1Curriculum in Neurobiology and 2Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Fickbohm, David J. and Alan L. Willard. Upregulation of calcium homeostatic mechanisms in chronically depolarized rat myenteric neurons. Perturbations of intracellular Ca2+ ion concentration ([Ca2+]i) have important effects on numerous neuronal processes and influence development and survival. Neuronal [Ca2+]i is, in large part, dependent on activity, and changes in activity levels can alter how neurons handle calcium (Ca). To investigate the ability of neuronal Ca homeostatic mechanisms to adapt to the persistent elevation of [Ca2+]i, we used optical and electrophysiological recording techniques to measure [Ca2+]i transients in neurons from the rat myenteric plexus that had been chronically depolarized by growth in culture medium containing elevated (25 mM) KCl. When studied in normal saline, neurons that had previously been chronically depolarized for 3-5 days had briefer action potentials than control neurons, their action potentials produced smaller, more rapidly decaying increases in [Ca2+]i, and voltage-clamp pulses with action potential waveforms evoked smaller Ca currents than in control neurons. Simultaneous voltage-clamp measurements and calcium imaging revealed that increases in the Ca handling capacities of the chronically depolarized neurons permitted them to limit the amplitudes of action potential-evoked [Ca2+]i transients and to restore [Ca2+]i to basal levels more rapidly than control neurons. Release of Ca from endoplasmic reticulum-based Ca stores made smaller contributions to action potential-evoked [Ca2+]i transients in chronically depolarized neurons even though those neurons had larger caffeine-releasable Ca stores. Endoplasmic reticulum-based Ca sequestration mechanisms appeared to contribute to the faster decay of [Ca2+]i transients in chronically depolarized neurons. These results demonstrate that when neurons experience prolonged perturbations of [Ca2+]i, they can adjust multiple components of their Ca homeostatic machinery. Appropriate utilization of this adaptive capability should help neurons resist potentially lethal metabolic and environmental insults.
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INTRODUCTION |
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Normal cell functioning requires that
intracellular homeostatic mechanisms maintain
[Ca2+]i within a narrow range that separates
normal signaling from toxicity and death. Different neuronal cell types
are likely to differ in their abilities to regulate
[Ca2+]i because they experience a broad range
of Ca influx depending on their patterns of synaptic inputs and
electrical activity. Neurons can regulate
[Ca2+]i by altering any or all of the
following: influx of extracellular Ca via ligand or voltage-gated
channels, release and/or sequestration of Ca by intracellular stores,
or buffering and efflux that occurs after Ca entry (Carafoli
1987; Kostyuk and Verkhratsky 1994
;
Miller 1991
). For the purposes of this paper, the Ca
homeostasis components are divided into two groups based on their time
scale and effect on activity-dependent
[Ca2+]i transients: fast Ca
handling mechanisms, including Ca influx through Ca channels, Ca
buffering by Ca-binding proteins, and Ca release from intracellular
stores, act on the millisecond to second time scale to determine the
amplitude of [Ca2+]i transients, whereas
slow Ca handling processes, such as Ca sequestration and
efflux, act over several seconds to control the recovery of
[Ca2+]i transients.
Understanding how neurons adapt their Ca homeostatic capabilities in response to perturbations of [Ca2+]i is important for gaining insight into the ability of neurons to withstand potentially toxic challenges that can occur as a result of trauma, disease, or oxygen deprivation. In addition, knowledge of the processes underlying the adaptive capabilities of Ca homeostatic mechanisms may help us understand the changes that may occur during the aging of the nervous system. However, at present, relatively little is known about how (or whether) the regulation of the diverse cellular Ca handling mechanisms is coordinated. For example, what determines whether a neuron's response to a physiologically significant perturbation of [Ca2+]i is altered influx, altered buffering, altered efflux, or some combination of these processes? To begin to address such questions, we have examined the ways in which neurons adjust their Ca homeostatic capacities in response to a sustained elevation of [Ca2+]i evoked by growing them in depolarizing concentrations of KCl.
Chronically depolarizing neurons by increasing the concentration of KCl
in their culture medium is a convenient means of experimentally achieving long-term perturbations of [Ca2+]i.
This manipulation is used commonly to enhance survival of a variety of
central and peripheral neurons (Collins et al. 1991; Franklin et al. 1995
; Galli et al. 1995
;
Nishi and Berg 1981
; Scott 1971
). The
mechanism(s) by which chronic depolarization promotes neuronal survival
is unknown. It may activate intracellular pathways that normally are
activated by synaptic activity and/or by neurotrophic factors, both of
which are eliminated or reduced when neurons are placed in cell cultures.
