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J. Biol. Chem., Vol. 282, Issue 20, 15302-15311, May 18, 2007

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NAADP Controls Cross-talk between Distinct Ca²⁺ Stores in the Heart^*

From the Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom

Received for publication, December 5, 2006 , and in revised form, March 16, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In cardiac muscle the sarcoplasmic reticulum (SR) plays a key role in the control of contraction, releasing Ca²⁺ in response to Ca²⁺ influx across the sarcolemma via voltage-gated Ca²⁺ channels. Here we report evidence for an additional distinct Ca²⁺ store and for actions of nicotinic acid adenine dinucleotide phosphate (NAADP) to mobilize Ca²⁺ from this store, leading in turn to enhanced Ca²⁺ loading of the SR. Photoreleased NAADP increased Ca²⁺ transients accompanying stimulated action potentials in ventricular myocytes. The effects were prevented by bafilomycin A (an H⁺-ATPase inhibitor acting on acidic Ca²⁺ stores), by desensitizing concentrations of NAADP, and by ryanodine and thapsigargin to suppress SR function. Bafilomycin A also suppressed staining of acidic stores with Lysotracker Red without affecting SR integrity. Cytosolic application of NAADP by means of its membrane permeant acetoxymethyl ester increased myocyte contraction and the frequency and amplitude of Ca²⁺ sparks, and these effects were inhibited by bafilomycin A. Effects of NAADP were associated with an increase in SR Ca²⁺ load and appeared to be regulated by -adrenoreceptor stimulation. The observations are consistent with a novel role for NAADP in cardiac muscle mediated by Ca²⁺ release from bafilomycin-sensitive acidic stores, which in turn enhances SR Ca²⁺ release by increasing SR Ca²⁺ load.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ca²⁺ plays a pivotal role in excitation-contraction coupling in cardiac muscle and is a multifunctional regulator of diverse cellular functions (1). During the cardiac action potential, cytosolic Ca²⁺ concentration rises as a consequence of Ca²⁺ flux across the sarcolemma via voltage-gated L-type Ca²⁺ channels, and by subsequent Ca²⁺ release from the SR via ryanodine receptors (RyRs) (2). Clearance of elevated cytosolic Ca²⁺ concentration involves at least four pathways: the Ca²⁺-ATPase on SR membranes (SERCA), the sarcolemmal Na⁺/Ca²⁺ exchanger, the sarcolemmal Ca²⁺-ATPase, and the mitochondrial Ca²⁺ uniporter. Discrete changes in the regulation of intracellular Ca²⁺ concentration can result in congestive heart failure or arrhythmia, though possible mechanisms remain controversial, and hence it remains important to understand the detailed mechanisms that control Ca²⁺ signaling in the heart.

Nicotinic acid adenine dinucleotide phosphate (NAADP)² is a newly discovered Ca²⁺ messenger with unique properties that has been extensively investigated in various tissues and cell lines (3-8). Cardiac tissue expresses high affinity binding sites for NAADP, and NAADP stimulates Ca²⁺ efflux from microsomal fractions derived from rat heart (9). NAADP is an endogenous molecule, with levels in mouse heart reported to be 0.4 nmol/mg protein (10). Accumulating data have confirmed that NAADP has an ability to release Ca²⁺ from intracellular Ca²⁺ stores, but whether NAADP releases Ca²⁺ from endoplasmic reticulum (ER) or non-ER remains unclear (11). The most intriguing behavior of this molecule is, perhaps, the specific self-induced inactivation of its Ca²⁺ releasing mechanism that is not seen to the same extent with any other intracellular messenger (12-14). Examples of this behavior are provided by the ability of subthreshold concentrations of NAADP to inactivate NAADP-induced Ca²⁺ release in invertebrates and plants (12, 13, 15), and by the ability of high concentrations of NAADP to cause profound self-desensitization in intact mammalian cells (16).

Here we report the first observations of NAADP actions in intact cardiac ventricular myocytes. Two methods were used in the majority of experiments to raise cytosolic levels of NAADP: photorelease of NAADP from a caged compound and loading of NAADP into the cytosol by rapid switch of the extracellular solution to one containing the membrane-permeant acetoxymethyl ester of NAADP (allowing rapid access of NAADP-AM to the cytosol and subsequent liberation of NAADP following action of intracellular esterases). NAADP applied by these methods in cardiac myocytes caused elevation of peak Ca²⁺ transient amplitude, enhancement of cell contraction and increases in the frequency and amplitude of Ca²⁺ sparks. Effects of NAADP were prevented by bafilomycin A1 (an H⁺-ATPase inhibitor that acts on acidic Ca²⁺ stores), by desensitizing concentrations of NAADP and by ryanodine and thapsigargin (to suppress SR function). The observations are consistent with NAADP-induced release of Ca²⁺ from acidic Ca²⁺ stores followed by uptake of additional Ca²⁺ by the SR, leading in turn to an enhanced Ca²⁺ transient. Actions of NAADP were also influenced by -adrenoreceptor stimulation in ways that are consistent with regulation of NAADP levels by this receptor pathway, providing support for a physiological role for NAADP in the heart.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Isolation—Guinea pig ventricular myocytes were isolated using methods described previously. Extracellular solution for superfusion of isolated cells contained (mM): NaCl 118.5, KCl 4.2, NaHCO₃ 14.5, NaH₂PO4 1.18, MgSO ^.₄6H₂O 1.18, CaCl₂ 2.5, glucose 11.1 (BDH Chemicals Ltd, Poole, UK.), gassed with 95% O₂, 5%CO₂ to maintain a pH of 7.4. All experiments were carried out at 36 °C.

