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 M. Seal resistances prior to breakthrough were always greater than 1 G. 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.

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).

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.

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, -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.