In many cell types, chronic depolarization causes a long-term decrease
in depolarization-evoked Ca influx through voltage-dependent Ca
channels (Delorme and McGee 1986; Feron and
Godfraind 1995
; Ferrante et al. 1991
;
Franklin et al. 1992
; Liu et al. 1994
). Chronic depolarization likely mimics the effects of electrical activity, which also causes a long-lasting reduction in Ca current in
vivo and in vitro (Hong and Lnenicka 1995
; Li et
al. 1996
; Lnenicka and Hong 1997
) and,
furthermore, influences [Ca2+]i regulation by
neurons (Lnenicka et al. 1998
).
Previously we have shown that the sustained, dihydropyridine-blockable,
elevation of [Ca2+]i caused by growth of rat
myenteric neurons in 25 mM KCl results in reduced densities of
voltage-dependent Ca currents and increased expression of a transient
K+ current but no changes in voltage-dependent Na current
(Franklin et al. 1992). In the present study, we have
examined how these changes in expression of ionic currents alter Ca
influx during action potential activity, and we have tested whether
other components of neuronal Ca homeostasis are altered in response to
prolonged elevation of [Ca2+]i. We report
that chronic depolarization increases fast Ca buffering and slow Ca
handling capacities in myenteric neurons, thereby increasing their
ability to reduce [Ca2+]i after electrically
evoked increases. We also have found evidence that chronic
depolarization alters the net action of the endoplasmic reticulum (ER)
associated Ca stores on electrically evoked
[Ca2+]i transients, reducing their
contribution to [Ca2+]i transient amplitudes
and increasing their role in regulating [Ca2+]i transient decay rates.
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METHODS |
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Cell culture
Myenteric neurons from the small intestines of Sprague-Dawley
rat pups were grown in cell cultures as described previously (Franklin and Willard 1993; Nishi and Willard
1985
) except that the cells were grown on glass or ACLAR
(Allied Signal Plastics, Pottsville, PA) coverslips coated with
poly-lysine and laminin instead of collagen-coated plastic. When
chronically depolarizing neurons, KCl was added to raise the final
concentration to 25 mM; these are called "25K cells." The
concentrations of other ions were not reduced to compensate for
osmolarity changes caused by raising the KCl concentration. The effects
of elevating the concentration of KCl do not appear to be due to
changes in osmolality because elevating the NaCl concentration by an
additional 20 mM has no effect on neuronal survival, expression of
voltage-dependent Ca currents, or voltage-dependent K currents
(Willard, unpublished data). 25K cells remain depolarized for the
entire period they are exposed to 25 mM KCl and repolarize rapidly to
normal levels on return to control medium (Franklin et al.
1992
). Cells grown in control medium, which contains 5.4 mM
KCl, are called "5K cells." Cells were recorded from within 1 wk
of plating to avoid contributions of morphological differences to the
observed phenomena; chronic depolarization increased cell body size and
number of processes of myenteric neurons as well as cell survival at
longer times in culture (data not shown). During the first week of
chronic depolarization, there were no overt morphological differences, such as the disposition of intracellular organelles, as viewed in
unstained cells with modulation contrast optics (×40 objective).
[Ca2+]i measurement instrumentation
[Ca2+]i was estimated using fura-2
(Grynkiewicz et al. 1985). Cells were illuminated with
light from a 75-W xenon lamp that passed through 340- and 380-nm
band-pass excitation filters in a computer-controlled filterwheel
(Metaltek Instruments, Research Triangle Park, NC) and neutral density
filters in a second filterwheel (to minimize photobleaching). The light
was transmitted by a quartz fiber optic light scrambler (G. W. Ellis, Woods Hole, MA) to an inverted microscope, reflected by a
dichroic mirror and focused onto cells with a ×100 objective (Nikon
Fluor, NA 1.3). Emitted light was filtered (510 nm), intensified
(GenIIsys, Dage-MTI, Michigan City, IN), and imaged with a CCD video
camera (CCD 72, Dage-MTI). Because the purpose of our experiments was
to accurately monitor changes in [Ca2+]i, the
dynamic range of the imaging setup was adjusted so that cytoplasmic
[Ca2+]i signals occupied the maximum range
possible. This procedure had implications for
[Ca2+]i calibration as discussed in the
following text. Images (4- or 8-frame averages/wavelength) were
digitized by Image-1/FL hardware and software (Universal Imaging, West
Chester, PA). Video images usually were captured every 3.5 s. For
a temporal resolution of 1.5-1.6 s/ratio, individual wavelength data
(4- or 8-frame averages) were written to files without capturing
images. Ratio images were formed in Image-1/AT (Universal Imaging) and
converted to [Ca2+]i images using calibration
values determined as described in a later section. The
[Ca2+]i for each cell in an image was
averaged for a region of its soma.
