* Instituto de Biología y Genética Molecular, Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de
Medicina, Universidad de Valladolid y Consejo Superior de Investigaciones Científicas, E-47005 Valladolid, Spain; and Instituto de Farmacología Teófilo Hernando, Departamento de Farmacología y Terapéutica, Facultad de Medicina,
Universidad Autónoma de Madrid, E-28029 Madrid, Spain
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Abstract |
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The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4,5-trisphosphate (InsP3)- producing agonists released only 60-80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.
Key words: endoplasmic reticulum; aequorin; chromaffin cells; calcium; ryanodine ![]() |
Introduction |
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AT present, the role played by ryanodine receptors
(RyR)1 in the homeostasis of intracellular Ca2+ in
nonmuscle cells is unclear. Mammalian tissues express three isoforms, RyR1, RyR2, and RyR3, encoded by
different genes. RyR1 and RyR2 are expressed predominantly in sarcoplasmic reticulum of skeletal muscle and
heart, respectively, where they have an essential role to
trigger muscle contraction (Sutko and Airey, 1996; Zucchi
and Ronca-Testoni, 1997
). RyR3 was originally identified in brain (Hakamata et al., 1992
), but in fact all three isoforms are actually expressed in brain, and the major brain
isoform appears to be RyR2 (McPherson and Campbell,
1993
; Sorrentino and Volpe, 1993
; Giannini et al., 1995
).
RyRs are widely distributed in many other different tissues (Giannini et al., 1995
; Mackrill et al., 1997
), including
the adrenal gland, and they are in many cases coexpressed
with one or more isoforms of inositol 1,4,5-trisphosphate receptors (InsP3R) (Walton et al., 1991
; Poulsen et al.,
1995
).
The reasons for the presence of multiple Ca2+ release
mechanisms in the same cell are not clear (for discussion
see Newton et al., 1994; Sutko and Airey, 1996
). A possible reason for the coexistence of both InsP3R and RyR in
the same cells could be that they might release Ca2+ from
different compartments, and with a different physiological significance. They may be modulated by different second
messengers, such as InsP3 in the case of InsP3R or cyclic
adenosine diphosphate ribose (cADPR) for the RyR (Lee,
1998
). However, although InsP3 is a well-established physiological activator of the InsP3R, the role of cADPR as activator or modulator of RyR in the presence of physiological concentrations of ATP remains controversial (Sutko and Airey, 1996
; Zucchi and Ronca-Testoni, 1997
). In neuronal cells, RyR could be activated via the classical Ca2+-induced Ca2+ release (CICR) mechanism after Ca2+ entry
through voltage-dependent Ca2+ channels. Studies in some
neuronal preparations have shown that depolarizing stimuli produce an increase in cytosolic [Ca2+] ([Ca2+]c) that
may be due in part to Ca2+ release from intracellular
stores (Verkhratsky and Shmigol, 1996
). However, little direct evidence has been presented for CICR in nonmuscle
cells; this is due to the difficulties in separating the contribution of Ca2+ entry and Ca2+ release to the [Ca2+]c signal and
to the nonspecificity of caffeine and other pharmacological agents used to activate RyR.
To study CICR in neuronal cells, it would be greatly advantageous to measure [Ca2+] specifically inside the Ca2+
stores. We have recently reported a method to measure
[Ca2+] in the lumen of the endoplasmic reticulum ([Ca2+]ER)
of intact cells, the main intracellular Ca2+ store, by using an
ER-targeted aequorin (Montero et al., 1995, 1997a
,b; Barrero et al., 1997
), which can be expressed in different types
of cells using a viral vector (Alonso et al., 1998
). This technique is ideal to study directly CICR, because Ca2+ release
can be measured independently of the variations in [Ca2+]c.
Here we have used this technique to monitor [Ca2+]ER in
chromaffin cells. These neuroendocrine cells have a potent caffeine-sensitive Ca2+ release mechanism (Cheek et al.,
1990
), and Ca2+ entry through several types of voltage-
dependent Ca2+ channels can be induced by K+ depolarization or more physiologically, using nicotinic agonists (Núñez
et al., 1995
; Lara et al., 1998). Direct evidence for a CICR
mechanism working under physiological conditions in
these cells has not been provided. However, we have reported recently that the caffeine-sensitive Ca2+ stores may
modulate catecholamine secretion induced by depolarization with high K+ in these cells. Catecholamine secretion
was reduced after store emptying with caffeine, and recovered as Ca2+ stores refilled during consecutive K+/Ca2+
pulses. The main conclusion from that work was that Ca2+
stores could have a double role, acting either as a sink or as a source of Ca2+, depending of their state of filling (Lara et
al., 1997
).
Additionally, several questions regarding the function of
RyR in chromaffin cells remain unanswered. For instance,
chromaffin cells have also InsP3R, which are at least in
part colocalized with RyR2 in the ER (Poulsen et al.,
1995). The presence of separate or overlapping Ca2+ pools
responsive to either InsP3, caffeine, or cADPR, their differential sensitivity to inhibitors of the ER Ca2+-pump
such as thapsigargin, and the physiological significance of
the different Ca2+ release mechanisms, has been a subject
of debate for many years (Cheek et al., 1991
; Liu et al.,
1991
; Robinson and Burgoyne, 1991
; Stauderman et al.,
1991
; Morita et al., 1997
). On the other hand, the mechanism of Ca2+ release induced by caffeine is quite particular
because increasing concentrations of caffeine release Ca2+
in a quantal manner, a phenomenon that has been suggested to indicate that the caffeine-sensitive Ca2+ pool is
composed of functionally discrete stores with heterogeneous sensitivities to caffeine (Cheek et al., 1993
, 1994a
).
Here we have monitored [Ca2+]ER in chromaffin cells to
investigate the mechanism of quantal Ca2+ release by caffeine, its relationship in terms of Ca2+ pools with InsP3-mediated Ca2+ release, and the presence of CICR triggered by Ca2+ entry. In brief, our results indicate that the
ER Ca2+ pools responding to caffeine and InsP3 mostly
overlap. The response to caffeine was also quantal when
studied from inside the ER, but this quantal response
could be explained by the control of caffeine-induced Ca2+
release by [Ca2+]ER, with no need for separate ER compartments with heterogeneous sensitivities to caffeine, as
proposed previously (Cheek et al., 1993, 1994a
). We show
that CICR can be induced by Ca2+ entry elicited either by
high K+ depolarization or by stimulation with nicotinic agonists. This is consistent with our previously proposed
model for the Ca2+ store as a modulator of secretion (Lara
et al., 1997
). Additionally, using fast confocal [Ca2+] measurements, we show that CICR participates in the generation and propagation of the Ca2+ wave induced by cell depolarization.