Electrophysiology—Electrophysiological experiments were performed using an Axoclamp 2B microelectrode system in voltage or current clamp mode as appropriate (Axon Instruments). Action potentials were stimulated by 2 ms current pulse injections (magnitude 3-5 nA) in bridge current clamp mode. Recordings were made in either the whole cell or permeabilized patch (200 µg/ml amphotericin B; Sigma-Aldrich) configurations using glass microelectrodes. The electrodes were pulled from 1.5 mm (external diameter), 1.17 mm (internal diameter) thin-walled, filamented borosilicate glass capillary tubing (GC150TF-15, Harvard Apparatus Ltd, Kent) using a vertical electrode puller (Narishige PE-2, Japan); electrode resistances were in the range 1.5-4 M when filled with patch pipette solution containing (mM): KCl 140, NaCl 5, MgCl₂ 2, K₂ATP 1, and HEPES 5 (pH 7.2). L-type Ca²⁺ currents were activated by step depolarizations from a holding potential of -40 mV to potentials between -30 mV and +50 mV for 200 ms at a frequency of 0.3 Hz. Ca²⁺ current was measured as the difference between peak current and current at the end of the pulse, using the Clampfit 9.0 (pClamp, Axon Instruments) software package.

Measurement of Ca²⁺ Transients—The loading of ventricular myocytes with fluo-4 was achieved by either inclusion of the dye in the patch pipette solution (75 µM) or incubation for 15 min with the acetoxymethyl ester of fluo-4 (fluo-4 AM, 5 µM). Coverslips were mounted in a static chamber on a confocal microscope system that consisted of a Leica TCS NT scanning head coupled to a Leica DMIRB inverted microscope with a 63x water immersion objective lens (1.2 NA, Leica). After the loading period, cells were washed and imaged in line-scan mode (2.6 ms per line; excitation at 488 nm, using an argon ion laser, Uniphase Ltd; emission at wavelengths longer than 515 nm using a longpass filter with cutoff at this wavelength).

Measurement of Cell Contraction—Images of single cardiac myocytes were captured by a PAL camera (COHU 4710 CCIR) mounted on a Leica DMIRB inverted microscope with a Leitz Wetzler x40 objective lens and recorded onto video tape (Panasonic AG6200 video recorder) for later analysis with an edge detection system (Brian Reece Scientific Newbury, UK); time resolution was 20 ms per point. Values were then captured by the software and translated into % of resting cell length under control and drug conditions.

Drug Application—Application of membrane-impermeant drugs to the cytosol was achieved by inclusion of the drug in the patch pipette solution, in the whole cell (ruptured) patch configuration. In the experiments investigating the effects of NAADP on cell contraction, control contraction measurements were taken in the permeabilized-patch configuration, and then NAADP was allowed access to the cytosol by physical rupture of the patch to achieve whole cell configuration. We have previously shown that, under control conditions (i.e. absence of any drug in the patch pipette solution), contractions are well maintained over a period of many minutes (34).

Synthesis of caged-NAADP was performed essentially in accordance to the procedures specified previously (7). Caged-NAADP (or caged-phosphate Molecular Probes Inc., Eugene, OR) was introduced to the cytosol via the patch pipette, and then an ultraviolet laser (Coherent Inc), which provided light at wavelengths of 351 nm and 364 nm, was applied for 500 ms to produce the active form.

Bafilomycin A (Calbiochem-Novabiochem Ltd., Nottingham, UK, stock solution dissolved in Me₂SO) and NAADP-AM were applied to ventricular cells by a rapid switch local perfusion system (Warner Instrument Corp) to ensure rapid application. A triple barrelled square glass capillary tube was positioned within 100 µm of the cell under study, such that the cell was bathed in solution (36 °C) flowing from only one barrel. Lateral movement of the glass capillary tube (driven by a step-per motor) caused the cell to be bathed with solution from a different barrel (changeover < 1s). Solution flow was 100 µl/minute through each barrel and was driven by a syringe pump (Palmer Instruments). Caffeine was also applied by this rapid switch system in experiments to test the Ca²⁺ load of the SR. When ryanodine and thapsigargin (Calbiochem-Novabiochem Ltd., Nottingham, UK) were applied in the solution superfusing the cells, a tap close to the inflow of the bath was used to switch to the drug-containing solution. The same perfusion system was used for experiments using isoproterenol.

NAADP-AM Synthesis—Synthesis and characterization of NAADP-AM was described.³

Imaging of Internal Organelles—Cells were loaded with 0.1 µM Bodipy-FL-X-ryanodine, 0.1 µM Bodipy-TR-X-thapsigargin, or 50 nM LysoTracker Red (stock solutions dissolved in Me₂SO) for 20 min at room temperature. Labeled cells were visualized (after removing excess dye in the bath) using a Zeiss LSM510 laser scanning confocal microscopy (Zeiss). Excitation light was provided by an argon laser for Bodipy-FL-X-ryanodine (excitation: 505 nm, emission: 513 nm), and by a HeNe laser for both Bodipy-TR-X-thapsigargin (excitation: 488 nm, emission: 513 nm) and LysoTracker Red (excitation: 568 nm, emission: 590 nm).

