Virginia Merrill Bloedel Hearing Research Center, Department of Otolaryngology-Head and Neck Surgery, University of Washington School of Medicine, Seattle, Washington 98195
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ABSTRACT |
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Kato, B. Maya and Edwin W Rubel. Glutamate regulates IP3-type and CICR stores in the avian cochlear nucleus. Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are activated by glutamate released from auditory nerve terminals. If this stimulation is removed, the intracellular calcium ion concentration ([Ca2+]i) of NM neurons rises and rapid atrophic changes ensue. We have been investigating mechanisms that regulate [Ca2+]i in these neurons based on the hypothesis that loss of Ca2+ homeostasis causes the cascade of cellular changes that results in neuronal atrophy and death. In the present study, video-enhanced fluorometry was used to monitor changes in [Ca2+]i stimulated by agents that mobilize Ca2+ from intracellular stores and to study the modulation of these responses by glutamate. Homobromoibotenic acid (HBI) was used to stimulate inositol trisphosphate (IP3)-sensitive stores, and caffeine was used to mobilize Ca2+ from Ca2+-induced Ca2+ release (CICR) stores. We provide data indicating that Ca2+ responses attributable to IP3- and CICR-sensitive stores are inhibited by glutamate, acting via a metabotropic glutamate receptor (mGluR). We also show that activation of C-kinase by a phorbol ester will reduce HBI-stimulated calcium responses. Although the protein kinase A accumulator, Sp-cAMPs, did not have an effect on HBI-induced responses. CICR-stimulated responses were not consistently attenuated by either the phorbol ester or the Sp-cAMPs. We have previously shown that glutamate attenuates voltage-dependent changes in [Ca2+]i. Coupled with the present findings, this suggests that in these neurons mGluRs serve to limit fluctuations in intracellular Ca2+ rather than increase [Ca2+]i. This system may play a role in protecting highly active neurons from calcium toxicity resulting in apoptosis.
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INTRODUCTION |
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The viability of brain stem neurons in the
cochlear nucleus of young animals is dependent on tonic stimulation by
the auditory nerve (see Lachica et al. 1996;
Rubel et al. 1990
for reviews). In the absence of
auditory nerve stimulation, immature cochlear nucleus neurons undergo a
number of cytological changes that culminate in cellular atrophy or
death. Using the avian cochlear nucleus, nucleus magnocellularis (NM),
we have studied the early events that precede cell atrophy and death
when afferent stimulation is eliminated or transiently blocked. Our
goal is to identify the intracellular signals that are activated by
afferent stimulation and that regulate the survival of NM neurons.
One of the earliest events seen in unstimulated NM neurons is a rise in
intracellular calcium ion concentration
([Ca2+]i) (Zirpel and Rubel
1996). We believe that the deregulation of
[Ca2+]i stimulates cellular events that are
characteristic of dying neurons. Thus it is possible that
[Ca2+]i imbalance may trigger a cascade of
cell death events such as those seen in excitotoxicity (Randall
and Thayer 1992
).
We have not determined what mechanisms are responsible for causing the
elevated [Ca2+]i seen in afferent-deprived NM
neurons. In neurons, [Ca2+]i can increase in
at least two ways: influx across plasmalemma channels or efflux from
the second-messenger regulated Ca2+ storing organelles.
Because deafferented NM neurons are no longer electrically active
(Born et al. 1991), contributions to the elevated [Ca2+]i by ligand- or voltage-operated
channels seem unlikely. Numerous studies indicate that Ca2+
released from intracellular Ca2+-storing organelles plays a
major role in neuronal pathologies caused by elevated
[Ca2+]i (e.g., Frandsen and Shousboe
1992
, 1993
). In addition, cytopathologic changes that are
associated with glutamate-induced excitotoxicity (presumed to be caused
by supernormal [Ca2+]i) can be corrected
partially in neurons that have been treated with agents that
preferentially block Ca2+ release from intracellular stores
(see e.g., Yoon et al. 1996
; Zhang et al.
1993
).
These observations led to the present study, which describes the
pharmacological regulation of intracellular Ca2+ stores in
NM neurons. Ca2+ liberation from organelles is a
ligand-gated event. Two distinct receptor channels have been
identified: one is sensitive to inositol trisphosphate
(IP3), a metabolite of phosphatidylinositol (PI) hydrolysis, and the other is sensitive to Ca2+.
Anatomically distinct IP3 receptor (IP3R)
channels and Ca2+-sensitive Ca2+ channels
(called CICRs, "Ca2+-induced Ca2+
release") have been identified. Receptor-binding studies indicate that the IP3R and CICR are unique proteins
(Supattapone et al. 1988). Furthermore the organelles
that they are attached to occupy spatially distinct sites in a neuron,
indicating that the IP3R and CICR are functionally unique
(Feng et al. 1992
; Kijima et al. 1993
;
Sharp et al. 1993
; Shoshan-Barmatz et al.
1991
; Walton et al. 1991
).
Glutamate is the excitatory neurotransmitter released from auditory
nerve endings onto NM neurons. Previously we demonstrated that
glutamate significantly can reduce voltage-dependent increases in
[Ca2+]i (Lachica et al. 1995).