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Materials and Methods |
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Preparation and Culture of Bovine Chromaffin Cells
Bovine adrenal medulla chromaffin cells were isolated following standard
methods (Livett, 1984) with some modifications (Moro et al., 1990
). Cells
were suspended in Dulbecco's modified Eagle's medium (DME) supplemented with 5% fetal calf serum, 10 µM cytosine arabinoside, 10 µM fluorodeoxyuridine, 50 IU ml
1 penicillin and 50 IU ml
1 streptomycin. For
secretion experiments, cells were plated in 5-cm-diam Petri dishes (5 × 106 cells per 5 ml of DME). For aequorin experiments, cells were plated
on 12- or 13-mm glass poly-D-lysine-coated coverslips (0.5 × 106 cells per
1 ml of DME). For measurements of [Ca2+]c transients by confocal microscopy and ionic currents, cells were plated on 2.5-cm-diam glass coverslips at a density of 5 × 104 cells per ml. Cultures were maintained at 37°C
in a humidified atmosphere of 5% CO2.
Preparation of Viral Stock and Infection of Cultures
Construction, packaging, and titering of the pHSVerAEQ amplicon vector and expression in chromaffin cells has been previously described
(Alonso et al., 1998). The EcoRI fragment of the erAEQmut cDNA was
subcloned into the pHSVpuc vector to generate the pHSVerAEQ. As a
helper virus, the herpes simplex virus type 1 (HSV-1) IE2 deletion mutant
5dl1.2 was used with a titer of 2 × 107 infectious virus units (ivu)/ml (Lim
et al., 1996
). Titers of viral stocks were determined by immunocytochemistry on PC12 cells. Infected cells were visualized by using a rabbit anti-
HSV-1 particle antibody (1:10,000 dilution; Dako) or a mouse anti-HA1
primary antibody (1:200 dilution; ) followed by an alkaline phosphatase-conjugated anti-mouse IgG antibody (1:200 dilution;
). The titers of the vector stock were 1.1 × 106 ivu/ml pHSVerAEQ
and 4.1 × 106 ivu/ml 5dl1.2. Chromaffin cell cultures (5 × 105 cells/0.5 ml)
were routinely infected with 1.2 × 104 ivu 1 d before measurements. The
percentage of cells expressing ER-targeted aequorin was usually ~20%.
Immunofluorescence revealed a typical nonnuclear reticular pattern (data
not shown), similar to that previously seen in HeLa cells (Montero et al.,
1995
). This pattern was not modified by the 1-h period of ER Ca2+ depletion required for ER aequorin measurements.
Measurements of [Ca2+]ER with Aequorin
For [Ca2+]ER studies, cells were infected after 1 d in culture with HSV-1
carrying the ER-targeted aequorin construct as described previously
(Alonso et al., 1998). Measurements of [Ca2+]ER were started ~16 h after
infection that were required to allow adequate expression of the targeted
photoprotein. Aequorin photoluminescence measurements were performed essentially as previously described (Barrero et al., 1997
). In brief,
cells were depleted of Ca2+ by incubation for 5-10 min at 37°C with the
sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor 2,5-di-tert-butyl-benzohydroquinone (BHQ) 10 µM in standard medium containing
145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 10 mM Hepes,
pH 7.4, supplemented with 3 mM EGTA. Cells were then incubated for
1 h at room temperature in standard medium containing 0.5 mM EGTA,
10 µM BHQ, and 1 µM coelenterazine n. The coverslip was then placed in
the perfusion chamber of a purpose-built thermostatized luminometer
and standard medium containing 1 mM Ca2+ was perfused to refill the ER
with Ca2+. Measurements were performed at 22°C and [Ca2+]ER values
were calculated from the luminescence records using a computer algorithm (Brini et al., 1995
) which follows the calibration curve reported before (Barrero et al., 1997
). In the experiments carried out with permeabilized cells, cells were placed in the luminometer as described above and
perfused for 1 min with intracellular-like medium (10 mM NaCl, 140 mM
KCl, 1 mM MgCl2, 1 mM KH2PO4, 2 mM ATP, 20 mM Hepes, pH 7) containing 2 mM EGTA and 20 µM digitonin. Then, intracellular medium
without digitonin and containing 100 nM EGTA-buffered Ca2+ was perfused for 3-5 min to refill the ER with Ca2+. The total number of counts
obtained ranged between 0.3 and 2 million.
Measurements of Single-cell [Ca2+]c
Single-cell measurements of [Ca2+]c were performed at room temperature
in fura-2-loaded cells as described previously (Núñez et al., 1995). Cells
were epi-illuminated alternatively at 340 and 380 nm and light emitted
above 520 nm was recorded by an extended ISIS-M camera (Photonic Science) and analyzed using an Applied Imaging Magical image processor
(Sunderland). 16 frames excited at every wavelength were averaged by
hardware, with a time resolution of ~7 s for each pair of images, and
[Ca2+]c was estimated from the ratio F340/F380 by comparison with fura-2 standards. Field electric stimulation (McIlwain and Rodnight, 1962
) was
performed through a pair of silver electrodes placed 7 mm apart and 1.5 mm
above the cells. Alternating positive-negative square pulses of 50-ms duration and 60-V intensity were applied at 10 Hz. The peak current was 100 mA.
Confocal [Ca2+]c Measurements and Electrophysiological Recordings
Electrical measurements and [Ca2+]c were recorded by using the whole-cell patch-clamp technique (Hamill et al., 1981) in combination with fluo-3
based microfluorometry. Cells were placed in an experimental chamber
that was mounted on the stage of an inverted microscope (Diaphot 200;
). Cells were loaded via the patch pipette with the pentaammonium
salt form of the fluorescent dye fluo-3 (100 µM). The dye was excited with
a Kr-Ar laser light at 488 nm and emission was detected at 522 nm (32-nm
band width). Cells were dialyzed with an intracellular solution containing
135 mM CsCl2, 8 mM NaCl; 1 mM MgCl2, 20 mM Hepes, 2 mM ATP, and
0.3 mM GTP, pH 7.3. The chamber was continuously perfused with
Krebs-Hepes medium. Line-scan images (0.33-µm width) of the intracellular Ca2+ distribution were acquired every 2 ms with a confocal microscope (MRC 1024; Bio-Rad), using an oil immersion, planapochromatic 60× objective (NA = 1.4 []). Changes in [Ca2+]c were inferred from
the intensity of fluo-3 fluorescence normalized to that in resting conditions (F/F0). Whole-cell currents were monitored with a DAGAN PC-ONE patch-clamp amplifier. Data were recorded and analyzed with Igor
Pro 3.02 (Wave Metrics).
Chemicals
Coelenterazine n, fura-2AM, and fluo-3 were obtained from Molecular Probes. InsP3 was from Research Biochemicals International. cADPR was obtained from and from . Other reagents were of the highest quality available from or Merck.
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Results |
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Overlap between Caffeine-, Ryanodine-, Histamine-, and Thapsigargin-sensitive Components of [Ca2+]ER
After aequorin reconstitution with coelenterazine, with
the ER completely depleted of Ca2+, the experiments
were started by perfusing the cells with medium containing 1 mM Ca2+ to refill the ER (Fig. 1 a). As in other cells
studied previously (Montero et al., 1995, 1997a
; Barrero et
al., 1997
; Alonso et al., 1998
), full refilling of the ER required 3-5 min and the steady-state [Ca2+]ER reached was
500-800 µM (Fig. 1 a). Addition of histamine produced a
rapid but partial (60-80%) Ca2+ emptying of the ER, a
new [Ca2+]ER steady-state being reached at ~200-300 µM.