Measurement of NAADP Levels in the Guinea Pig Cardiac Tissue—Experiments were carried out on guinea pig tissue extracts. Guinea pigs were sacrificed by stunning and cervical dislocation and the heart removed and placed on a standard Langendorff apparatus. Control hearts were perfused for 10 min with a Ca²⁺-containing solution, composition in mM: NaCl 137, KCl 5, NaHCO₃ 12, CaCl₂ 1.8, glucose 5, sodium pyruvate 1, NaH₂PO₄ 0.4, MgCl₂ 1, NaOH 1, EGTA 0.1, pH 7.4, gassed with 95% O₂/5% CO₂. Isoproterenol-treated hearts were initially perfused for 5 min with physiological salt solution (as above) and then perfused for 5 min with solution containing 60 nM isoproterenol (this time period allows a maximal response to this concentration of isoproterenol to be achieved). The hearts were removed from the Langendorff apparatus and the ventricles were isolated and snap frozen in liquid nitrogen. Samples were then stored at -80 °C until ready for the acid extraction process. Frozen guinea pig tissue was first powdered in a pestle and mortar under liquid nitrogen, and then acid extraction of NAADP was performed. NAADP levels were determined using a sea urchin egg homogenate bioassay, based on the ability of NAADP to induce release of Ca²⁺ in this system (27).

View larger version (38K): [in this window] [in a new window]   FIGURE 1. Effects of photorelease of NAADP on Ca2+ transients. A, line scan images of a representative guinea pig ventricular myocyte loaded with fluo-4, showing Ca2+ transients before and 60 s after photorelease of NAADP (5 µM caged NAADP in the patch pipette). B,Ca2+ transient profiles calculated from the line scan images shown in A; timescale is the same as in A. C, series of Ca2+ transient profiles (taken at selected intervals from the same cell) showing the time course of the development of the enhancing effect of photoreleased NAADP. D, bar graph showing the concentration and time dependence of the effects of NAADP on Ca2+ transients. The augmenting actions of photoreleased NAADP were concentration-dependent. No significant effects were observed on photolysis of caged phosphate in the same range of concentrations.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Actions of NAADP Photoreleased from a Caged Analogue on Whole Cell Ca²⁺ Transients—In the first series of experiments presented here, NAADP was photoreleased from a caged analogue to test whether or not NAADP could exert any effect on whole cell Ca²⁺ transients accompanying action potentials in cardiac ventricular myocytes. Guinea pig ventricular myocytes were loaded with fluo-4 together with various concentrations of caged-NAADP through a cell-attached patch electrode. Cells were then electrically stimulated at 0.5 Hz to fire action potentials. The rises in Ca²⁺ accompanying the action potential were compared before and after photorelease of NAADP (with a single 500-ms pulse of UV light) using laser scanning confocal microscopy. Fig. 1A shows line-scan images of Ca²⁺ transients in a representative cell before (as a control) and 60 s after photorelease of NAADP from caged-NAADP (5 µM in the patch pipette). Fig. 1B presents Ca²⁺ transient profiles calculated as the integrated fluorescence along the scanned line at each time point in Fig. 1A. It may be seen from these Figs that photorelease of NAADP resulted in an enhancement in the magnitude of the Ca²⁺ transient. The increases in the Ca²⁺ transient induced by photolysis of caged-NAADP showed progressive changes with time and were greater at 60 s than at 20 s after photolysis (Fig. 1C). Photolysis of caged-NAADP (5 µM in the patch pipette) caused a significant increase in the Ca²⁺ transient amplitude 60 s after photolysis (Fig. 1B); the mean increase in 8 cells was 41 ± 10% (n = 8, p < 0.05). Under these conditions, photorelease of NAADP also increased background Ca²⁺ fluorescence (data not shown). When the concentration of caged-NAADP was reduced from 5 to 1.5 µM, there were smaller but still significant increases in the amplitudes of the Ca²⁺ transients following photolysis of caged-NAADP (Fig. 1D, 19 ± 5% at 1.5 µM, n = 4, p < 0.05). Significant changes in Ca²⁺ transient magnitude were not detected at a concentration of 0.5 µM caged-NAADP (Fig. 1D). The specific effect of photoreleased NAADP was confirmed by comparison with effects of photolysis of the same concentrations of caged-phosphate (Fig. 1D). Photoreleased phosphate failed to increase whole cell Ca²⁺ transients at all concentrations tested.

Effects of NAADP Applied via a Patch Pipette on Myocyte Contraction—In another series of experiments, guinea pig ventricular myocytes were stimulated to fire action potentials at a frequency of 1 Hz and NAADP was introduced to the cytosol at various concentrations by diffusion of NAADP from the patch pipette solution. Control contractions were measured in the permeabilized patch configuration before physical rupture of the patch by suction that allowed NAADP access to the cytosol. NAADP applied in this way failed to cause any significant consistent changes in contraction amplitude when applied at a range of concentrations from 50 nM to 100 µM. There was, however, a significant reduction in contraction magnitude in the presence of NAADP at concentrations higher than 500 µM. 1 mM NAADP caused a significant decrease in contraction of 26 ± 5% 3 min after gaining access to the cytosol following physical rupture of the patch (n = 10, p < 0.05). This reduction may reflect the profound self-inactivation mechanism that has been previously reported in a variety of mammalian systems exposed to higher concentrations of NAADP (16-19). Evidence that this action of high concentrations of NAADP is not simply a nonspecific toxic effect is presented in a later section. Because the discovery of the self-inactivation mechanism of NAADP in the sea urchin egg (12, 13), this property of NAADP has been used as the best tool to abolish NAADP signals in several systems such as mouse pancreatic acinar cells (14). The self-inactivation mechanism provides a possible reason as to why no increase in contraction was detected in experiments in which low concentrations of NAADP were applied via the patch pipette: diffusion may be too slow to allow sufficiently speedy access of low concentrations of NAADP to the cytosol to detect an increased contraction, and intermediate concentrations may provoke competing activating and inactivating mechanisms. The self-inactivation mechanism was further tested and put to good use in the next series of experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Self-inactivating property of NAADP. A, bar graph illustrating the effects of photoreleased NAADP (5 µM caged NAADP in the patch pipette) on Ca2+ transients in the absence and presence of high concentrations of NAADP (0.1 mM or 1 mM in the patch pipette) (bar (1)).: Photorelease of 5 µM caged-NAADP greatly increased whole cell Ca2+ transient amplitude (by 41 ± 10%, n = 8, p < 0.05) (bar (2)). In the presence of 0.1 mM NAADP, photorelease of NAADP increased the Ca2+ transient by only 12 ± 4% (n = 5, p < 0.05) (bar (3)).1 mM NAADP completely abolished the effect of photoreleased NAADP (0.3 ± 6%, n = 6, p < 0.05). B,Ca2+ transients from a representative cell showing a lack of effect of photoreleased NAADP when 1 mM NAADP was present in the patch pipette.