Further studies showed that glutamate also attenuates elevations in
[Ca2+]i that are attributed to CICR stores
(Kato et al. 1996
). One of the specific aims of the
present report was to determine whether glutamate modulates
Ca2+ increases stimulated by IP3R-activating
agents. In addition, we wanted to begin investigating the intracellular
pathways involved in glutamate regulation of intracellular stores. We
hypothesize that the elevated [Ca2+]i seen in
sensory-deprived NM neurons is partly due to the absence of
glutamate-dependent inhibition of release from intracellular Ca2+ stores.
Using video-enhanced fluorometric techniques, the present study shows that IP3- and Ca2+-sensitive intracellular stores are present in NM neurons. We demonstrate that glutamate activation of metabotropic receptors reduces changes in [Ca2+]i due to activation of IP3R- and CICR-regulated stores. Interestingly, changes in [Ca2+]i due to IP3R and CICR stores appear to be regulated by second-messenger systems that do not attenuate the Ca2+ increases carried by voltage-operated channels.
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METHODS |
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Tissue preparation
White leghorn chicken embryos, aged 17-18 days, were removed from their shells and decapitated. The brain stems were rapidly dissected out and blocked above and below the level of the cochlear nucleus. Specimens were placed into 5% low-melting-point agarose dissolved in artificial cerebrospinal fluid (ACSF) for 3 min. The ACSF was composed of (in mM) 125 NaCl, 5 KCl, 1.25 KH2PO4, 1.3 MgCl2, 26 NaHCO3, 10 dextrose, and 3.1 CaCl2. When the agarose solidified, a square block containing the brain stem was cut out and glued to the stage of a vibratome. The entire block then was immersed in cold ACSF.
Coronal brain stem slices (300 µM) through NM were made. Slices were loaded with 5 µM FURA-2 acetoxymethyl ester (FURA-2), dissolved in a 0.1% dimethylsulfoxide, 0.025% pluronic acid, ACSF solution. The tissue slice approximately mid way through NM was incubated in the oxygenated FURA-2/ACSF solution at 40°C for 25 min. The slice then was rinsed in fresh ACSF and prepared for imaging. Imaging experiments were conducted at room temperature.
Drug application
FURA-2-loaded brain stem slices were placed in a Leiden-style chamber on an inverted microscope stage and anchored to the floor of the chamber with a stainless steel net. Pharmacological agents were applied to the slices in bath application via a stainless steel capillary inlet tube (1-mm diam) positioned on top of the net. A second capillary tube connected to suction rested on the floor of the chamber, establishing laminar flow of the superfusate at a rate of ~3 ml/min. Specimens were superfused continuously with oxygenated solution throughout the experiment. Media reservoirs containing the solutions were attached to the capillary tube delivering the superfusate by a length of plastic tubing ~30 cm long. As a result, there was a delay between the time the solutions were changed at the level of the reservoir and the time that the solution in the test chamber containing the specimen was completely changed. This delay was ~30 s and is not corrected for in the figures.
Pharmaceuticals
Fresh solutions of ACSF and Ca2+-free ACSF were made daily. The Ca2+-free ACSF was identical in composition to the normal ACSF with the exception that it lacked CaCl2 and contained 1 mM EGTA and 4.4 mM MgCl2. At the beginning of each experiment, neurons typically were depolarized pharmacologically with 60 mM KCl in ACSF to assess cell viability. All other drugs were delivered in Ca2+-free ACSF. These agents can be grouped into six general categories as follows: drugs that stimulate Ca2+ release from intracellular stores: homo-bromo-ibotenate (HBI) and caffeine; agents that block Ca2+ release from stores: TMB-8 hydrochloride (TMB8) and ryanodine; glutamate and its receptor agonists: trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), trans-azetidine dicarboxylic acid (t-tADA), L(+)-2-amino-3-phosphonopropionic acid (AP3), L(+)-cysteine sulfinic acid (L-CSA), kainic acid (KA); glutamate receptor antagonists: (±)-a-methyl-4-carboxyphenylglycine (MCPG); protein kinase activators: Sp-cAMPS triethylamine (Sp-cAMPS) and phorbol 12-myristate 13-acetate (PMA); and protein kinase inhibitors: H7 dihydrochloride (H7) and H8 dihydrochloride (H8).
Microfluorometry
NM neurons were excited alternately by 340 and 380 nm
wavelengths from a xenon source (Ushio). Excitation wavelengths were obtained using interference filters from Chroma Technology
(Brattleboro, VT). Emitted light was passed through a ×40 fluor
oil-immersion objective (Nikon) attached to a Nikon Diaphot inverted
microscope, through a 480-nm long-pass exit filter, and finally into an
image intensifier coupled to a CCD camera (Hamamatsu, Japan). Cells were exposed to UV light, attenuated to 3% its normal intensity by
neutral density filters, during data-collection periods only. Exposure
time for each wavelength was 500-750 ms controlled via a
computer-controlled shutter and filter wheel (Sutter Instruments, Novato, CA). Paired images were captured every 3 s. Data were obtained by comparing the intensity of fluorescent emission to 340- and
380-nm excitation wavelengths. The difference in emitted fluorescence
was expressed as a ratio (F340:F380) that was compared with a standard
curve for free Ca2+ constructed from solutions of known
Ca2+ and FURA-2 concentrations. As a result, ratios of
fluorescent intensity were translated directly to Ca2+
concentrations using software designed by Universal Imaging (West Chester, PA). The Kd of hydrolyzed FURA-2 for
Ca2+ was assumed to be 224 nM (Grynkiewicz et al.
