COMMUNICATION:
Cyclic ADP-ribose Enhances Coupling between Voltage-gated Ca2+ Entry and Intracellular Ca2+ Release*

(Received for publication, May 22, 1997, and in revised form, June 11, 1997)

Ruth M. Empson Dagger and Antony Galione

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Ca2+ release from intracellular stores can be activated in neurons by influx of Ca2+ through voltage-gated Ca2+ channels. This process, called Ca2+-induced Ca2+ release, relies on the properties of the ryanodine receptor and represents a mechanism by which Ca2+ influx during neuronal activity can be amplified into large intracellular Ca2+ signals. In a differentiated neuroblastoma cell line, we show that caffeine, a pharmacological activator of the ryanodine receptor, released Ca2+ from intracellular stores in a Ca2+-dependent and ryanodine-sensitive manner. The pyridine nucleotide, cyclic ADP-ribose, thought to be an endogenous modulator of ryanodine receptors also amplified Ca2+-induced Ca2+ release in these neurons. Cyclic ADP-ribose enhanced the total cytoplasmic Ca2+ levels during controlled Ca2+ influx through voltage gated channels, in a concentration-dependent and ryanodine-sensitive manner and also increased the sensitivity with which a small amount of Ca2+ influx could trigger additional release from the ryanodine-sensitive intracellular Ca2+ stores. Single cell imaging showed that following the Ca2+ influx, cyclic ADP-ribose enhanced the spatial spread of the Ca2+ signal from the edge of the cell into its center. These powerful actions suggest a role for cyclic ADP-ribose in the functional coupling of neuronal depolarization, Ca2+ entry, and global intracellular Ca2+ signaling.


INTRODUCTION

Calcium-induced calcium release (or CICR),1 is a means of amplifying intracellular cytosolic Ca2+ signals. Ca2+ entering a cell, for example through voltage-sensitive channels, can trigger the release of further Ca2+ from the intracellular stores (1-3). The mechanism relies upon the distinct properties of the ryanodine receptor, in particular its ability to control Ca2+ release from the store and the sensitivity of its gating mechanism to Ca2+ (4, 5). A wide variety of neurons, both cultured and acutely isolated, possess ryanodine-sensitive intracellular Ca2+ stores (6-9) and also express ryanodine receptors (9-12). A number of recent studies have emphasized the importance of Ca2+ release from intracellular stores during physiological neuronal activity (13), during changes in synaptic efficacy that may be involved in learning processes (14-16), as part of the mechanism of neurotransmitter release (17), and even during neuronal development (18). Caffeine (4) and cyclic ADP-ribose, the novel Ca2+-mobilizing pyridine nucleotide originally discovered in the sea urchin egg (19), both release Ca2+ from intracellular stores via modulation of the ryanodine receptor (20, 21). In the last few years cyclic ADP-ribose has begun to emerge as a potential physiological regulator of ryanodine-sensitive Ca2+-dependent processes in a number of intact mammalian systems. Cyclic ADP-ribose modulates excitation-contraction coupling in the heart (22), it alters excitability of pancreatic acinar cells (23) and dorsal root ganglion cells (24), and stimulates Ca2+ release from the intracellular stores of T-lymphocytes (25). Using a combination of electrophysiology and Ca2+ imaging we show here, in intact mammalian cultured neuroblastoma cells (26), that release from ryanodine-sensitive intracellular stores is coupled to Ca2+ influx via voltage-activated channels and potentiated by cyclic ADP-ribose applied through the recording electrode.


EXPERIMENTAL PROCEDURES

NG108-15 neurons, a mouse neuroblastoma × rat glioma hybrid culture (obtained from the European Collection of Cell Cultures, Porton, UK), were cultured as described previously (26, 27).

