaza-cADPR showed a significantly acceleration in decay. This effect of 3-Deaza-cADPR was blocked by thapsigargin, indicating a specific action on SERCA.

Although the extent of the acceleration of Ca²⁺ clearance by cADPR appears relatively modest (< 2-fold; Fig. 3), it must be remembered that SERCA is only one of several mechanisms (including sequestration by mitochondria; plasma membrane pumps and exchangers) removing Ca²⁺ from the cytosol. In particular, the enormous volume of the Xenopus oocyte in conjunction with the peripheral organization of InsP₃-mediated Ca²⁺ release sites make passive diffusion into the interior of the cell a major factor contributing to the decline in cytosolic free [Ca²⁺] adjacent to the plasmalemma [43]. Indeed, application of thapsigargin to control oocytes caused little detectable change in decay rate, suggesting that SERCA plays only a minor role in Ca²⁺ removal when measured in this way under basal conditions. Thus, the thapsigargin-sensitive speeding of decay induced by 3-Deaza-cADPR implies a much stronger regulatory action on SERCA than is immediately apparent from our records, and its effect on Ca²⁺ dynamics would be more prominent in cells of ‘normal’ size.

The decay of global InsP₃-evoked Ca²⁺ signals was accelerated more prominently by cADPR than was the clearance of Ca²⁺ entering across the plasma membrane, suggesting an action on the termination of Ca²⁺ liberation through InsP₃R in addition to its effects on cytosolic Ca²⁺ clearance. cADPR has been shown to interact directly with the InsP₃R (in particular the type 1 InsP₃R present in Xenopus oocytes)[32], but an alternative explanation may be that InsP₃R function is indirectly modulated by elevated [Ca²⁺] in the ER lumen resulting from accelerated SERCA activity [44].

The mechanism by which cADPR exerts its action on SERCA remains unclear. Observation that photorelease of cADPR causes a small but significant acceleration of Ca²⁺ sequestration within < 8 s would be consistent with a direct molecular interaction, but is not so rapid as to rule out a pathway involving intermediate proteins that may interact with SERCA after binding cADPR. In this context it is, however intriguing that unidentified cADPR-binding proteins apparently distinct from the RyRs have similar molecular weights (100 and 140 kDa) to isoforms of SERCA [45].

Modulation of SERCA activity has been intensively studied in the heart, where Ca²⁺ re-uptake into the SR is the predominant mechanism of sequestration from the cytosol [15]. SERCA activity in the heart is known to be enhanced by phosphorylation of phospholamban by cAMP-dependent protein kinase (PKA) [15]. Interestingly it has been indicated the existence of an SR-associated ADP-ribose cyclase in guinea-pig heart activated by PKA-mediated phosphorylation [46, 47]. Moreover, in hydroid [46] it has been reported that PKA-mediated phosphorylation associates ADP-ribose cyclase activity, adding further weight to the notion that cADPR may serve as a parallel pathway for SERCA regulation. Our results were obtained in oocytes which express type 2b and 3 isoforms of SERCA rather than the type 2a isoform in cardiac tissue, the observation that cADPR enhances pump rate by cardiac microsomes [29] suggests that this action is conserved across SERCA isoforms.

In summary, ample evidence indicates that a major role of cADPR is to modulate or directly activate RyRs in those tissues where RyRs are expressed [9]. We show that cADPR also modulates Ca²⁺ clearance in Xenopus oocytes where RyRs are not present. Thus, cADPR plays dual, apparently opposing roles in modulating the ‘ins and outs’ of Ca²⁺ signalling, by both potentiating Ca²⁺ liberation through RyR and accelerating its subsequent sequestration by SERCA. Most studies of cellular Ca²⁺ signalling have tended to emphasize the fast dynamic release of Ca²⁺ into the cytosol through ion channels as the predominant factor shaping cytosolic Ca²⁺ signals. However, there is a growing appreciation that Ca²⁺ clearance mechanisms such as SERCA are also an important site of modulation [48, 49], and that disruptions in their physiological regulation may be implicated in diseases as diverse as heart failure [48–50] and Alzheimer’s [51].

Abbreviations

cADPR

cyclic ADP-ribose

SERCA

sarco/endoplasmic reticulum Ca²⁺ -ATPase

ER

endoplasmic reticulum

nAChR

nicotinic acetylcholine receptor

PKA

protein kinase A

RyR

ryanodine receptor