Influence of Bafilomycin A on the Enhancement of Whole Cell Ca²⁺ Transients by Photoreleased NAADP—It has been reported that NAADP may release Ca²⁺ from an acidic compartment in several mammalian cells (20-24). In such systems, a vacuolar proton pump inhibitor, bafilomycin A has been used to collapse the H⁺ gradient across the organelle membrane and this subsequently results in an inhibition of Ca²⁺ uptake (25, 26). As a consequence, bafilomycin A has been applied as a useful experimental tool to test for the involvement of acidic stores in the control of Ca²⁺ signals in a variety of cell types (20-24, 27, 28).

The effects of bafilomycin A on whole cell Ca²⁺ transients were therefore investigated in guinea pig ventricular myocytes. Cells were loaded with fluo-4 AM and field stimulated to produce whole cell Ca²⁺ transients. When such cells were exposed to bafilomycin A (1 µM for 3 min), peak whole cell Ca²⁺ transients showed a 21 ± 4% reduction compared with control levels (n = 6, p < 0.05). In cells that were loaded with fluo-4 AM and superfused with an identical solution lacking bafilomycin A, there was no significant change in peak Ca²⁺ transient magnitude over the time course of the experiments. In a second series of experiments, the effects of bafilomycin A were investigated in cells loaded with fluo-4 and 5 µM caged NAADP, and stimulated via the patch pipette to elicit Ca²⁺ transients. Exposure to 1 µM bafilomycin A again caused a significant reduction in the magnitude of the whole cell Ca²⁺ transient (red trace in Fig. 3B, 17 ± 5%, n = 6, p < 0.05) that was similar to that seen in the field stimulated cells. Subsequent photorelease of NAADP from caged-NAADP in cells exposed to bafilomycin A failed to cause a significant increase in the whole cell Ca²⁺ transient (measured 60 s after photolysis, blue trace in Fig. 3A, 6 ± 2%, n = 6, p > 0.05). These observations are consistent with the proposal that the actions of photoreleased NAADP on whole cell Ca²⁺ transients require a bafilomycin-sensitive acidic store.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Interplay between the SR and the acidic compartment in mediating the actions of NAADP. A, whole cell Ca2+ transients recorded from a representative cell. The black trace shows the Ca2+ transient under control conditions. The red trace represents the Ca2+ transient 3 min after application of bafilomycin (1 µM) showing 17 ± 5% reduction from controls (n = 6, p < 0.05). The blue trace shows Ca2+ transients observed 60 s after photorelease of NAADP (5 µM in the patch pipette) in the presence of bafilomycin A; under these conditions, there was no significant increase the Ca2+ transient (6 ± 2%, n = 6, p > 0.05). B, whole cell Ca2+ transients recorded from a representative cell. The black trace shows the Ca2+ transient under control conditions. Application of a mixture of ryanodine (2 µM) and thapsigargin (2 µM) significantly reduced Ca2+ transient magnitude (red trace). Subsequent photorelease of NAADP (5 µM caged NAADP; blue trace) in the presence of ryanodine and thapsigargin failed to increase the Ca2+ transient. C, mean data from several cells. In the presence of (2) bafilomycin A (1 µM) or (3) a mixture of ryanodine (2 µM) and thapsigargin (2 µM), photorelease of NAADP (5 µM caged NAADP) was without significant effect on Ca2+ transient amplitude. D, photorelease of NAADP (5 µM caged NAADP) increased the Ca2+ content of the SR (by 20 ± 4.1%, n = 7, p < 0.01), as assessed from the magnitude of the Ca2+ transient elicited by rapid application of caffeine (10 mM). E, photorelease of NAADP (5 µM caged NAADP) was without significant effect on L-type Ca2+ currents activated by step depolarizations from -40 mV to potentials in the range -30 to +50 mV.

Involvement of the SR in the Actions of NAADP—Next, a possible involvement of the SR in the actions of NAADP were investigated by testing the effects of NAADP on whole cell Ca²⁺ transients in the presence of agents that inhibit SR function. Ryanodine and thapsigargin were used to suppress SR function by respectively blocking the release of Ca²⁺ from the SR via ryanodine receptors and preventing Ca²⁺ uptake by the Ca²⁺-ATPase. When cells were incubated with 2 µM ryanodine together with 2 µM thapsigargin (Ry/Thaps) for 5 min, the peak whole cell Ca²⁺ transient was reduced to 38 ± 9% of control (Fig. 3B, red trace, n = 4, p < 0.05). In such cells, subsequent photorelease of NAADP from caged-NAADP (5 µM in the patch pipette) failed to cause any significant change in the amplitudes of whole cell Ca²⁺ transients (measured 60 s after photolysis, blue trace in Fig. 3, B and C, 3 ± 3%, n = 4, p > 0.05).