For Ca2+ measurements in intact cells, the cells were loaded with Fura-2 using the acetoxymethyl ester loading technique for a maximum loading period of 15 min. Before commencing experiments the cells were washed three times in the perfusion buffer containing 130 mM NaCl, 10 mM HEPES, 5 mM KCl, 5 mM CaCl2, 1 mM MgCl2, 25 mM glucose, on the stage of the upright microscope (Zeiss, Oberkochen, Germany). To depolarize the cells, 30 mM KCl (and an equimolar reduction in Na+ ions) was switched into the perfusion flow (rate varied between 1 and 1.5 ml/min). Changes in Ca2+ were measured every 4 s in these experiments using ratiometric determinations of image intensity following excitation with 340- and 380-nm wavelength light supplied by a TILL Photonics monochromator (Planegg, Germany) controlled by Improvision (Leicester, UK) "Ionvision" software (as described previously (28)). An in vitro calibration using the Ionvision software was used to analyze average Ca2+ changes, over the whole cell, in individual cells from the pseudocolor images, as described previously. The same experimental set up and analysis was used to determine Ca2+ changes during the electrophysiological experiments, except that an image was captured every 0.8-1.2 s. Variations in intracellular Ca2+ in different regions of the cell were also measured with the same calibrations and software using small rectangular regions of interest. Electrophysiological recordings were made in the whole cell patch clamp mode using an Axopatch 200A (Axon Instruments, Foster City, CA) with pipettes of resistance 2-6 MOmega . Seal resistances prior to breakthrough were always greater than 1 GOmega . Cells settled and filled with Fura-K+ for approximately 10 min after breakthrough. Current and voltage signals were digitized using an ITC-16 A/D converter (Instrutech Corp., Great Neck, NY) and the experiments controlled using the Axodata program (Axon Instruments) through the same interface. The intracellular solution contained 135 mM CsCl, 10 mM HEPES, 1 mM Mg-ATP, 100 µM Fura-2-pentapotassium salt, and extracellular solution contained 130 mM NaCl, 10 mM HEPES, 5 mM KCl, 5 mM tetraethylammonium chloride, 5 mM CaCl2, 5 mM 4-aminopyridine, 1 mM MgCl2, 25 mM glucose, and 1 µM tetrodotoxin. Both intra- and extracellular solutions were designed to block K+ and Na+ channels, so that during cellular depolarization the area beneath the inward current, measured with Axograph (Axon Instruments), an indication of the charge entering the cell, represented the entry of Ca2+ and not other cations. Ca2+ (or charge) entry was controlled by changing the duration of the +60- or +80-mV voltage step evoked from a holding potential of -70 or -90 mV. Voltage steps were applied in a random order, every 20-30 s to avoid excessive run-down of the Ca2+ current with linear on-line leak subtraction. For each voltage step the peak Ca2+ change was measured and divided by the area beneath the current trace (pA × s or pC) to express the unit Ca2+ transient (29), Ca2+/pC (pA × s) charge entering the cell. Three voltage steps were used to calculate a mean value of the unit Ca2+ transient at each step duration. All experiments were conducted at room temperature. All values are compared with a Student's t test, and values are means ± standard error of the mean. All materials were obtained from Sigma (Poole, UK) except Fura-2, which was from Molecular Probes.


RESULTS AND DISCUSSION

Initial studies showed that these differentiated neuroblastoma cells responded to high concentrations of caffeine, 20-50 mM, with small increases in intracellular Ca2+ (Fig. 1A). The responses persisted in the absence of extracellular Ca2+, indicating that these cells possess an intracellular, caffeine-sensitive Ca2+ store. If the NG108-15 cells were first depolarized with 30 mM K+, we observed a rise in intracellular Ca2+ as observed previously (30), consistent with influx of Ca2+ through N and L-type voltage-sensitive Ca2+ channels present on the plasma membrane (open bar, Fig. 1B). Application of caffeine, immediately after the depolarization, then gave a fast and large intracellular Ca2+ rise (Fig. 1, B and C). The responses resembled those seen in bullfrog and rat sympathetic neurons and also in rodent central neurons (6, 7, 31). The caffeine responses were blocked by ryanodine (5-10 µM), an antagonist of the ryanodine receptor at these concentrations (see legend to Fig. 1). This result shows that Ca2+ entry by prior depolarization sensitized the ryanodine receptors on the intracellular stores to subsequent activation by caffeine and represented a form of CICR. A concentration response curve (Fig. 1D) shows a steep response to caffeine in the presence of the prior depolarization, strongly indicating the operation of an amplification process.