Subsequent addition of caffeine (50 mM) induced a further emptying to near background aequorin luminescence.
The effects were reversible by washing, this allowing refilling of the ER that was completed within 3-5 min. Addition of caffeine at that point, when the stores were completely refilled, triggered a rapid and complete emptying
of the ER. Histamine was then unable to produce any further effect. These results suggest that essentially the whole
ER Ca2+ pool is sensitive to caffeine and a large part of it
is also sensitive to InsP3 producing agonists such as histamine. Similar results were obtained using 1 µM bradykinin
instead of histamine (data not shown).
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To obtain more information about the nature of the caffeine-sensitive Ca2+ pool, we studied the ability of the ER
to refill in the presence of caffeine, ryanodine, or the Ca2+-ATPase inhibitor thapsigargin. Fig. 1 b, left panel shows
that when Ca2+-depleted cells were incubated with Ca2+-
containing medium but in the presence of 50 mM caffeine,
refilling was almost abolished until caffeine was washed
away. The small increase observed in [Ca2+]ER in the presence of caffeine was insensitive to histamine. Ryanodine
has been reported to lock open irreversibly the RyR in a
use-dependent manner, that is, when ryanodine is present while the channels have been opened by caffeine (Ehrlich
et al., 1994). If all the ER had functional caffeine and ryanodine-sensitive RyR, we would then expect that pretreatment of the cells with caffeine and ryanodine would inhibit
also refilling of the ER with Ca2+. Fig. 1 b, middle panel,
shows that this is the case. Cells were treated with five
pulses of 50 mM caffeine and 10 µM ryanodine, and then
the drugs were removed before aequorin reconstitution. Addition of 1 mM Ca2+ to these cells produced only an
small increase in [Ca2+]ER, that was little sensitive to caffeine or bradykinin. The same results were obtained (Fig.
1 b, right panel) if the cells were pretreated with the
SERCA inhibitor thapsigargin (1 µM). Similar effects
were obtained using lower (20 nM) thapsigargin concentrations (data not shown). Therefore, the whole ER has
caffeine- and ryanodine-sensitive RyRs, and refills with
Ca2+ via thapsigargin-sensitive Ca2+ pumps.
Fig. 2 illustrates the time course of the use-dependent
effect of ryanodine on RyR. Fig. 2 a shows that consecutive additions of 50 mM caffeine produced comparable decreases in [Ca2+]ER if an interval of 3-5 min was left between two consecutive additions to allow refilling with
Ca2+ of the ER. If 10 µM ryanodine was present during
the caffeine pulses (Fig. 2 b), the first pulse was identical
to the control but then the ER became progressively unable to refill. After four pulses, the ER remained at near
background [Ca2+]ER levels, and only the increase in
[Ca2+]c elicited by depolarization with 70 mM K+ was able
to activate the Ca2+ pump and produce a small and transient increase in [Ca2+]ER. The effect of caffeine was not
inhibited by incubation with 20 µM dantrolene (data not
shown), an inhibitor of RyR that is particularly effective
on the skeletal muscle RyR1 (Van Winkle, 1976).
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Quantal Effect of Caffeine
The effect of caffeine has been reported to be quantal
(Cheek et al., 1993, 1994a
), meaning that low caffeine concentrations release only part of the caffeine-sensitive pool.
The same phenomenon was observed here. Fig. 3 a shows
that addition of submaximal caffeine concentrations induced a rapid but partial emptying of the ER, leading
within 30 s to a new lower steady-state of [Ca2+]ER. At that
point, only the addition of a higher caffeine concentration was able to produce further emptying of the ER. Similar
quantal effects were also observed during refilling of the
ER when it was carried out in the presence of caffeine.
Fig. 3 b shows that the ER did not refill in the presence of
50 mM caffeine, but refilled about halfway once the caffeine concentration was dropped to 5 mM, and completely
when caffeine was washed away. Subsequent addition of 5 and 50 mM caffeine released Ca2+ and reached the same
[Ca2+]ER levels obtained during refilling in the presence of
these caffeine concentrations. The degree of emptying induced by a particular caffeine concentration was quite reproducible in consecutive additions. Fig 3 c shows that
consecutive additions of 5 mM caffeine produced always
~50% emptying of the ER, and only the addition of a
higher caffeine concentration was able to produce further
emptying. Fig. 3 d shows the effect of ryanodine added in
the presence of a submaximal dose of caffeine. We can see
that the first pulses were identical to the control, but again
here the ER refilled progressively more slowly after each
new caffeine addition. In this case, in contrast to the experiment shown in Fig. 2, finally a [Ca2+]ER steady-state
corresponding to about half-filling was reached, in fact, the
same [Ca2+]ER obtained initially after addition of 5 mM
caffeine. Once at this point, addition of a maximal dose of
caffeine was required to induce emptying of the remaining
portion of the ER.
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The experiments of Fig. 3 are consistent with previous
results obtained looking at [Ca2+]c in the same cell preparation by Cheek et al., 1994a. Those experiments led the
authors to suggest that there should be different compartments within the ER having different sensitivities to caffeine. This hypothesis would explain why ryanodine only
empties the pool sensitive to 5 mM caffeine but leaves the
rest of the pool untouched, which could be only released
with a higher caffeine dose. However, there is an alternative explanation for these results, based on the regulation
of caffeine-induced Ca2+ release by the lumenal [Ca2+]. In
this hypothesis, the results can be explained with only one
ER compartment if we assume that submaximal caffeine
concentrations can only release Ca2+ until [Ca2+]ER is reduced to a certain level. The higher the caffeine concentration, the lower the [Ca2+]ER level attained. Both alternative hypotheses lead to different predictions under some
experimental conditions. In particular, if we obtain half-filled stores by different procedures, e.g., by emptying
them with an agonist acting via InsP3 production or by refilling the stores only halfway, the hypothesis of several compartments predicts that we should have all of
them half-filled. Therefore, a submaximal dose of caffeine
should still release Ca2+ from half of them. On the contrary, if the release was directly controlled by [Ca2+]ER, we
would expect the effect of 5 mM caffeine to be independent of the procedure used to reach that half-filling. The
experiments shown in Fig. 4 indicate that the last hypothesis is the correct one. In Fig. 4 a, half-filling was obtained
by emptying the ER with histamine. After that, 5 mM caffeine had no effect. Instead, if 5 mM caffeine was added
with the ER full of Ca2+, it was able to empty it exactly
down to the same point. Fig. 4 b shows the effect of half-filling the ER by reducing the time of refilling. Again, the
effect of 5 mM caffeine was strictly dependent on the level
of [Ca2+]ER reached at the point it was added. It had no effect at half-filling, but released 50% of the pool when the
Ca2+ stores were completely filled. Fig. 4 c shows a similar
approach but made in cells preloaded with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to slow the refilling. We can observe again
that the effect of 5 mM caffeine was strictly dependent on
the [Ca2+]ER at the moment of addition. In addition, this
experiment also shows that Ca2+ release induced by caffeine requires only resting [Ca2+]c. Fura-2 measurements
performed in parallel showed that in cells loaded with
BAPTA, the [Ca2+]c changes induced by caffeine were almost abolished (data not shown). This result points out
also that quantal Ca2+ release by caffeine is due to the regulation of Ca2+ release by the lumenal [Ca2+], and suggests that changes in [Ca2+]c do not play a major role in the
development of the quantal effect.