To examine how NAADP interacts with SR Ca²⁺ handling, the Ca²⁺ content of the SR was measured before and after photorelease of NAADP. The SR Ca²⁺ content was evaluated by rapid application of 10 mM caffeine to release the stored Ca²⁺, and the resultant Ca²⁺ transient was measured (29). When assessed 45 s after photolysis of 5 µM caged-NAADP, the Ca²⁺ signal caused by rapid application of 10 mM caffeine was significantly increased by 21 ± 4% (Fig. 3D, n = 7, p < 0.01) as compared with control measurements before photolysis of caged-NAADP, indicating that photoreleased NAADP resulted in an enhancement in the Ca²⁺ content of the SR. In addition, the possibility that NAADP may enhance sarcolemmal L-type Ca²⁺ current was excluded by experiments in which photolysis of caged-NAADP had no effect on Ca²⁺ currents measured under voltage-clamp conditions (Fig. 3E, n = 5, p > 0.05).

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Effects of NAADP-AM on contractions and Ca2+ sparks in ventricular myocytes. A, mean amplitude of cell contractions measured in guinea pig ventricular myocytes. Under control conditions, no changes in contraction were observed over the time period studied (left panel). Rapid application of NAADP-AM (60 nM; center panel) increased contraction of guinea pig ventricular myocytes (by 28 ± 5% at 20 s, n = 8, p < 0.05); the first 10 contractions shown in the figure were recorded before switch to NAADP-AM. This effect was inhibited by pretreatment with bafilomycin A (1 µM, 5 min; right panel) (-1 ± 1% at 20 s, n = 6, p < 0.05). B, inrat ventricular myocytes, application of NAADP-AM (60 nM, 20 s) increased spark frequency from 2.67 ± 1.64 to 4.52 ± 1.97 events/s/100 µm, spark amplitude by 37 ± 6% and resting fluorescence level by 25 ± 5% (n = 6, p < 0.05); all these actions were prevented by pretreatment with bafilomycin A. Bafilomycin A itself had no significant effects on the characteristics of the Ca2+ sparks (n = 6, p > 0.05).

Effects of NAADP-AM on Ca²⁺ Sparks in Rat Ventricular Myocytes—Ca²⁺ sparks were recorded in rat ventricular myocytes in preference to guinea pig cells (see Ref. 30), because under our experimental conditions, guinea pig ventricular myocytes fail to exhibit Ca²⁺ sparks. 20 s after application of 60 nM NAADP-AM by rapid switch, both the amplitude and frequency of Ca²⁺ sparks were significantly increased (Fig. 4B;Ca²⁺ spark frequency: from 2.67 ± 1.64 to 4.52 ± 1.97 sparks/s/100 µm; amplitude: increase above control of 37.0 ± 6.3%, n = 6 p < 0.05). The background Ca²⁺ level, as indicated by the baseline fluorescence, was also increased by 24.5 ± 5.4% (n = 6, p < 0.05). The effect of NAADP on Ca²⁺ spark amplitude is particularly interesting since this observation is thought to provide additional evidence that the Ca²⁺ load of the SR is increased under these conditions. The effects of NAADP-AM on the frequency and amplitude of Ca²⁺ sparks, and on background Ca²⁺ levels were all suppressed by bafilomycin A: in cells pretreated with bafilomycin A (1 µM) for 3 min, rapid application of 60 nM NAADP-AM in the continued presence of bafilomycin A failed to cause any significant effect in a spark amplitude, frequency and background Ca²⁺ level (measured 20 s after application of NAADP-AM, Fig. 4B;Ca²⁺ spark frequency: from 3.52 ± 1.03 to 3.87 ± 0.94 sparks/s/100 µm, amplitude: -1.2 ± 8.4%, background Ca²⁺ level: 4 ± 2.4%, n = 7, p > 0.05).

Specific Effect of Bafilomycin A on Acidic Compartments—The specificity of the action of bafilomycin A on NAADP-sensitive stores was tested by labeling targeted organelles with LysoTracker Red, a weak base that selectively accumulates and labels acidic compartments. Bodipy-FL-ryanodine and Bodipy-TR-thapsigargin were used as markers for the SR. The upper panels of Fig. 5 show confocal images of guinea pig ventricular myocytes labeled with Bodipy-FL-ryanodine, Bodipy-TR-thapsigargin, and LysoTracker Red. The LysoTracker Red labeling of the acidic compartments (Fig. 5A) showed a punctate staining pattern throughout the cell that was clearly different from that seen with SR staining using either Bodipy-FL-ryanodine (Fig. 5B) or Bodipy-TR-thapsigargin (Fig. 5C). Upon application of bafilomycin A (1 µM for 20 min), a dramatic reduction in LysoTracker Red staining was observed (Fig. 5A, lower panel). In contrast, the Bodipy-FL-ryanodine or Bodipy-TR-thapsigargin labeling were unaffected by bafilomycin A (Fig. 5, B and C, lower panels). The data demonstrate that bafilomycin A acts specifically on the acidic store labeled by LysoTracker Red without affecting the integrity of the SR.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. Specific effect of bafilomycin A on acidic compartments. Upper panels show typical staining of (A) Bodipy-FL-ryanodine, (B) Bodipy-TR-thapsigargin, and (C) Lysotracker Red, before application of bafilomycin A. Note the difference in the pattern of staining between Lysotracker Red and either Bodipy-FL-ryanodine or Bodipy-TR-thapsigargin. Lower panels were imaged 20 min after application of bafilomycin A (1 µM). Bafilomycin A did not affect either Bodipy-FL-ryanodine (A) or Bodipy-TR-thapsigargin (B) labeling, whereas cells labeled with LysoTracker Red showed a substantial reduction in staining level.