Fig. 1. A, intracellular Ca2+ levels were increased by 50 mM caffeine, indicated by the filled bar. B, depolarization of the cells with 30 mM external K+, as shown by the open bar, lead to an increase in Ca2+ levels through the opening of N and L-type voltage-sensitive Ca2+ channels (30). Subsequent application of 50 mM caffeine (filled bar) leads to a marked increase in Ca2+ levels, as shown for all cells in C. In the absence of extracellular Ca2+, as shown by the dotted line, depolarizations did not lead to significant rises in intracellular Ca2+, nor did it enhance caffeine-induced Ca2+ release, rather Ca2+ changes were not significantly different to the results when caffeine was applied alone, 172 ± 20 nM Ca2+ (n = 5) p = 0.24, t test. These responses were reduced by extracellular application of 10 µM ryanodine, mean caffeine-induced Ca2+ rises were 658 ± 113 reduced to 91 ± 25 nM Ca2+ (n = 8, p = 0.004, paired t test). D, concentration response curve showing the sharp increase in the predepolarization caffeine-induced intracellular Ca2+ rises in response to raised caffeine concentrations, values are means ± S.E. of the mean for at least three separate experiments and between five and nine cells.
[View Larger Version of this Image (23K GIF file)]

Having established that these neurons possessed a ryanodine-sensitive CICR capability, we next sought to estabish whether cADPR, a positive modulator of the ryanodine receptor, could potentiate CICR in these cells. Since cADPR is not membrane-permeable, we applied it to the cells via a whole cell patch pipette and simultaneously used voltage clamp to control the membrane potential of the cell. This allowed us to control Ca2+ influx through voltage-sensitive Ca2+ channels. Previous studies have successfully used a method called the unit Ca2+ transient to relate the change in intracellular Ca2+ levels directly to the amount of charge entering the cell (29, 32, 33). The amount of charge entering the cell was estimated from the area beneath the current trace that represented Ca2+ entry, since K+ and Na+ channels were blocked. By dividing the peak Ca2+ rise simultaneously recorded during the voltage step, by the area beneath the current trace, we calculated a unit Ca2+ transient expressed as nanomolar Ca2+ release per picocoulomb of charge entry. As shown in Fig. 2A, increasing the length of the voltage step from 100 to 1000 ms allowed the Ca2+ channels to stay open for longer, thus allowing more Ca2+ to enter the neuron. The cell in Fig. 2 was recorded using a pipette containing 10 µM cADPR, and it can be seen that relative to a rather modest increase in the area beneath the Ca2+ current traces, the longer voltage steps evoked a large increase in the intracellular, simultaneously measured Ca2+ level (Fig. 2B). This was consistent with extra Ca2+ being released from the intracellular stores triggered by the initial Ca2+ entry and had the effect of increasing the absolute value of the unit Ca2+ transient, above that seen in a control cell (Fig. 2C). Note also that the red Ca2+ trace (Fig. 2B) showed an additional later Ca2+ release following the initial peak, a common occurrence in cells treated with cADPR. The effects of cADPR present in the patch pipette are illustrated by the images of two cells with similar charge entry during a depolarizing pulse (Fig. 2D). Particularly notable is the larger Ca2+ rise in the cADPR-treated cell compared with control. Also apparent, after application of the depolarizing pulse, was the rise in Ca2+ at the edge of the cell before Ca2+ rose in the center of the cell, and a striking difference was the relatively larger elevation in Ca2+ at the center of the cADPR-treated cells compared with control. By placing two small regions of interest over the edge and center of the cells and using the Ca2+ rises in these regions to calculate a unit Ca2+ transient, we observed a 120% increase in the unit Ca2+ transient in the center of cADPR-treated cells compared with the center of control cells (n = 9). This suggested that cADPR increased the likelihood with which elevated Ca2+ levels close to the plasma membrane propagated to the center of the cell to give a global Ca2+ signal over the whole of the cell (34).