1985). Numerical values reported are an average of >50
adjacent pixels for each cell. All results are presented as mean change
in [Ca2+]i ± 1 SE. The total number of
neurons and number of slices that were tested in each experiment are
indicated parenthetically. Typically 10-13 neurons were studied in
each slice. For statistical comparisons, a conservative approach of
computing the mean value of all neurons in a slice, and using that
value as a single observation was used.
Materials
FURA-2 was obtained from Molecular Probes (Eugene, OR), glutamate was purchased from Sigma, and L-CSA and HBI were acquired from Tocris-Cookson (St. Louis, MO). All other pharmaceuticals were bought from Research Biochemicals (Natick, MA) and the remaining reagents were of analytic grade.
Statistics statistical analyses were performed using Student's t-test or one-way ANOVA, as required. Post hoc analyses were computed using Scheffe's test.
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RESULTS |
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NM neurons possess two types of intracellular Ca2+ stores: IP3-sensitive stores and CICR stores
At least two pharmacologically distinct types of intracellular stores have been described: IP3-sensitive stores and Ca2+-sensitive CICR stores. In this study, HBI, a metabotropic glutamate receptor agonist linked to the phospholipase C (PLC) signal transduction cascade, was used to stimulate calcium release from IP3-sensitive stores, and caffeine was used to stimulate CICR stores.
Figure 1 shows the changes in
[Ca2+]i seen in six NM neurons stimulated
with 500 µM HBI followed by 100 mM caffeine. The doses used have been
shown to reliably stimulate large increases in [Ca2+]i in NM neurons (Kato et al.
1996; Lachica et al. 1998
). Before stimulation,
the cells were given a 3-min washout period in Ca2+-free
ACSF with 1 mM EGTA. HBI and caffeine were delivered in Ca2+-free ACSF. Therefore elevations in
[Ca2+]i elicited by these agents can be
attributed to Ca2+ mobilized from intracellular stores and
are not due to calcium influx through the plasma membrane.
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On the average (72 neurons, n = 6 slices), 500 µM HBI
increased [Ca2+]i by 530 ± 55 nM. The
mean [Ca2+]i (75 neurons,
n = 6 slices) produced by caffeine was 331 ± 51 nM. Both of these values differ significantly from fluctuations in
basal [Ca2+]i levels in these neurons, which
varies ~100 nM. We observed that HBI-and caffeine-stimulated
Ca2+ responses differed in their spatial and temporal
characteristics. Elevations in [Ca2+]i evoked
by HBI developed slowly to a maximum and were buffered over a
protracted period of time. These HBI-stimulated increases in
[Ca2+]i developed and decayed synchronously
in response to the presentation and withdrawal of the HBI stimulus. In
contrast, the caffeine-stimulated Ca2+ responses occurred
asynchronously between NM neurons in a single brain stem slice. These
Ca2+ transients rose rapidly and were buffered quickly in
the presence of continued caffeine superfusion. The caffeine-stimulated
Ca2+ responses usually were distinguished by a
"postcaffeine undershoot"
a period immediately after the
caffeine-stimulated
[Ca2+]i where
Ca2+ levels sink below resting levels (Kato et al.
1996
; Usachev et al. 1993
). The HBI-treated
neurons never demonstrated this undershoot.
The effects of two agents that block Ca2+ release from
intracellular stores, TMB8 and ryanodine, were tested on HBI- and
caffeine-stimulated cells. HBI-stimulated Ca2+ increases
(Fig. 2) were attenuated significantly
([Ca2+]i = 85 ± 7 nM; P < 0.001) when neurons were pretreated with 100 µM TMB8 for 5 min (17 neurons, n = 3 slices). However, exposure to 100 µM
ryanodine did not significantly affect HBI-stimulated Ca2+
responses ([Ca2+]i = 400 ± 47 nM; 42 neurons = 4 slices). Another set of cells was stimulated with
caffeine after exposure to 100 µM concentrations of either ryanodine
or TMB8. Although both agents significantly attenuated
caffeine-stimulated Ca2+ responses, the actions of
ryanodine were greater than those of TMB8. The average
[Ca2+]i seen in ryanodine-treated,
caffeine-stimulated neurons was 57 ± 16 nM (71 neurons,
n = 6 slices). This is much less than the
[Ca2+]i to caffeine alone
(P < 0.0001). The average
[Ca2+]i seen in TMB8-treated
caffeine-stimulated neurons was 103 ± 27 nM, and it is
significantly smaller than in slices treated with caffeine alone
(t = 8.4; P < 0.01).