The effects of bafilomycin A and of high self-inactivating concentrations of NAADP to reduce Ca²⁺ transients and contractions by about 20% could be taken to indicate that there is a background level of NAADP in the absence of -adrenoreceptor stimulation with effects that are suppressed by either bafilomycin A or high concentrations of NAADP. The possibility that NAADP may exist endogenously in guinea pig hearts, and that the levels of NAADP might be enhanced following -adrenoreceptor stimulation were investigated using a bioassay for NAADP based on the ability of NAADP to release Ca²⁺ from sea urchin egg homogenate. When guinea pig hearts were perfused with physiological saline (in the absence of any drugs) on a Langendorff apparatus, the endogenous level of NAADP was found to be of 0.220 ± 0.027 nmol/mg protein (n = 10). 60 nM isoproterenol produced a significant elevation in the NAADP level 5 min after application (Fig. 6A, 0.337 ± 0.043 nmol/mg protein, n = 9, p < 0.05).

These possibilities were further tested by comparing the effects of photoreleased NAADP and of self-inactivating concentrations of NAADP in the absence and presence of -adrenoreceptor stimulation. The upper panel of Fig. 6B presents a control whole cell Ca²⁺ transient and the lower panel shows the whole cell Ca²⁺ transient in the same cell after 1 min application of 5 nM isoproterenol (measured with fluo-4 using laser scanning confocal microscopy). Fig. 6C presents Ca²⁺ transients calculated as the integrated fluorescence along the scanned line at each time point. In the presence of 5 nM isoproterenol, there was a large increase in the peak of the whole cell Ca²⁺ transient (Fig. 6, B and C,67 ± 15%, n = 5, p < 0.05). In addition to the increase in peak Ca²⁺ transient magnitude there was also marked shortening of the duration of the transient (red trace in Fig. 6C). This is presumably due to the effect of -adrenoreceptor stimulation on the Ca²⁺ uptake mechanism following phosphorylation of phospholamban that in turn increases the activity of the Ca²⁺-ATPase (32). In the continued presence of 5 nM isoproterenol, photolysis of caged-NAADP (5 µM in the patch pipette) did cause a significant increase in the Ca²⁺ transient by 18 ± 7% 60 s after photolysis (Fig. 6, D and E, n = 7, p < 0.05), but this increase was notably smaller than that observed following photorelease of NAADP in the absence of -adrenoreceptor stimulation (Fig. 1A, 1,41 ± 10%; p < 0.05). These observations are consistent with a higher endogenous level of NAADP following activation of -adrenoceptors, so that the effects of exogenous photoreleased NAADP are less than would be the case in the absence of -adrenoreceptor stimulation (when the photoreleased NAADP would give a larger increment of NAADP over the lower baseline endogenous level).

Effects of high, self-inactivating concentrations of NAADP on myocyte contraction were also investigated during -adrenoreceptor stimulation. Contractions were measured from the video image of cells using an edge detection technique. Treatment of single guinea pig ventricular myocytes with 2 nM isoproterenol caused significant increases in the amplitude of contraction (Fig. 6F, bars 1 and 2,45 ± 8% increase over that in the absence of isoproterenol, n = 6, p < 0.05). In these experiments, 1 mM NAADP was included in the patch pipette solution but could not enter the cytosol until the cell membrane under the patch pipette was ruptured (though the permeabilized patch conditions allowed electrical stimulation). When the membrane under the patch pipette was physically ruptured to allow access of NAADP to the cytosol during the continued activation of -adrenoceptors by isoproterenol, 1 mM NAADP caused a very marked reduction in the magnitude of contraction (40 ± 6% decrease of the amplitude of contraction; Fig. 6F, bars 3 and 4, n = 6, p < 0.05). This reduction of contraction was substantially larger than the reduction caused by 1 mM NAADP in the absence of 2 nM isoproterenol (Fig. 6, D-F, (bar 2) 26 ± 5%, n = 10, p < 0.05) (Fig. 6F, bars 1-4). These observations are again consistent with an effect of -adrenoreceptor stimulation to increase endogenous levels of NAADP: the self-inactivating concentration of NAADP (1 mM) would be expected to have a greater effect on the actions of endogenous NAADP when levels are raised following -adrenoreceptor stimulation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we have provided the first observations concerning NAADP actions in intact cardiac myocytes. The most direct observations are provided by experiments in which NAADP was photoreleased from a caged analogue. It is clear that NAADP applied to the cytosol in this way increased the amplitude of whole cell Ca²⁺ transients. It is interesting that the effects were accompanied by a small but significant increase in the resting level of cytosolic Ca²⁺ and that the effects were slow to develop, taking tens of seconds. The effects were not artifacts of photorelease since they showed a dependence on the concentration of caged NAADP and were not seen following photorelease of the same concentrations of phosphate.