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Ca2+ Entry Activates CICR
The next step in this study was to investigate the presence
of CICR activated by Ca2+ entry through the plasma
membrane Ca2+ channels. Depolarization with high K+
medium or by stimulation with nicotinic acetylcholine agonists such as 1,1-dimethyl-4-phenyl-piperazinium iodide
(DMPP), produced large [Ca2+]c peaks (Núñez et al.,
1995), that were not significantly modified by previous
Ca2+ depletion of the ER with caffeine or with the ER
Ca2+ pump inhibitor thapsigargin (see below). Therefore,
the possible contribution of CICR to these [Ca2+]c peaks
cannot be estimated from conventional [Ca2+]c studies,
and direct measurement of [Ca2+]ER becomes essential.
Fig. 5 a shows that 10-s pulses of depolarization with high
K+ medium induced a transient Ca2+ release from the
ER, which could be triggered repetitively by consecutive
pulses. The [Ca2+]ER decrease was of 60-100 µM (10-15%
of the steady-state [Ca2+]ER). Therefore, in spite of the
large increase in [Ca2+]c, the activation of CICR produced
a much smaller [Ca2+]ER decrease than treatment with caffeine. Increasing the duration of the high K+ pulse increased the magnitude of Ca2+ release little. Fig. 5 b illustrates the effect of depolarization with longer (1 min) high
K+ pulses. In this case, the first high K+ pulse produced
the same effect as in Fig. 5 a, and then [Ca2+]ER increased
more rapidly, probably as a result of the prolonged stimulation of the ER Ca2+ pump by the sustained high [Ca2+]c
levels. This led to a new [Ca2+]ER steady-state at ~800-900
µM. After that, subsequent K+ pulses induced somewhat
larger [Ca2+]ER decreases of 150-200 µM, but corresponding still to only 20% of the steady-state [Ca2+]ER. To investigate if increased Ca2+ pumping could be responsible for
the incomplete Ca2+ release, the effect of K+ depolarization was tested in the presence of the ER Ca2+ pump inhibitor ciclopiazonic acid (CPA). Fig. 5 c shows that CPA
itself induces a slow Ca2+ release from the ER, and that simultaneous addition of high K+ medium induced a fast
initial Ca2+ release of ~20% of the [Ca2+]ER, followed by
a slower release at a rate comparable to that induced by
CPA alone. This suggests that CICR induced by a maximal K+ depolarization is able to produce only a decrease
of ~20% of the [Ca2+]ER, even in the absence of Ca2+
pumping. CICR, however, was potentiated by simultaneous addition of a low caffeine concentration. Fig. 5 d
shows that addition of 1 mM caffeine produced little effect
by itself, but strongly potentiated the effect of K+ depolarization, that was now able to release rapidly ~50% of the
stored Ca2+. Finally, CICR could also be triggered in a
more physiological way using an agonist for the nicotinic
acetylcholine receptor. Addition of DMPP induced a rapid
and partial Ca2+ release from the ER, very similar to that
shown above for K+ depolarization, and which was also
potentiated by low concentrations of caffeine (data not
shown).
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The effect of a low caffeine concentration as positive
modulator of CICR might reproduce the action of a physiological modulator of this phenomenon. Phosphorylation
by cAMP-dependent protein kinase and production of the
-NAD+ metabolite cyclic ADP ribose (cADPR) have
been reported to act as physiological modulators for RyR
in bovine chromaffin cells (Morita et al., 1997
). However,
incubation for 3-5 min with the adenylate cyclase activator
forskolin (20 µM) had neither any significant effect on the
sensitivity to caffeine of Ca2+ release nor on the magnitude of high K+ depolarization-induced CICR (data not
shown). Regarding cADPR, it has been reported that acetylcholine, high K+ depolarization, and forskolin all stimulate its synthesis by ADP ribosyl cyclase in bovine chromaffin cells (Morita et al., 1997
). Therefore, production of
this mediator should already be stimulated in our CICR
experiments. Nevertheless, to study directly the effect of
cADPR on Ca2+ release from the ER, we performed experiments in permeabilized cells. Cells were depleted of
Ca2+ and reconstituted with coelenterazine as usual. Recording of luminescence was started and the cells were
permeabilized by perfusion with intracellular-like medium
containing 20 µM digitonin and 2 mM EGTA for 1 min.
Then, intracellular-like medium containing 100 nM Ca2+
(buffered with EGTA) and 2 mM ATP-Mg was perfused.
Fig. 6 a shows that [Ca2+]ER increased in digitonin-permeabilized cells with very similar kinetics to that found in intact cells after addition of 1 mM extracellular Ca2+. The
steady-state [Ca2+]ER reached was also similar (compare with
Fig. 1 a). Fig. 6 a also shows that 2 µM InsP3 and 50 mM
caffeine produced a rapid and near complete release of
Ca2+ from the ER whereas 5 µM cADPR had no effect.
Two different commercial sources of cADPR were tested
with the same results. In some experiments, cADPR was
added in the presence of 1 µM calmodulin and no effect was found either. A possible explanation for the discrepancy among our results and those of Morita et al. (1997)
would be that cADPR releases Ca2+ from a different
(non-ER) Ca2+ pool. In fact, these authors report that
InsP3 releases Ca2+ from a pool sensitive to 20 nM thapsigargin, whereas cADPR and caffeine release Ca2+ from a
pool only sensitive to >200 nM thapsigargin. In our hands, refilling of the ER was completely inhibited by either 20 nM
or 1 µM thapsigargin in both intact (see above) and permeabilized cells (data not shown). Therefore, in order to
make compatible our own results and those of Morita et al.
(1997)
, cADPR and caffeine should be able to release
Ca2+ from an additional non-ER Ca2+ pool in the presence of 20 nM thapsigargin. We then performed single-cell
fura-2 imaging experiments in intact cells looking at the effect of caffeine on [Ca2+]c in cells pretreated with 20 nM
thapsigargin. Fig. 6 b shows that addition of caffeine or
histamine in Ca2+-free medium produced no increase in
[Ca2+]c under these conditions (the observed decrease is
due to the perfusion of Ca2+-free medium), whereas K+
depolarization still produced the usual [Ca2+]c peak due to
Ca2+ entry.