Fig. 2. A, whole cell voltage clamp electrophysiological recording of a cell showing the characteristic inward Ca2+ current traces evoked upon depolarization of the membrane by 60 mV from a holding potential of -70 mV. Note the initial larger amplitude transient current, O, and the smaller sustained inward current, 255. Both components were reduced by the L-type Ca2+ channel antagonist diltiazam (10 µM) by 44 ± 7% and 68 ± 9%, respectively (n = 6). This confirms previous studies, which also suggest that the initial fast inactivating current is a result of the activation of an N-type channel (41, 42), while the later component results from the opening of an L-type Ca2+ channel (42). B, below the current records are the simultaneously measured Ca2+ levels inside the cell. The colors of the Ca2+ traces correspond to the colors of the current traces. The arrow shows the time the voltage step was applied, and note the different time scales of the Ca2+ changes and the current responses. The pipette used to record this cell contained 10 µM cADPR that potentiated the measured Ca2+ rises when longer voltage steps, leading to prolonged inward currents (red, green, and yellow) triggered an enhanced Ca2+ release. By measuring the area beneath the current trace, pA multiplied by seconds, we obtained a direct indication of the charge, pC, entering the cell. Since all channels except voltage-sensitive Ca2+ channels were pharmacologically blocked, this charge entry represented Ca2+ entry. This charge was then expressed as a function of the Ca2+ rise measured inside the cell, to give the unit Ca2+ transient. In C, the unit transient calculated for this cell at each duration voltage step (from three separate voltage step applications) is plotted against the duration of the step. Also shown, in black, are values from a representative control cell. D, pseudocolor images of a control cell and a cell treated with 10 µM cADPR. Images are sequential left to right and taken at 1.4-s intervals, the white arrow represents the time at which the voltage step was applied. The charge entry was similar in both cells. The pseudocolor images show that the edges of the cells were always the first regions to increase, presumably as Ca2+ entered the cell following the depolarization. In the cADPR-treated cell there was a significant rise of Ca2+ in the center of the cell after the influx, whereas in the control cell the rise in Ca2+ in the center was less apparent and more confined to the edges. Calibration of the pseudocolor is shown and ranges from 0 to 1400 nM Ca2+, blue to red. The pipette is attached to the cell at the top right-hand corner of each cell. Mean whole cell resistance was 783 ± 70.3 MOmega (n = 38), and there was no significant difference in the whole cell input resistance between control cells and cells treated with cADPR (p = 0.5, t test) or in the areas beneath the evoked Ca2+ currents, in cADPR-treated cells compared with controls (p = 0.34, t test).
[View Larger Version of this Image (50K GIF file)]

When Ca2+ levels over the whole cell were measured and compared for all cells, as the duration of the voltage step was increased, the increase in the unit transient was greatest in the cells treated with cADPR (Fig. 3A). Controls also demonstrated a rise in the unit transient (33) as more Ca2+ entered the cell, and it is tempting to suggest that this was due to low levels of endogenous cADPR found in a number of brain preparations (35). In response to the short 100-ms duration voltage step, the unit Ca2+ transient was approximately 1.4 when charge (or Ca2+) entry was 25.4 ± 3.6 pC, rising to a value of around 2.5 (see Fig. 3A), indicating additional release of Ca2+ from the internal stores, as the amount of charge (or Ca2+) entry was increased to 101.6 ± 14.5 pC as the length of the voltage step increased to 1000 ms. Addition of 10 µM cADPR to the patch pipette increased the unit Ca2+ transient in two ways. First it increased the value of the unit Ca2+ transient, compared with control, even following a short, 100-ms duration voltage step (Fig. 3, filled circles compared with open squares, p = 0.007, t test). A direct comparison shows that for a similar charge (or Ca2+) entry to the controls, 30.7 ± 11.6 pC (p = 0.24, t test), during the shortest 100-ms duration voltage step, the presence of cADPR in the pipette gave rise to a unit Ca2+ transient of approximately 2.8, consistent with release of Ca2+ from intracellular stores. Since a similar charge entry in controls failed to elicit Ca2+ release from the intracellular stores, but 100 pC could, our result indicates that cADPR reduced, by approximately 3-fold, the amount of Ca2+ entry required to trigger additional Ca2+ release from the internal stores. Second, as the length of the voltage step was increased and more Ca2+ entered the cell, the presence of cADPR resulted in a relatively larger increase in the unit Ca2+ transient, compared with the controls (even though the absolute values of charge entry were similar between control and cADPR-treated cells, p = 0.34, t test). This suggested that cADPR allowed the extra Ca2+ entry evoked by depolarizations longer than 100 ms, to trigger even further release of Ca2+ from the intracellular stores. We noted that the values of the unit transient were similar to those observed in isolated dorsal root ganglion cells (33, 36), but approximately 10 × larger than those observed in bullfrog neurons (29). This difference could relate to the much larger amplitude of the Ca2+ currents in the bullfrog neurons and may reflect a greater degree of Ca2+ buffering of the larger Ca2+ entry compared with the smaller currents evoked in these mammalian neurons.