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Glutamate modulates Ca2+ release from intracellular stores
In vivo, Ca2+ mobilized from IP3-sensitive
or CICR stores should be linked to the release of glutamate from the
auditory nerve terminal. However, in NM, glutamate does not appear to
stimulate a [Ca2+]i at the concentrations
that have been shown to reliably stimulate increases
[Ca2+]i in other neurons (Lachica et
al. 1995
, 1998
). Millimolar concentrations are required to
raise [Ca2+]i in NM neurons, and even at
these concentrations, glutamate-stimulated elevations in
[Ca2+]i due to the influx or efflux of
Ca2+ are variable (Lachica et al. 1998
;
Zirpel et al. 1995b
). We previously demonstrated that
glutamate significantly attenuates increases in
[Ca2+]i in KCl-stimulated NM neurons
(Lachica et al. 1995
), and it is possible that glutamate
has a similar effect on Ca2+ mobilized from intracellular
stores. The remainder of this report is directed at evaluating this
glutamate effect on IP3R stores and further analyzing its
effect on CICR-stores (see Kato et al. 1996
).
Figure 3A shows that large
increases in [Ca2+]i were elicited repeatedly
in NM neurons by multiple exposures to 500 µM HBI. Thus
IP3R stores are not labile and the release of
Ca2+ from IP3R stores is not dependent on the
store being "reloaded" with Ca2+. When 1 mM glutamate
is applied to the slice, the HBI-stimulated [Ca2+]i transients are reduced markedly (Fig.
3B) This effect lasts for several minutes. Figure
4 shows that the suppressive effect of
glutamate on Ca2+ responses from intracellular stores is
dose dependent. At a concentration of 1.0 mM, glutamate maximally
reduced HBI-stimulated Ca2+ changes by 460 nM (Fig. 4); a
half-maximal effect was produced by 100 µM glutamate. Glutamate also
attenuates caffeine-stimulated changes in
[Ca2+]i (see Kato et al. 1996,
Fig. 2C). This effect is also dose dependent. The
caffeine-stimulated
[Ca2+]i was reduced
most efficaciously by 100 µM glutamate. Interestingly, 1 and 10 mM
glutamate did not suppress the caffeine response to the same degree as
100 µM glutamate. In fact, 10 mM glutamate did not have a
statistically significant effect on caffeine-stimulated calcium
responses (see also Kato et al. 1996
, Fig.
2D).
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Glutamate effects on Ca2+ responses produced by HBI and
caffeine were mimicked by the mGluR agonist t-ACPD. The t-ACPD
reduction of HBI-stimulated transients were also dose dependent (Fig.
4). Maximal reduction of the HBI response was elicited by 100 µM
t-ACPD. The [Ca2+]i seen in HBI-stimulated
neurons (54 neurons; n = 4 slices) exposed to 100 µM
t-ACPD was only 57 ± 11 nM. This same concentration of t-ACPD was
also the most effective in reducing caffeine-stimulated transients. The
[Ca2+]i of caffeine-stimulated neurons (41 neurons; n = 4 slices) treated with 100 µM t-ACPD was
108 ± 19 nM. These dose-dependent effects of glutamate and t-ACPD
on Ca2+ stores may be due to receptor downregulation,
receptor desensitization, or to other second-messenger activation.
Again, it is important to note that higher concentrations of t-ACPD did
not have an inhibitory effect on HBI- or caffeine-stimulated responses.
For example, 1 mM t-ACPD potentiated the
[Ca2+]i stimulated by HBI.
Caffeine-stimulated responses neither were potentiated nor inhibited by
1 mM t-ACPD. The fact that the higher concentrations of glutamate and
t-ACPD do not inhibit HBI- and caffeine-stimulated responses is
important. These findings suggest that the suppressive effect of
glutamate and t-ACPD is not due to competitive binding at the level of
the mGluR.
To determine whether the suppressive effect of glutamate on
intracellular Ca2+ stores was modulated specifically by a
metabotropic receptor, the effect of the ionotropic receptor agonist,
kainic acid (KA; 50 µM), was tested on a separate set of neurons
stimulated with HBI (37 neurons; n = 3 slices) or
caffeine (45 neurons; n = 4 slices). This dose of
kainate used has been shown to effectively stimulate the
KA/-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
without being toxic to the cell (Lachica et al. 1998
).
The HBI- and caffeine-stimulated Ca2+ responses of the
KA-treated neurons did not differ from normal (data not shown).
The mGluR antagonist, MCPG, partially reversed glutamate effects on
IP3R and CICR stores (Fig. 5,
A and B). These results are shown in Fig. 5,
A and B, respectively. Fifty-two neurons (n = 4) were stimulated with 500 µM HBI immediately
after the coapplication of 1 mM MCPG and 1 mM glutamate. The mean
[Ca2+]i measured in these neurons was
246 ± 22 nM. Although this value was much smaller than the
response to HBI in untreated neurons (Scheffe's test,
P < 0.0001), it was significantly greater than the
rise in
[Ca2+]i stimulated by HBI in
neurons exposed only to glutamate (P < 0.01).
Therefore it could be argued that the mGluR antagonist, MCPG, partially
blocks the suppressive effect of glutamate. The effects of MCPG on
glutamate inhibition of caffeine responses were tested on a separate
group of cells (43 neurons; n = 4 slices). The mean
[Ca2+]i measured in these neurons was
205 ± 31 nM. Although this value was not statistically different
from the response to caffeine seen in untreated neurons, it also did
not differ significantly from the responses of caffeine-stimulated
cells that were exposed only to glutamate.