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Effect of Ryanodine on CICR
On the basis of the use-sensitive action of ryanodine, illustrated here for caffeine stimulation (Figs. 2 and 3), we should expect also to find a use-sensitive inhibition of ER refilling after repeated stimulation with high K+ pulses inducing CICR. Under this rationale, experiments similar to those shown in Fig. 5, but in the presence of 10 µM ryanodine, were performed. In these experiments we were not able to detect any significant effect of ryanodine on the [Ca2+]ER decrease induced by four or five consecutive K+ pulses (data not shown). However, as aequorin consumption limits the sensitivity of the measurement at the end of these experiments, we also decided to perform single-cell imaging experiments looking at the magnitude of the [Ca2+]c peak induced by caffeine after several K+ pulses in the presence of ryanodine. Fig 7 a shows that the [Ca2+]c increase induced by caffeine was little modified after five consecutive 30-s K+ pulses given in the presence of ryanodine (compare with the initial three caffeine additions). It is interesting to note that the second caffeine addition after the K+ + ryanodine pulses produced no [Ca2+]c increase, even though caffeine was always added in the absence of ryanodine. In fact, perfusion with ryanodine for a short period (30-60 s) before any caffeine addition was effective in promoting the typical use-dependent inhibition of ER refilling on application of caffeine (without ryanodine) 30-60 min later. This means that, even though ryanodine by itself does not produce any apparent effect on ER filling, it remains within the cells after washing and acts later, on stimulation of Ca2+ release (see below). Depolarization can also be produced using the nicotinic acetylcholine agonist DMPP instead of K+. Again here, consecutive pulses of 10 µM DMPP in the presence of ryanodine did not have any effect on a later [Ca2+]c peak induced by caffeine (data not shown). An alternative and perhaps more physiological depolarizing maneuver is field electric stimulation. Fig. 7 b shows the effect of several consecutive 10-s pulses at 10 Hz, before and after the addition of ryanodine. Again, we can observe that 5 mM caffeine produced the same [Ca2+]c peak after and before field electric stimulation. As in Fig. 7 a, consecutive additions of caffeine produced inhibition long after washing of ryanodine. The lack of effect of ryanodine in these experiments may be attributed to the much smaller activation of Ca2+ release by high K+-induced Ca2+ entry compared with that induced by caffeine.
|
An alternative possibility to activate RyR by Ca2+
would be to release Ca2+ directly from the ER using an
InsP3-producing agonist in order to produce a big increase
in [Ca2+]c just besides the RyR. In fact, it has been reported that histamine-induced Ca2+ release could be inhibited partially with ryanodine after five consecutive
stimulation pulses (Stauderman and Murawsky, 1991). In
our hands, stimulation by five consecutive pulses of histamine and ryanodine had little effect on the first subsequent
[Ca2+]c peak obtained by caffeine stimulation (Fig. 7 c). Histamine was always added in the absence of extracellular
Ca2+, as it also activates Ca2+ entry (Cheek et al., 1994b
).
Similarly, caffeine was added also in Ca2+-free medium to
avoid the Ca2+ entry induced by caffeine, similar to that
reported in GH3 pituitary cells (Villalobos and García-Sancho, 1996
). As before, the second caffeine stimulation
after histamine and ryanodine pulses was completely abolished.
Effects of [Ca2+]ER on CICR
In spite of the lack of inhibition by ryanodine, the potentiation by caffeine of Ca2+ release induced by K+ depolarization (as shown in Fig. 5 d) suggests that CICR may take place through the same RyR activated by caffeine. An important property of the RyR activated by caffeine is the regulation by lumenal [Ca2+] shown in Figs. 3 and 4. In the case of CICR, data of Fig. 5 b also suggest that Ca2+ release may be stronger at higher lumenal [Ca2+] since the first stimulation with high K+, carried out when the ER was only half-filled, was less efficient than the subsequent ones. To obtain more evidence on this point, we investigated the effects of 10-s pulses of high K+/Ca2+-containing medium at different [Ca2+]ER levels (Fig. 8). At the beginning of the experiment the ER was completely depleted of Ca2+ and the first two K+ pulses induced an increase in [Ca2+]ER. Since the cells were kept in EGTA-containing medium during the intervals between the K+/Ca2+ pulses, refilling of the ER took place only from Ca2+ entering into the cells during the pulses. Subsequent K+ pulses produced also [Ca2+]ER increase, although progressively smaller, reaching finally a steady-state [Ca2+]ER at ~300 µM, where the K+/Ca2+ pulses had almost no effect. We then completely refilled the ER with Ca2+ by perfusing the cells with Ca2+-containing medium, thus reaching the usual [Ca2+]ER steady-state levels at ~700 µM. At that point, we restarted the protocol of repeated stimulation with 10-s high K+/Ca2+ pulses, using EGTA-containing medium for the intervals. The pulses produced now a rapid Ca2+ release that could be clearly distinguished from the slower [Ca2+]ER decrease induced by the lack of extracellular Ca2+. After three K+ pulses, [Ca2+]ER was reduced again to ~300 µM, and at that point the last pulse produced little effect. This result shows clearly that Ca2+ release induced by K+ depolarization, similarly to that induced by caffeine, is strictly dependent on the [Ca2+]ER.
|
Differential Inhibition of CICR by Type-specific Ca2+ Channel Inhibitors
We have tested the effect of several inhibitors of Ca2+ entry
on both refilling of the ER and high K+ depolarization-
induced CICR. A combination of inhibitors of all the voltage-dependent Ca2+ channels including 1 or 2 h of preincubation with -conotoxin GVIA (1 µM),
-conotoxin
MVIIC (3 µM), and
-agatoxin IVA (1 µM), together
with preincubation and perfusion along the experiment with 3 µM nisoldipine, had little effect on the rate of refilling of the ER with Ca2+. Instead, refilling was almost completely blocked by perfusion of 100 µM Cd2+ (data not
shown). Next, the effects of the inhibitors of voltage-gated Ca2+ channels on CICR were tested. For these purposes,
we stimulated the cells with high K+ medium in the presence of 1 mM caffeine (as in the experiments shown in Fig.
5 d) in order to potentiate the mechanism and increase the
sensitivity of the measurements. The decreases in [Ca2+]ER
observed, normalized as percentage of those obtained in
the controls, were (mean ± SEM): control, 100 ± 11 (n = 6); 3 µM nisoldipine, 103 ± 6 (n = 5); 1 µM
-conotoxin
GVIA, 93 ± 6 (n = 6); 3 µM
-conotoxin MVIIC, 42 ± 6 (n = 7); 1 µM
-agatoxin IVA, 41 ± 9 (n = 5); a combination of all three toxins and nisoldipine, 20 ± 6 (n = 5). Toxins and nisoldipine were preincubated with the cells
for 1 or 2 h before the measurements. When the effect of
nisoldipine was tested, this inhibitor was also perfused
along the experiment.
Ryanodine Treatment Modifies the [Ca2+]c Wave Induced by Short Depolarizations as Visualized by Confocal Microscopy
Under physiological conditions, cell stimulation is triggered by short depolarizations lasting a few milliseconds.