Fig. 3. A, mean changes in the unit Ca2+ transients with increasing length of the voltage step, in control cells (square ) compared with those recorded with pipettes containing 10 µM cADPR (black-diamond ). The absolute values of the mean unit Ca2+ transient in the cADPR-treated cells were significantly increased at all pulse lengths compared with control (p < 0.05, t test, all duration pulses, n = 5), and a large increase was seen as the pulse length increased. In the control cells the longer pulse lengths also evoked an increase in the unit Ca2+ transient consistent with an extra release of Ca2+ from the intracellular stores (n = 7 cells, p < 0.05, t test, comparing 100 ms pulse duration with 1000-ms pulse duration). Note also that the unit Ca2+ transient evoked by a 100-ms duration pulse in the cADPR-treated cells was significantly increased compared with the control, p < 0.05, t test. There were no differences in the basal Ca2+ levels observed in control cells compared with those treated with 10 µM cADPR, mean values were 278 ± 24 nM for controls and 277 ± 25 nM for cADPR-treated cells, p = 0.48, t test. 10 µM ryanodine reduced the change in the unit Ca2+ transients in both control and cADPR-treated cells (n = 3 for both, there being no significant difference in the effects of ryanodine in the two conditions, p = 0.33, t test). Unit Ca2+ transients at 1000 ms were also effectively reduced by the inclusion of 20 µM ruthenium red into the pipette and also in separate experiments by 10 mM Mg2+ (43), mean values were 1.5 ± 0.5 and 0.8 ± 0.8, n = 4 and 3, respectively. B, concentration response curve for the actions of cADPR, showing the increase in the value of the unit Ca2+ transient calculated during a 1000-ms voltage step, with different concentrations of intracellularly applied exogenous cADPR. Values are taken from between 5 and 8 cells at each concentration and represent a larger data set than shown in Fig. 3A.
[View Larger Version of this Image (18K GIF file)]

Inositol trisphosphate also enhances release of Ca2+ from intracellular stores in a Ca2+-dependent manner, so called inositol trisphosphate-induced Ca2+ release (IICR) (2, 34). However we did not observe any effect on the unit Ca2+ transient when 50 µM InsP3 was included in the patch pipette, even though a number of studies suggest that these neuroblastoma cells express InsP3-sensitive intracellular Ca2+ stores (37-39) (mean value in the presence of InsP3 following a 1-s pulse was 2.6 ± 1.0 compared with 2.8 ± 0.7 for control, p = 0.4, t test).

10 µM ryanodine, an inhibitor of CICR, decreased the unit Ca2+ transient during the shorter, 100-ms voltage step and also blocked the increase in the unit Ca2+ transient with increasing pulse duration in both control cells and in cells treated with 10 µM cADPR (Fig. 3, open triangles) A similar blockade occurred when either 10 mM Mg2+ or 20 µM ruthenium red were included in the pipette solution (see legend to Fig. 3A). All these treatments indicate that exogenously applied cADPR, acting on or close to the ryanodine receptor, increased the sensitivity of CICR in these neurons. cADPR did this in two ways: first, it reduced the amount of Ca2+ entry required to trigger additional Ca2+ release from the internal stores and second, by increasing the value of the unit Ca2+ transient, it increased the total amount of Ca2+ released from intracellular stores for a given controlled Ca2+ influx. This increase in the unit Ca2+ transient occurred in a concentration-dependent manner (Fig. 3B). 50 and 100 µM cADPR increased the unit Ca2+ transient to a level 3-fold over control, indicating that cADPR action reached a maximum.

Our results show that this neuronal cell line possessed ryanodine- and caffeine-sensitive intracellular Ca2+ stores. Cyclic ADP-ribose, in the presence of Ca2+ influx, increased the amount of Ca2+ released from the intracellular store in these cells and also increased the sensitivity with which Ca2+ entry triggered additional Ca2+ release leading to a global intracellular Ca2+ rise. These actions of cADPR suggest a requirement for powerful regulatory mechanisms to control the production of cytosolic levels of cADPR from its ubiquitous precursor, beta -NAD+ (40). As Ca2+ release from internal stores becomes more widely recognized as part of neuronal Ca2+ homeostasis, an increased understanding of the factors controlling endogenous levels of cADPR in neurons is now required.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 44-1865-271890; Fax: 44-1865-271853; E-mail: ruth.empson{at}pharm.ox.ac.uk.
1   The abbreviations used are: CICR, Ca2+-induced calcium release; cADPR, cyclic ADP-ribose; InsP3, inositol trisphosphate; pA, picoamps; pC, picocoulomb(s).

ACKNOWLEDGEMENTS

We acknowledge the support of the Wellcome Trust and also thank Dr. A. A. Genazzani for helpful discussions.


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