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Effects of kinase inhibitors and activators on HBI-stimulated responses
Metabotropic glutamate receptors act mainly by modulating the
effectors of one of two pathways: the phospholipid/Ca2+
signaling pathway or the adenylate cyclase/cyclic AMP transduction system. Preliminary attempts have been made to determine the signaling pathways underlying mGluR regulation of calcium-storing organelles. We
examined the Ca2+ responses of HBI-stimulated neurons after
they were exposed to the following conditions: coapplication of 1 mM
glutamate and 100 µM H7, an inhibitor of protein kinase C or
coapplication of glutamate and 100 µM H8, an inhibitor of protein
kinase A. The doses of H7 and H8 have been proven to be effective in
inhibiting protein kinases (e.g., Chik et al. 1995;
Glaum and Miller 1995
). In addition, we examined
responses to HBI in the presence of 1 µM PMA, a C-kinase-activating
phorbol ester and in the presence of 1 mM sp-cAMP, an analogue of
A-kinase-activating cAMP. These agents were tested in the absence of
glutamate stimulation. The results of these experiments are summarized
in Fig. 6.
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HBI-stimulated [Ca2+]i was examined in 60 neurons (n = 4 slices) exposed to glutamate and H7 and
in 66 neurons (n = 4 slices) exposed to glutamate and
H8 (Fig. 6A). The mean
[Ca2+]i
seen in Glu-H7-treated neurons was 313 ± 36 nM, and the mean
[Ca2+]i seen in Glu-H8 treated neurons was
42 ± 5 nM. It is important to note that the responses of
Glu-H7-treated slices were greater than the
[Ca2+]i of HBI-stimulated neurons exposed
solely to glutamate (P < 0.0001). In contrast, the
Ca2+ responses of Glu-H8-treated neurons did not differ
significantly from the responses of neurons treated with glutamate
alone. These data indicate that H7 partially reversed the effects of
glutamate. An alternative explanation is that HBI-stimulated
Ca2+ responses may be attenuated by an increase in C kinase
activity. As a control, additional slices were tested with H7 or H8
alone (without glutamate) and were not found to alter resting
[Ca2+]i (data not shown).
The actions of PMA and Sp-cAMPs on HBI-stimulated
[Ca2+]i were also very different (Fig.
6B). PMA reduced HBI-stimulated responses by ~90% to
48 ± 6 nM (59 neurons; n = 4 slices). Sp-cAMPS,
however, did not significantly alter HBI-stimulated
[Ca2+]i. On the average, HBI evoked a
455 ± 51 nM
[Ca2+]i in Sp-cAMPs
treated neurons (54 neurons; n = 4 slices).
Effects of kinase inhibitors and activators on caffeine-stimulated responses
We performed a parallel set of experiments to assess the effects
of the protein kinase inhibitors and activators on caffeine-stimulated [Ca2+]i. Caffeine-stimulated responses
were examined in 77 neurons (n = 4 slices) treated with
H7 alone and 67 neurons (n = 4 slices) treated with H8
alone. The
[Ca2+]i in H7-treated neurons
was 135 ± 19 nM and 151 ± 14 nM in H8-treated cells. These
values did not differ from one another nor did they differ from the
Ca2+ measured in caffeine-stimulated neurons treated
only with glutamate. These results suggest that both H7 and H8, acting
by themselves, attenuate caffeine-stimulated Ca2+ responses
as effectively as glutamate.
Fifty-two neurons (n = 4 slices) were exposed to
glutamate and H7 and 68 neurons (n = 4 slices) were
exposed to glutamate and H8 before stimulation with caffeine (Fig.
7A). The
[Ca2+]i measured in Glu-H7-treated neurons
was 163 ± 28 nM. This value is 50% smaller than that measured in
the cells treated only with caffeine and is not significantly different
from the
[Ca2+]i in caffeine-stimulated
neurons exposed to glutamate or H7 alone. The
[Ca2+]i measured in Glu-H8-treated neurons
was 27 ± 7 nM. This is significantly smaller than the
caffeine-stimulated
[Ca2+]i in cells
treated with glutamate (P < 0.0001), demonstrating that H8 potentiates the inhibitory effects of glutamate on CICR stores.
This suggests that glutamate and H8 may act through different mechanisms to suppress Ca2+ release from CICRs.
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The Ca2+ responses of 61 caffeine-stimulated neurons
(n = 4 slices) that were exposed to 1 µM PMA were
compared with Ca2+ responses of 63 caffeine-stimulated
neurons (n = 4 slices) exposed to 1 mM Sp-cAMPS. The
actions of PMA and Sp-cAMPS on caffeine-stimulated Ca2+
responses were nearly identical (Fig. 7B). Both agents
suppressed caffeine-stimulated responses. The mean caffeine-stimulated
[Ca2+]i seen in PMA-treated neurons was
122 ± 17 nM. The
[Ca2+]i in neurons
treated with Sp-cAMPS and caffeine was 128 ± 12 nM.