To estimate the contribution of CICR to the Ca2+ transient under these conditions, we have compared the rate of diffusion of the Ca2+ wave induced by a short (100 ms) cell
depolarization both in control cells or in cells in which
the Ca2+ stores had been blocked by previous treatment
with caffeine and ryanodine. We combined the whole-cell
patch-clamp technique with fluo-3-based microfluorimetry using a confocal microscope. Cells were line-scanned
along 100-ms square depolarizing pulses from a holding
potential of 70 to +10 mV. The recorded inward currents showed two typical components: a initial transient
peak (INa) followed by a slow inactivating phase (ICa) (data
not shown). The ryanodine treatment did not affect the total stimulated Ca2+ entry, calculated as the integral of the
last 90 ms of the recorded inward current (mean ± SEM:
control cells, 7.15 ± 0.42 pC [n = 34]; ryanodine-treated
cells, 6.68 ± 1.03 pC [n = 21]). In spite of this, line scan images representing [Ca2+]c showed clear differences between control and ryanodine-treated cells. Fig. 9 a shows
the spatiotemporal pattern of [Ca2+]c increase in control
cells, codified in pseudocolor. [Ca2+]c increased first near
the plasma membrane and then the Ca2+ wave propagated
intracellularly. Fig. 9 b shows the results obtained in cells
with the Ca2+ stores previously emptied by treatment with
caffeine and ryanodine. In this case, the [Ca2+]c increase
was smaller and the propagation of the Ca2+ wave delayed. Fig. 9, panels c-e detail the behavior of several parameters that quantify the phenomenon described above
in terms of peak [Ca2+]c rise (Fig. 9 c), maximum rate of
[Ca2+]c increase (Fig. 9 d), and time required to increase
fluorescence by 10% (Fig. 9 e) at different intracellular locations. Fig. 9 c shows that the maximum fluo-3 fluorescence (indicating the maximum [Ca2+] peak) was reached
near the plasma membrane. An 80% increase was found in
control cells compared with only a 40% increase in ryanodine-treated cells. The fluorescence peaks were smaller
as we move deep inside the cell, but the difference among
control and ryanodine-treated cells was maintained. Fig. 9 d
shows that the maximum rate of fluorescence increase was
located near the plasma membrane and decreased steeply
as we move into the cell. Again here, the rates were two to
three times faster in the control cells than in the ryanodine-treated ones. Fig. 9 e shows the time required for the
fluorescence to be increased by 10% at different locations.
This parameter is very sensitive to the intracellular propagation of the [Ca2+]c wave. We find that the [Ca2+]c wave
propagates about twice as fast in control cells than in cells
treated with ryanodine. These results indicate that CICR significantly contributes to the Ca2+ signal induced by cell
depolarization during a short, more physiological stimulation.
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Discussion |
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Most of previous research on CICR has been performed
by inferring the changes in [Ca2+]ER from the evolutions of
[Ca2+]c. Much of the value of the present study comes
from the fact that, for the first time, CICR has been
looked at from the inside of the ER using targeted aequorin. Such an approach provides unambiguous evidence
for the existence of CICR in adrenal chromaffin cells and
reveals several previously unrecognized features on the
mechanisms and the regulation of this important phenomenon. We find that essentially all the stored Ca2+ can be
released by stimulation with a maximal dose of caffeine and that refilling was almost completely prevented by both
caffeine and ryanodine. This means that RyR must be homogeneously distributed within the ER or, alternatively,
Ca2+ release at some specific points is able to empty rapidly subcompartments lacking RyRs, that is, the ER behaves as a continuum. We can not distinguish at present
between these two possibilities. InsP3-producing agonists
such as histamine or bradykinin also produced a major release of Ca2+ from the ER, although smaller than caffeine.
This may suggest that the InsP3-sensitive pool is smaller
than the caffeine-sensitive one, as previously suggested
(Liu et al., 1991). In permeabilized cells, however, direct
addition of InsP3 produced Ca2+ release as big as that induced by caffeine. This suggests that the incomplete releasing effect of histamine or bradykinin in intact cells may
be due to desensitization phenomena, either at the plasma
membrane receptors or at the InsP3 receptor, or to other
mechanisms that may limit InsP3-mediated Ca2+ release
such as inhibition by [Ca2+]c (Bezprozvanny et al., 1991
;
Finch et al., 1991
; Barrero et al., 1997
; Montero et al.,
1997a
). In conclusion, our results support the idea that the
bulk of the ER is able to release Ca2+ either via InsP3 receptors or via RyR. In addition, the whole ER was also
sensitive to inhibition of the ER Ca2+ pump with thapsigargin, even at low concentrations (20 nM).
These results are consistent with those of Poulsen et al.
(1995), showing that the distribution of thapsigargin-sensitive Ca2+ pumps was parallel to that of InsP3R and RyR in
subcellular fractions of bovine chromaffin cells. On the
other hand, our results contrast with those of several authors working also with bovine chromaffin cells. Cheek
et al. (1991)
concluded using permeabilized cells that the
caffeine-sensitive Ca2+ store is largely distinct from the
InsP3-sensitive one. The presence of separate pools exclusively sensitive to InsP3 or to caffeine, or sensitive to both
has also been reported from studies with permeabilized
cells by Stauderman et al. (1991)
, although in the same
study the caffeine and the InsP3-sensitive pools overlapped more than 90% in intact cells. Robinson and Burgoyne (1991)
, also using permeabilized cells, suggested
that there are two distinct nonoverlapping Ca2+ stores sensitive to InsP3 or to caffeine, and only the InsP3-sensitive one was emptied by thapsigargin. Finally, Morita et al.
(1997)
have shown recently that in permeabilized cells,
cADPR and caffeine release Ca2+ from a compartment insensitive to 20 nM thapsigargin, a concentration that, however, abolished InsP3-mediated Ca2+ release. We have
no explanation for the discrepancies among the different
studies. Nevertheless, we should make clear that, in the aequorin experiments, we are measuring [Ca2+] specifically
inside the ER, whereas in all the other studies there may
be a contribution to Ca2+ release from other Ca2+-storing
organelles. Using mitochondrially targeted aequorin in chromaffin cells, we have seen that mitochondria have a
resting [Ca2+] similar to that in the cytosol (our unpublished observations), as it has been also reported in other
cell types (Rizzuto et al., 1993
); therefore they cannot release Ca2+ under resting conditions. An alternative possibility would be the secretory granules, which can accumulate large amounts of Ca2+ by a mechanism not requiring
Ca2+-ATPase (Pozzan et al., 1994
). It has been claimed
that InsP3 may release Ca2+ from chromaffin granules
(Yoo and Albanesi, 1991), but evidence against the presence of InsP3R in the secretory granules of the closely
related PC12 pheochromocytoma cells has also been
reported (Fasolato et al., 1991
; Zachetti et al., 1991). Pancreatic acinar secretory granules have also been shown to
release Ca2+ on stimulation with either InsP3 or cADPR
(Gerasimenko et al., 1996
), but these results have been
also questioned later (Yule et al., 1997
). Moreover, Pouli
et al. (1998)
, have just reported in PC12 cells, using an aequorin chimera targeted to the outer side of the granule
membrane, that both the agonist- or the K+-induced increases in [Ca2+] are identical in the cytosol and in the
outer side of the granule membrane, suggesting that there
is no Ca2+ release from the granules during physiological
stimulation. Our results are consistent with the last view
since even low concentrations of thapsigargin (20 nM)
were able to abolish the Ca2+ release induced by both histamine or caffeine in fura-2 single-cell imaging experiments (Fig. 6 b). Perhaps some of the discrepancies among
the different studies may come from the modifications
generated by permeabilization in the organellar structure.