View larger version (57K): [in this window] [in a new window]   FIGURE 6. Effects of NAADP in the absence and presence of -adrenoreceptor stimulation. A, isoprenaline (60 nM, 5 min) induced significant increases in NAADP levels from a resting level of 0.220 ± 0.027 nmol/mg protein (n = 10) to 0.337 ± 0.043 (n = 9, p < 0.05). B, linescan images of Ca2+ transients from a representative cell in the absence and presence of isoproterenol (5 nM). C,Ca2+ transient profiles calculated from the linescan images shown in B; timescale is the same as in B. Application of isoproterenol increased the Ca2+ transient by 67 ± 15% (n = 5, p < 0.05). D, linescan images of Ca2+ transients from a representative cell before and after photorelease of NAADP (5 µM caged NAADP) in the continued presence of isoproterenol (5 nM). E,Ca2+ transient profiles calculated from the linescan images shown in D; timescale is the same as in D. Under these conditions, photoreleased NAADP caused a smaller increase in the Ca2+ transient (18 ± 7% n = 7, p < 0.05) than was observed in the absence of isoproterenol. F, cytosolic application of a high self-inactivating concentration of NAADP (1 mM, 3 min) caused a greater reduction of contraction amplitude in the presence of 2 nM isoprenaline (40 ± 6%; n = 6, p < 0.05) than in its absence (26 ± 5%; n = 10, p < 0.05).

Effects of NAADP were also seen when the acetoxymethyl ester of NAADP was applied to the extracellular solution. The extracellular concentration of NAADP-AM used was 60 nM and effects were seen within 30 s, so although the AM esters can lead to accumulation of free drug within the cytosol at a higher concentration than that of the extracellular acetoxymethyl form (because a concentration gradient for entry of the AM ester is maintained when, in this case, NAADP is liberated intracellularly by action of endogenous esterases), it again seems likely that the effective concentration of NAADP in the cytosol is in the tens of nanomolar range, as has been found with other mammalian cells (9, 16, 18). It should be noted that, in our experience, cardiac myocytes take up AM esters and liberate the free compound (such as Ca²⁺ probes and Ca²⁺ chelators) remarkably quickly, particularly at physiological temperature.

The observations with bafilomycin A are particularly important. This substance prevented all effects of NAADP observed, whether these involved whole cell Ca²⁺ transients or myocyte contractions. Furthermore, bafilomycin A prevented the actions of both photoreleased NAADP and NAADP-AM, two differing methods of application of NAADP to the cytosol. Because bafilomycin A is known to inhibit Ca²⁺ uptake into acidic organelles by collapse of the proton gradient generated by the H⁺-ATPase, it has been used extensively as an experimental tool in the field of NAADP signaling to support an involvement of acidic stores (20-24, 27, 28). It appears that in cardiac myocytes, as in many other mammalian cell types, bafilomycin-sensitive acidic stores may again be involved in the actions of NAADP. In this context it is particularly important that the SR remained structurally intact (as indicated by Bodipy-FL-ryanodine and Bodipy-TR-thapsigargin labeling) during exposure to 1 µM bafilomycin A, while under the same conditions the LysoTracker Red staining was greatly reduced, as expected if bafilomycin A collapses the proton gradients in the acidic organelles where LysoTracker Red is accumulated.

View larger version (24K): [in this window] [in a new window]   FIGURE 7. Scheme showing the proposed mechanism by which NAADP increases Ca2+ transients in cardiac myocytes. NAADP mobilizes Ca2+ from a bafilomycin-sensitive acidic Ca2+ store. This released Ca2+ is taken up by the SR, leading to increased loading of the SR with Ca2+. This in turn enhances Ca2+ release from the SR during excitation-contraction coupling, thereby increasing the Ca2+ transient and cell contraction.

The enzyme responsible for synthesis of NAADP is thought to be ADP ribosyl cyclase working by base exchange with NADP as the substrate. Interestingly, the same enzyme catalyzes the synthesis of cADP-ribose, using NAD as the substrate. As with the observations reported here for NAADP, photorelease of cADP-ribose from a caged analogue increases whole cell Ca²⁺ transients and increases the frequency of Ca²⁺ sparks, but in the case of cADP-ribose (at least for short times of exposure of 3 min) there is no accompanying increase in the Ca²⁺ load of the SR (because the Ca²⁺ signal accompanying emptying of the SR by caffeine is unchanged, and the amplitude of Ca²⁺ sparks is not increased) (30). There is therefore a very interesting contrast between the actions of cADP-ribose and NAADP, even though both can increase Ca²⁺ transients and spark frequency, and both could potentially be synthesized by action of the same enzyme.

The effects of NAADP on whole cell Ca²⁺ transients were suppressed by application of ryanodine and thapsigargin, providing strong evidence that a functioning SR is required for the effect. However, the observations with bafilomycin A show that disruption of the acidic store without an effect on the integrity of the SR is sufficient to prevent NAADP actions. A hypothesis that combines these observations is that NAADP leads to Ca²⁺ release from the bafilomycin-sensitive acidic store and this released Ca²⁺ is in turn taken up by the SR, giving an increased Ca²⁺ load of the SR (Fig. 7). A functional SR is therefore required for the effect, but the primary action of NAADP is to release Ca²⁺ from the acidic store. Such a mechanism would therefore represent an interaction between two different Ca²⁺ pools, with NAADP effectively enlarging the SR Ca²⁺ pool by mobilizing Ca²⁺ from acidic organelles.