Effects of other mGluR-stimulating agents
mGluRs can be activated by a number of agents. We evaluated the
effects of L-cysteine sulfinic acid (L-CSA),
L-2-amino-3-phosphonopropionic acid (L-AP3),
and trans-azetidine dicarboxylic acid (t-ADA) on HBI and
caffeine-stimulated Ca2+ responses. L-CSA has
been shown to stimulate phospholipase D and cyclic AMP activities
(Boss and Boaten 1995; Boss et al. 1994
). L-AP3 has been shown to block and stimulate mGluRs
(Irving et al. 1990
; Lonart et al. 1992
;
Manev et al. 1993
; Saugstad et al. 1995
).
t-ADA is believed to be a selective agonist of a mGluR coupled to
phosphoinositide hydrolysis. Each agent was tested at concentrations of
1, 100, and 1,000 µM. HBI- and caffeine-stimulated Ca2+
responses were not inhibited by any of these agents.
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DISCUSSION |
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It is well known that tight regulation of intracellular free
calcium levels is essential to cellular survival. Neurons and other
excitable cells are challenged particularly in this respect because
their responses to extrinsic stimuli result in calcium influxes due to
activation of membrane channels and calcium or release from internal
stores. Fluctuation in levels of intracellular calcium resulting from
short periods of high levels of stimulation usually are bound,
sequestered, or efficiently extruded from the cell. In very active
neurons, however, calcium levels could rise rapidly to toxic levels
without cellular machinery limiting its accumulation. Auditory system
neurons of birds and mammals are known for their high levels of
intrinsic activity. For example, many eighth nerve neurons have
spontaneous firing rates of 50-100 spikes/s in the absence of acoustic
stimulation (Liberman 1978) and rates may rise to levels
four to five times that level during an acoustic stimulus. Nucleus
magnocellularis neurons also show very high rates of ongoing activity,
averaging ~100 spikes/s in the absence of stimulation and 300-400
spikes/s during stimulation (Rubel and Parks 1975
;
Stopp and Whitfield 1961
; Warchol and Dallos 1990
). Furthermore we have shown previously that in the absence of eighth nerve activity, intracellular-free calcium levels in NM
neurons rise three- to fourfold within 3 h (Zirpel and
Rubel 1996
; Zirpel et al. 1995a
). This time
period corresponds to deprivation-induced degenerative events that
result in cell death and atrophy (Kelley et al. 1997
;
Rubel et al. 1990
). Consequently, we recognized that NM
neurons, and presumably other highly active neurons, need specialized mechanisms for limiting calcium accumulation and that these mechanisms should respond to synaptic stimulation. In a previous study, we showed
that a pathway involving one or more metabotropic glutamate acting
through A kinase inhibits calcium entry through low-voltage-activated channels (Lachica et al. 1995
). This study investigates
intracellular Ca2+ storing organelles in NM neurons and
their regulation by glutamate. It describes the mechanisms underlying
glutamate regulation of IP3R-type Ca2+-storing
organelles. In addition, this study further analyzes glutamate
modulation of CICR-type Ca2+-storing organelles (see
Kato et al. 1996
), focusing on the intracellular pathways involved in this regulation.
NM neurons possess both IP3R- and CICR-type Ca2+-storing organelles. Changes in [Ca2+]i linked to the activation of IP3Rs and CICRs differ in a number of ways. Ca2+ released from IP3R stores is reduced by TMB8 but not by ryanodine. CICR stores are sensitive to the effects of both of these agents but are particularly sensitive to ryanodine. The temporal characteristics of Ca2+ responses attributable to IP3Rs and CICRs are also different. Within a sample of neurons treated with HBI, [Ca2+]i increased and decreased in conjunction with the presence and withdrawal of HBI. On the other hand, it was difficult to predict exactly when a given NM neuron would respond to caffeine. HBI-stimulated Ca2+ transients were buffered over a protracted period of time, whereas caffeine-stimulated transients were buffered rapidly and the [Ca2+]i transiently sank slightly below prestimulus basal [Ca2+]i.
Calcium increases stimulated by HBI and caffeine were both inhibited by
glutamate, the neurotransmitter released at the auditory nerve ending,
and by t-ACPD, a mGluR agonist (Favaron et al. 1993). The inhibitory effect of glutamate and t-ACPD on HBI- and
caffeine-stimulated responses were long lasting, reversible and dose
dependent. These results suggest that stimulation of one or more mGluRs
triggers intracellular events that attenuate calcium increases
originating from calcium-storing organelles. t-ACPD usually activates
an IP3-dependent increase in cell calcium by stimulating
the hydrolysis of PIP2 (Palmer et al. 1988
).
However, at the concentrations used to inhibit IP3R and
CICR responses, t-ACPD does not cause a
[Ca2+]i in NM neurons (Lachica et
al. 1998
). This finding indicates that t-ACPD does not
exclusively stimulate phospholipase C activity in NM neurons. The
identity of the effectors that are mGluR modulated in NM neurons is not
yet known.
It could be argued that the glutamate effect on HBI-stimulated
transients is due to receptor desensitization, given that these two
agents may act on the same family of binding sites. There are several
observations that suggest that this cannot explain the results
presented. First, HBI repeatedly stimulates calcium increases (Fig.
3A). Second, glutamate and t-ACPD have similar inhibitory
effects on caffeine-stimulated responses (which are not linked to a
membrane-affiliated receptor). Third, the dose-dependent effects of
glutamate and t-ACPD are not linearhigher concentrations of glutamate
and t-ACPD were less efficacious than lower concentrations (Fig. 4).