In our experiments with permeabilized cells (Fig. 6 a), we
have attempted to minimize this problem by performing a
very fast (1 min) on-line permeabilization procedure.
Regarding Ca2+ release from the ER by caffeine, our experiments confirm previous results by Cheek et al. (1993
and 1994a), showing that Ca2+ release by increasing concentrations of caffeine is quantal in nature. We can add a
new insight to this effect, since our results suggest that this
quantal effect is due to the control of Ca2+ release by
[Ca2+]ER rather than to the existence in the ER of different Ca2+ pools with varying sensitivity to caffeine. Fig. 4
shows that when [Ca2+]ER was at the half-filling level, obtained either using an InsP3-producing agonist (Fig. 4 a) or
after incomplete refilling of the ER (Fig. 4 b), 5 mM caffeine had no effect. These results could be compatible with
the hypothesis of heterogeneous Ca2+ pools only if we
assume that (a) half-maximal quantal release induced by
InsP3 involves the same compartments than that induced by 5 mM caffeine, and (b) refilling of the ER is sequential
and starts first by those compartments having the lowest
sensitivity to caffeine. The first condition cannot be ruled
out conclusively, given that it is not known if both receptors may share the same mechanism for quantal release.
However, we consider the second one highly improbable
because the distribution of Ca2+-ATPases in chromaffin
cells has been reported to be parallel to that of InsP3 receptors and RyR (Poulsen et al., 1995
). In addition, Fig. 3
b shows that the rate of refilling is slower in the presence
of 5 mM caffeine than in the absence of caffeine, suggesting that compartments sensitive to 5 mM caffeine refill early in control cells. Therefore, our results clearly suggest that 5 mM caffeine does not release Ca2+ from one half
of the stores, but releases Ca2+ from all of them until
[Ca2+]ER reaches half-filling. The same type of control of
Ca2+ release by [Ca2+]ER was evidenced when it was triggered by Ca2+ entry. This suggests that Ca2+ release through
RyR is similarly regulated in both cases, stimulation by
caffeine or CICR. Control of Ca2+ release through both
InsP3R and RyR by the lumenal [Ca2+] has been suggested
previously (Missiaen et al., 1992
; Berridge, 1993
; Hidalgo
and Donoso, 1995
; Tanimura and Turner, 1996
) and we
provide here the first direct demonstration in intact cells, obtained by measuring [Ca2+] specifically into the ER.
The mechanism for this regulation remains unknown. The
fact that the same quantal phenomena were observed in
BAPTA-loaded cells suggests that [Ca2+]c is not involved
in the regulation. Since the intralumenal portion of RyR is
quite small and it has no apparent Ca2+-binding sites, it is
likely that intralumenal low affinity Ca2+-binding proteins
participate in the modulation of Ca2+ release by [Ca2+]ER
(Hidalgo and Donoso, 1995
).
One of the main aims of this study was to investigate the
presence of CICR elicited by the activation of plasma
membrane Ca2+ channels. Results in Fig. 5 demonstrate
for the first time that CICR is operative in bovine chromaffin cells, and that it can be potentiated by caffeine. This
suggests that CICR takes place through the same RyR activated by caffeine. However, we have been unable to
show inhibition of CICR by ryanodine after several pulses of K+ depolarization, stimulation by DMPP or field electrical stimulation. A possible reason may be that stimulation of RyR by CICR is much smaller than stimulation by
caffeine and therefore should require a much longer activation before inhibition is achieved, because of the use
dependence. In fact, inhibition by ryanodine was much slower on stimulation with 5 mM caffeine than with 50 mM
caffeine (Figs. 3 and 7). Similarly, the fact that in our
hands Ca2+ release induced by histamine was not sensitive
to ryanodine (Fig. 7 c), does not necessarily mean that
RyR have not been activated by Ca2+ released by InsP3R.
Regarding this point, it has been reported that histamine-induced Ca2+ spikes are inhibited by ryanodine in bovine
chromaffin cells (Stauderman and Murawsky, 1991), although inhibition required at least five consecutive stimuli
with histamine and was relatively small and variable. In
conclusion, ryanodine locks open RyR Ca2+ channels in a
use-dependent manner, and both the extent and the rate
of this effect depend on the magnitude of the stimulus
(caffeine or K+). Therefore, a lack of or very slow effect of
ryanodine should not be taken as a conclusive proof
against the involvement of RyR. We also find remarkable
that ryanodine required the presence of caffeine to lock
the channels open, but not to bind to the channels. As seen
in Fig. 7, ryanodine produced no effect when it was added
in the absence of caffeine, but even after extensive washing it was rapidly effective much later provided that caffeine was added to open the channels. This kind of memory could be best explained if ryanodine binds irreversibly
to RyR even in a closed state, but produces no effect until
the channels open.
Low concentrations of caffeine produce a clear potentiation of CICR and therefore it would be extremely important to see if there may be a physiological modulator able
to produce similar effects. Here we have studied the effects
of several possible modulators of RyR, such as cAMP-
mediated phosphorylation or cADPR. We did not find any
effect of forskolin on caffeine- or K+-induced Ca2+ release
and cADPR did not release Ca2+ from the ER in permeabilized cells. The lack of effect of cADPR contrasts with
the effects recently reported by Morita et al. (1997). These
authors find mobilization to the extracellular medium, where Ca2+ appearance is measured, of ~2 nmole/107 digitonin-permeabilized cells upon maximal stimulation with
cADPR. However, Morita et al. (1997)
report under the
same conditions, a catecholamine secretion amounting ~2
µg (120 nmole)/107 cells. For a ratio of catecholamine to
Ca2+ of about 1:20 inside the secretory granules (von Grafenstein and Powis, 1989
) the exocytotic mobilization of intragranular Ca2+ could amount as much as 6 nmole/107 cells,
more than enough to explain the reported Ca2+ mobilization by cADPR. Therefore, this Ca2+ could come from a
non-ER Ca2+ pool and would not even pass through the
cytosol in the intact cells.