An additional point of interest is that bafilomycin A and NAADP (photoreleased, or applied as NAADP-AM) have opposite effects on Ca²⁺ transients, despite the fact that both are believed to discharge the acidic Ca²⁺ stores. This apparent paradox may arise from differences in timing of Ca²⁺ release from the lysosome-related stores and, perhaps, from differences in the precise site of release of Ca²⁺ in relation to uptake sites on the SR. NAADP is expected to cause rapid local release of Ca²⁺, permitting a sufficiently large rise in Ca²⁺ close to the SR uptake sites to overcome local Ca²⁺ buffering mechanisms and provoke the SR Ca²⁺-ATPase to take up this extra Ca²⁺, thereby enhancing SR Ca²⁺ load. Because mechanisms to maintain Ca²⁺ levels in the acidic stores would still be operation, NAADP would be able to provide a maintained release of Ca²⁺ from these stores that could be taken up by the SR. In contrast, bafilomycin A (under these conditions) may cause a much slower leak of Ca²⁺ from the lysosome-related compartments, with the result that Ca²⁺ in the vicinity of the SR uptake sites does not rise sufficiently to cause additional Ca²⁺ uptake. Ca²⁺ buffering mechanisms may play an important role in preventing a significant rise in cytosolic free Ca²⁺ concentration in the presence of such a slow leak of Ca²⁺ from the acidic stores.

In addition, the site of Ca²⁺ release provoked by NAADP and bafilomycin A may differ. In the case of NAADP, release sites may be strategically placed in close apposition to the SR Ca²⁺ uptake sites, thereby favoring SR Ca²⁺ uptake. In contrast, the leak sites for Ca²⁺ following exposure to bafilomycin A may be more diffusely located on the acidic store membrane, so that Ca²⁺ buffering and mechanisms for removal of Ca²⁺ across the sarcolemma may operate to limit the amount of Ca²⁺ that the SR can take up.

The above arguments provide an explanation for the opposite actions of NAADP and bafilomycin A observed in our experiments. We propose that bafilomycin can depress Ca²⁺ transients by slow depletion of Ca²⁺ from the acidic stores, which in turn would prevent an action of NAADP to release Ca²⁺ from this store. If, as we suggest, there are endogenous levels of NAADP that continuously provoke Ca²⁺ release from the acidic stores, it is not surprising that bafilomycin can prevent these actions and therefore reduce Ca²⁺ transients. Consistent with these arguments is the observation that self-inactivating concentrations of NAADP (see below) have a similar effect on cell contraction to that of bafilomycin.

The self-inactivation property of NAADP, in which high concentrations of NAADP inhibit its actions on the whole cell Ca²⁺ transient and the magnitude of contraction, was seen in cardiac myocytes. This is consistent with previous reports in other mammalian cells (16-19). This self-inactivating property has been useful in that high concentrations of NAADP can be used as a specific antagonist to the effects of lower concentrations of this molecule. This was illustrated by the reduction of the effects of photoreleased NAADP by 100 µM NAADP and by the prevention of these effects by 1 mM NAADP. It seems unlikely that the effects of 1 mM NAADP result from a simple nonspecific toxic action since there was no further effect of this concentration of NAADP in cells pretreated with bafilomycin A.

The reduction in contraction amplitude of 20% seen with either bafilomycin A or NAADP might be taken to indicate that there is an ongoing influence of a NAADP-dependent pathway supported by endogenous NAADP acting via the bafilomycin-sensitive Ca²⁺ store. Further support for this proposal is provided by evidence for endogenous levels of NAADP in cardiac tissue provided here and elsewhere (9).

Our observations using Langendorff perfused intact hearts show that the endogenous NAADP level can be elevated upon application of the -adrenoreceptor agonist isoproterenol. This would not be surprising if NAADP were synthesized in cardiac muscle by the action of ADP ribosyl cyclase, because Higashida et al. (31, 33) have reported that sympathetic stimulation may up-regulate the activity of this enzyme that is also responsible for synthesis of cADPR. Functional evidence to support elevated levels of NAADP during -adrenoreceptor stimulation is provided by our observations: while there was a significant increase in peak whole cell Ca²⁺ transient magnitude with photorelease of NAADP in the presence of isoproterenol, this increase was significantly reduced compared with the effects of photoreleased NAADP in the absence of -adrenoreceptor stimulation (Fig. 6, B-E). In addition, during -adrenoreceptor stimulation, high concentrations (1 mM) of patch-applied NAADP caused a decrease in the amplitude of contraction in ventricular myocytes that was significantly larger than that seen in the absence of -adrenoreceptor stimulation (Fig. 6F). This is consistent with the proposal that -adrenoreceptor stimulation regulates endogenous levels of NAADP, such that there is effectively more NAADP present, so that there is a smaller increment of NAADP when exogenous NAADP is added, yet a greater effect of an antagonising influence. These observations therefore support the hypothesis that NAADP is an endogenous regulator of cardiac function with a physiological role.

In summary, our experiments provide the first description of the regulation by NAADP of cardiac excitation-contraction coupling. NAADP causes an increase in the whole cell Ca²⁺ transient by enhancing the Ca²⁺ loading of the SR. The ability of NAADP to release Ca²⁺ from a bafilomycin A-sensitive acidic Ca²⁺ store, together with evidence that NAADP production is regulated by -adrenoreceptor stimulation, strongly indicate that NAADP may control an important novel mechanism underlying cardiac inotropy.

	FOOTNOTES

 
^* This work is supported by The Wellcome trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 44-1865-271614; Fax: 44-1865-271853; E-mail: michiko.yamasaki{at}pharmacology.oxford.ac.uk.

² The abbreviations used are: NAADP, nicotinic acid adenine dinucleotide phosphate; SR/ER, sarco/endoplasmic reticulum.

³ R. Parkesh, A. M. Lewis, P. K. Aley, A. Arredouani, S. Rossi, R. Tavares, S. R. Vasudevan, D. Rosen, A. Galione, J. Dowden, and G. C. Churchill, submitted manuscript.

	ACKNOWLEDGMENTS

 
We thank Dr. Paul A. Mattick and Thomas Collins for technical support.

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