Finally, previous studies have shown that HBI stimulates phosphoinositide hydrolysis by binding to mGluRs other than those sensitive to ACPD (Chung et al. 1994
; Littman et
al. 1995
; Thomsen et al. 1994
).
We recently have conducted and presented the results of preliminary
experiments using caged IP3 to directly stimulate calcium
release from IP3-sensitive stores (Kato and Rubel
1998). Brain stem slices were incubated in caged IP3 and calcium fluxes were studied with Oregon Green. All
experiments were performed in calcium-free media, buffered with 1 mM
EGTA. In most neurons, photolysis of the cage resulted in large
increases in the intensity of the fluorophore, indicating an elevation
in [Ca2+]i elicited by release of
IP3. In the presence of 1 mM glutamate, these calcium
elevations were suppressed dramatically. These preliminary data support
the results presented above and provide further support for the concept
that the glutamate-stimulated cascade directly inhibits calcium release
from IP3-sensitive stores.
Protein kinase C inhibits IP3 responses in NM
Preliminary studies examining the roles of A and C kinase pathways in the inhibition of the IP3 response have led to a relatively clear picture. Ca2+ responses attributable to IP3Rs were reduced by PMA, a C kinase activator. They were unaffected by Sp-cAMPS, a cyclic AMP analogue that activates cyclic AMP-dependent protein kinase I and II. The role of C kinase is strengthened by experiments using the kinase inhibitor, H7. Glutamate reduced HBI-stimulated responses by 86%; when coapplied with H7, responses were reduced by only 41%. Because H7 did not alter HBI responses by itself, it can be concluded tentatively that the glutamate inhibitory effects on IP3Rs are reversed partially when C kinase activity is attenuated.
The effects of protein kinase C (PKC) on HBI-stimulated responses is of
interest because PKC is activated by diacylglycerol (DAG), the second
major metabolite of PIP2 hydrolysis. PMA reduces the HBI
response by 90%. Conventionally, C kinase activation is thought to
result from a collaborative interaction between DAG and
Ca2+ released from IP3R stores. The
collaboration is synergistic but is not obligatory. In NM neurons, the
DAG branch of the cascade may be particularly sensitive. This may
explain why IP3-regulated Ca2+ transients are
so rarely observed in glutamate-stimulated NM neurons, as suggested by
the large concentrations of glutamate required to elicit an increase in
[Ca2+]i in calcium-free superfusate
(Zirpel et al. 1995b).
The inhibitory effects of PKC on IP3-regulated
Ca2+ signaling have been observed previously in a number of
cell types, including neurons (Shimizu et al. 1993). PKC
can alter [Ca2+]i in several ways. Because
the modulatory domain of the IP3 receptor is a substrate
for several protein kinases (Supattapone et al. 1988
),
including PKC (Ferris et al. 1991
), PKC may attenuate
Ca2+ efflux via receptor phosphorylation. PKC also may act
farther down stream in the cascade by promoting Ca2+
sequestration (Nishi et al. 1994
; Shivan and
Alexander 1995
; Werth et al. 1996
). PKC also
acts up stream, preventing PIP2 hydrolysis (Luo et
al. 1995
; Shimizu et al. 1993
).
A reduction in the rate of IP3 production appears to be the
least likely explanation for the glutamatergic inhibition of
Ca2+ signaling. Previous studies of NM neurons demonstrated
that concentrations of glutamate and t-ACPD that do not themselves
affect [Ca2+]i stimulate an increase in
the levels of PI metabolites (Zirpel et al. 1994
,
1995b
).
Kinase modulation of CICR responses in NM neurons
Clearly, additional pharmacological studies are needed to understand the regulation of CICR stores in NM neurons. Our preliminary experiments yielded a confusing picture. Every agent used in this study that modulated kinase activity inhibited CICR-transients by ~60%, which is equivalent to the amount of reduction affected by glutamate and t-ACPD. The paradoxical combinatory effects of glutamate and H8 suggest that CICRs in NM are subject to modulation by multiple mechanisms.
It is difficult to draw any conclusions from these data, as these
results are incompatible with existing information on the CICRs in
neurons. For example, in other neurons, caffeine-stimulated responses
have been potentiated by PKA phosphorylation (Yoshida et al.
1992). In NM, the A kinase activator Sp-cAMPS inhibited caffeine responses. The inhibitory effects of PMA on CICRs were equally
surprising and may be explained by Ca2+ sequestration, not
receptor modulation, because the brain CICR is thought to be a poor
substrate for PKC phosphorylation (Guerrini et al. 1995
;
Takasago et al. 1991
; Witcher et al.
1992
; Yoshida et al. 1992
).
Complicating matters further is the fact that caffeine, independent of
its Ca2+-liberating abilities, reduces phosphodiesterase
activity (Sawynok and Yaksh 1993). Thus all of the
manipulations could be confounded by this effect. Ultimately, our
understanding of the signaling pathways responsible for the CICR
regulation may be dependent on the discovery of agents that exclusively
stimulate the Ca2+ release from CICRs without directly
activating other second-messenger activity.
Glutamate attenuates any increase [Ca2+]i via multiple mGLuRs
We previously have shown that glutamate inhibits
[Ca2+]i increases stimulated by KCl and KA.