CICR may have multiple physiological functions in
chromaffin cells. It could amplify the effect of a brief Ca2+
entry through the plasma membrane, generating a wave of
[Ca2+]c that might be required at the earlier steps of exocytosis, i.e., vesicle transport to exocytotic sites (von Rüden
and Neher, 1993; Neher, 1998). The observed [Ca2+]ER decrease of 100-200 µM could correspond to a mean increase in [Ca2+]c of 1-2 µM, assuming that the ER constitutes ~10% of the cell volume and that 90% of the ER
Ca2+ and 99% of the cytosolic Ca2+ are bound (Pozzan et
al., 1994
; Meldolesi and Pozzan, 1998
). This [Ca2+]c increase is well in the range required for stimulation of vesicle transport to exocytotic sites (von Rüden and Neher,
1993; Neher, 1998
). Additionally, CICR could also be designed to amplify subcellular Ca2+ gradients resulting from
agonist-induced Ca2+ release via InsP3 receptors (Berridge, 1998
). The question now arises as to what may be
the importance of the K+-stimulated Ca2+ release observed here for catecholamine secretion. We have reported that, when the Ca2+ stores had been depleted with
caffeine, a subsequent K+ depolarization elicits smaller secretion than in cells with full Ca2+ stores (Lara et al.,
1997
). Consistently, we show here (Fig. 8) that after full
depletion of the ER Ca2+ pool, the first two or three initial
depolarizations contribute to refill the ER with Ca2+, and
therefore the ER behaves as a sink, reducing the amount of Ca2+ available for secretion. After several K+ pulses,
the [Ca2+]ER reached the threshold for release, ~300 µM,
and was not able to take up more Ca2+ upon the following
depolarizations. This leads to more Ca2+ available for secretion and probably to the increase in catecholamine secretion. On the other hand, when the ER is allowed to refill completely, it would be able to contribute with Ca2+ to
secretion via CICR. These results are consistent with the model we proposed previously for the caffeine-sensitive
store acting as a sink or source of Ca2+ that modulates K+-evoked secretion depending on its filling state (see Fig. 10 in Lara et al., 1997
).
The importance of the ER as a possible source of Ca2+
for secretion under physiological conditions was investigated in the fast confocal experiments, that showed that
CICR contributes very significantly to the generation and
propagation of the intracellular Ca2+ wave induced by
100-ms depolarizations (Fig. 9). Depolarization-induced Ca2+ influx has been reported previously in confocal imaging studies to release Ca2+ from ryanodine-sensitive stores
in several types of neurons (Hua et al., 1993; Kocsis et al.,
1994
; Usachev and Thayer, 1997
). In experiments performed at low time resolution (2 Hz), Usachev and Thayer
(1997)
have shown that CICR facilitates all-or-none inward propagation of the [Ca2+]c signal from the plasmalemma to the nucleus in rat sensory neurons. However, in
these experiments CICR had to be facilitated with 5 mM
caffeine to be evidenced. Here we present records of
[Ca2+]c at high-time resolution in order to study whether
CICR affects Ca2+ signaling during more physiological
brief stimulations and in the absence of caffeine. It is remarkable that the presence of functional Ca2+ stores increased not only the rate of [Ca2+]c increase at points deep
inside the cell, but also doubled the increase in [Ca2+]c
near the plasma membrane (Fig. 9 c). This may indicate
the presence of RyRs coupled to voltage-dependent Ca2+
channels, a mechanism that reminds the behavior of cardiac RyR (Cheng et al., 1996
) and would make possible
that stimuli as short as the action potentials could induce
CICR. The existence of local subplasmalemmal CICR has
been proposed previously from the ryanodine sensitivity
of Ca2+-activated chloride currents (Ivanenko et al., 1993
).
The rise of [Ca2+]c was also faster in the control than in the
ryanodine-treated cells (Fig. 9 d) and, probably as a consequence of this, the Ca2+ wave moved into the interior of
the cells at greater speed in the control cells (Fig. 9 e).
Thus CICR makes the Ca2+ signal larger in magnitude and
faster to reach the inner parts of the cells. CICR may
therefore play an important role in catecholamine release
under physiological conditions, both by providing directly the Ca2+ required to trigger exocytosis and by facilitating
Ca2+-dependent early steps such as vesicle transport to the
exocytotic sites. Additionally, CICR could have functional
implications not directly related to secretion but important
for the cell physiology such as gene expression or vesicle
transport (Berridge, 1998
).
Finally, additional evidence for the physiological role of
CICR in secretion came from the finding that the pattern
of inhibition of both CICR and K+-induced secretion by
several Ca2+ channel inhibitors was similar. In bovine
chromaffin cells, Ca2+ channels are expressed in the following proportions: 10% P-type, 20% L-type, 30% N-type,
and 40% Q-type (Lopez et al., 1994; Albillos et al., 1996
).
The L- and Q-type Ca2+-channels appear to be more
closely coupled to the exocytotic machinery than the N- or
P-type ones (Lopez et al., 1994
; Lomax et al., 1997
). In addition, functional evidence suggests that the Q-type Ca2+
channels are located closer to the secretory sites than the
L-type ones (Lara et al., 1998). We find here that a combination of inhibitors of all voltage-dependent Ca2+ channels inhibited CICR by 80%, even though the initial rate of refilling of the ER was little modified. The lack of effect on the rate of refilling suggests that the small fraction of
Ca2+ entry that takes place through voltage-independent
channels is enough to sustain a normal refilling. This is not
surprising since, as shown in Fig. 4 c, the ER can refill even
without noticeable change of [Ca2+]c in BAPTA-loaded
cells. A similar behavior has been reported in non-excitable cells (Montero et al., 1997a
; Hofer et al., 1998
). In contrast, CICR appears to require rapid Ca2+ entry through
voltage-dependent Ca2+ channels, particularly P-/Q-type, as
it was inhibited ~60% by
-conotoxin MVIIC (N-/P-/Q-type
Ca2+ channel inhibitor) and
-agatoxin IVA (P-/Q-type Ca2+
channel inhibitor at the concentration used here). On the
other hand,
-conotoxin GVIA (N-type Ca2+ channel inhibitor) and nisoldipine (L-type Ca2+ channel inhibitor) had
no significant effect. The fact that the same channels that
are closer to the secretory sites (Q-type, Lara et al., 1998)
are also those responsible for triggering CICR suggests
that CICR may occur preferentially near the secretory sites. This again suggests that CICR may be important for
the secretory response under physiological conditions.
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Footnotes |
---|
Address correspondence to J. Alvarez, Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Ramón y Cajal 7, E-47005 Valladolid, Spain. Tel: (34) 983-423085. Fax: (34) 983-423588. E-mail: jalvarez{at}ibgm.uva.es
Received for publication 14 September 1998 and in revised form 4 December 1998.
M.J. Barrero was supported by a predoctoral fellowship from the University of Valladolid and I. Cuchillo is a fellow from the Fundación Teófilo Hernando. Financial support from Fondo de Investigaciones Sanitarias to J. Alvarez (96/0456) and to M.T. Alonso (96/1443), Junta de Castilla
y León to J. García-Sancho (VA 87/96), Dirección General de Enseñanza
Superior (PB97/0474) to J. García-Sancho and Dirección General de Investigación Científica y Técnica (PB94/0150) to A.G. García is gratefully acknowledged.
M.T. Alonso and M.J. Barrero contributed equally to this work.
We thank J. Fernández (University of Valladolid, Valladolid, Spain) for excellent technical assistance.
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Abbreviations used in this paper |
---|
[Ca2+]c, cytosolic [Ca2+]; [Ca2+]ER, ER [Ca2+]; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; cADPR, cyclic adenosine diphosphate ribose; CICR, Ca2+-induced Ca2+ release; CPA, ciclopiazonic acid; DMPP, 1,1-dimethyl-4-phenyl- piperazinium iodide; HSV-1, herpes simplex virus type 1; InsP3R, InsP3 receptor; InsP3, inositol 1,4,5-trisphosphate; ivu, infectious virus units; RyR, ryanodine receptor.
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References |
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