(Received for publication, May 22, 1997, and in revised form, June 11, 1997)
From the Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom
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.
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.
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.
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.
We acknowledge the support of the Wellcome Trust and also thank Dr. A. A. Genazzani for helpful discussions.