It is important to note that the [Ca2+]i
increases produced by KCl and KA are carried by different channels. Voltage-dependent changes in [Ca2+]i are
carried mainly by dihydropyridine sensitive voltage-operated channels
(Lachica et al. 1995). Changes in
[Ca2+]i stimulated by KA are carried by a
Ca2+ permeable
non-N-methyl-D-aspartate (non-NMDA) receptor
channels (Lachica et al. 1996
). Interestingly, the
increases in [Ca2+]i carried voltage operated
calcium channels or Ca2+ permeable non-NMDA receptor
channels are reduced by agents that stimulate protein kinase A
activity. Phorbol esters, which inhibit [Ca2+]i increases from stores, do not affect
KCl- or KA-stimulated responses. Two conclusions may be drawn from
these results when they are paired with the observations described in
the present report. First, NM neurons must possess at least two
distinct mGluRs, one that activates adenylate cyclase and a second that
activates phospholipase C. Second, NM neurons use somewhat separate,
parallel pathways to inhibit increases in Ca2+ originating
from the exterior and the interior of the cell. Currently, mGluRs have
been linked directly to the PLC, adenylate cyclase, and phospholipase D
(PLD) transduction systems. L-CSA, an agent that
potentiates PLD and cAMP activities (Boss and Boaten
1995
; Boss et al. 1994
), did not inhibit
Ca2+ responses stimulated by HBI or caffeine. We have not
determined whether L-CSA alters transmembrane-related
[Ca2+]i.
Ca2+ stores may contribute to supranormal [Ca2+]i levels in dying neurons
The present study was stimulated by the observation that
[Ca2+]i rapidly increases in NM neurons
deprived of excitatory afferent activity (Zirpel et al.
1995a) and that this effect is prevented by orthodromic
stimulation of the auditory nerve (Zirpel and Rubel 1996
). We previously have demonstrated that manipulations that prevent this increase also prevent the early events associated with
deprivation-induced cell death and neuronal atrophy (Hyson and
Rubel 1989
, 1995
). We hypothesize that changes in
[Ca2+]i are an obligatory step in these and
other deprivation-induced neuronal changes
(Hartlage-Rübsamen and Rubel 1996
; Hyson
and Rubel 1989
; Lachica et al. 1995
;
Zirpel and Rubel 1996
).
We do not know why [Ca2+]i increases in
deafferented neurons. A number of possible explanations exist.
[Ca2+]i may increase as a result of
deregulation of intracellular Ca2+ stores. This may be due
to the loss of inhibition of Ca2+ release from these stores
that normally is effected by glutamate. In deafferented NM neurons,
Ca2+ stores still may be activated via the projections from
the superior olivary nuclei, which contain GABAergic neurons
(Lachica et al. 1994). GABA depolarizes NM neurons
(Hyson et al. 1995
) and stimulates increases in
[Ca2+]i (Lachica et al. 1998
).
In NM, as in other neurons, the GABA-stimulated
[Ca2+]i is sensitive to dihydropyridines
and agents that block Ca2+ release from intracellular
stores (Berninger et al. 1995
; Ito et al.
1995
; Nilsson et al. 1993
; Parramon et
al. 1995
; Spergel et al. 1995
) In fact, a large
component of the GABA-stimulated rise in Ca2+ can be
eliminated by the IP3R antagonist, heparin (Lachica
et al. 1998
). Thus in deafferented neurons, GABA-linked
IP3R-stimulated Ca2+ changes could increase to
levels that typically are kept in check by a mGluR. The
IP3-associated Ca2+ increase could be
potentiated further by CICR stores, which also would be
"glutamate-liberated" in deafferented neurons, resulting in a
supernormal level of [Ca2+]i that triggers
cytopathologic events.
The central role assigned to the Ca2+ storing organelles in
the scenario just described is not unusual. It has become apparent that
pathological increases in cell Ca2+ are not always caused
by Ca2+ influx. Ca2+ mobilization from stores
triggers apoptosis in HL-60 promyelocytic leukemia cells, and MCF-7
estradiol-receptor sensitive breast tumor cells (Vandewalle et
al. 1995; Zhu and Loh 1995
). Agents that
specifically block Ca2+ release from stores have been shown
to rescue or prevent glutamate toxicity in cortical neurons
(Frandsen and Schousboe 1992
, 1993
) and ameliorate the
effects of ischemia and other toxic agents in hippocampal neurons
(Yoon et al. 1996
; Zhang et al. 1993
). Clearly the role of intracellular stores in the regulation of [Ca2+]i and their contribution to the
cytopathology exhibited by deafferented NM neurons requires further investigation.
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ACKNOWLEDGMENTS |
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The authors thank Dr. Edward Lachica for many helpful discussions and technical help and M. Russo for editing and clerical help.
This research was supported by National Institute of Deafness and Other Communications Disorders Grants DC-00520 and DC-00018.
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FOOTNOTES |
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Address for reprint requests: E. W Rubel, The Virginia Merrill Bloedel Hearing Research Center, University of Washington, Box 357923, Seattle, WA 98195-7923.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 November 1997; accepted in final form 18 December 1998.
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REFERENCES |
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