Dye loading
Cells were incubated for 30 min at 37°C in a 5%
CO2 atmosphere in 1 ml Eagle's minimum essential medium
(supplemented with 20 mM KCl for 25K cells) containing 3 µM fura-2
AM, 0.3% dimethyl sulfoxide (DMSO), and 200 µg/ml Pluronic. After
incubation with fura-2 AM, the 5K or 25K cells were rinsed with 5K or
25K standard external solution, respectively, and incubated at room
temperature, in darkness, for 15 min. 5K and 25K neurons achieved
similar levels of fluorescence, indicating that overall dye loading was similar in the two groups. There was no obviously discernible difference in fura-2 localization in the two groups.
[Ca2+]i calibration
Conversion of video images to images of estimated
[Ca2+]i was done in Image-1/FL. For
measurements of electrically stimulated [Ca2+]i transients, we adjusted the camera,
intensifier, and gain settings on a cell-by-cell basis to optimally use
the linear range of the camera; signals from electrically evoked
[Ca2+]i transients typically occupied >80%
of the system's dynamic range. Therefore measurement of
Rmax
(F340/F380 at saturating Ca) (Grynkiewicz et al. 1985) was not possible because
F340 exceeded the fluorescence intensity limit
of the system. We used a standard curve generated with commercially
obtained stock solutions (Calcium Calibration Kit II, Molecular Probes,
Eugene, OR) to calibrate our system. The calibration solutions
consisted of mixtures of K2EGTA and CaEGTA, yielding 0, 17, 38, 65, 100, 150, 225, and 351 nM free Ca2+ in 100 mM KCl
and 10 mM MOPS, pH 7.2 (10 mM EGTA final concentration). The ionic
concentrations of the calibration solutions were not matched to
concentrations in situ (see following text). Droplets (0.5 µl) of the
calibration solutions and fura-2 pentapotassium salt were placed
beneath H2O-saturated mineral oil on glass or ACLAR
coverslips as appropriate. The fura-2 pentapotassium salt concentrations ranged from 5 to 25 µM, depending on the apparent amount of dye loading in the cells and the camera and intensifier shutter settings and the gain settings used to image the cells. The
ratio of the emitted intensities for 340 nm (360 nm for experiments involving estimation of Ca binding capacity, see following text) and
380 nm excitation
(F340/F380) was
calculated for each Ca2+ concentration. The ratios for the
calibration standards were fitted to curves by linear interpolation and
the estimated [Ca2+]i values in cells were
extrapolated from the curves. Because the purpose of these experiments
was to compare Ca homeostasis of control and chronically depolarized
cells rather than to determine the "true" absolute value of
[Ca2+]i, we did not correct for differences
in the behavior of fura-2 in situ and in buffered in vitro solutions.
As discussed in Franklin et al. (1992)
, we have made
extensive efforts to use in situ calibration protocols, but we find
such methods to be very unreliable for cultured myenteric neurons.
Nevertheless the in vitro calibration method provided a reliable
standard and yielded consistent estimates of
[Ca2+]i changes in similarly treated cells.
Accordingly, although we do not wish to imply that we have determined
the absolute values of [Ca2+]i, values for
changes in [Ca2+]i are reported in units of nanomolar.
Evoking [Ca2+]i transients with field electrodes
Neuronal [Ca2+]i transients were
elicited by field stimulation with a pair of platinum wire electrodes.
The wires were 10 mm long and were separated by 6 mm. The wires were
positioned just above the coverslip, and the stimulated cells were
centered in the field. A Grass SIU5 stimulus isolation unit and a Grass
S88 stimulator were used to deliver either single 150-V, 2.5-ms pulses or 25-Hz trains of 2-16 pulses. A similar system for stimulation has
been reported to effectively evoke action-potential (AP)-induced [Ca2+]i transients in cultured rat
hippocampal neurons (Jacobs and Meyer 1997).
Electrophysiology
Tight-seal whole cell recording techniques and tight-seal
perforated-patch recording techniques were used. There were no
significant differences between values obtained with the two recording
methods, and the results generally are reported without reference to
the exact method used. Patch pipettes were made from 1.5-mm Kimax-51 glass capillary tubing (Fisher) with tips and shanks coated with dental
wax (Kerr Sticky Wax, Emeryville, CA). Tips were heat-polished and had
resistances of 2-6 M when filled with recording solutions. Such
electrodes typically formed 2-5 G
seals with myenteric neuronal membranes. The higher resistance electrodes were used for experiments in which fura-2 was introduced to slow the rate of diffusion into the
cells. The bathing solution was grounded via a 0.9% NaCl agar bridge.
Experiments were performed at room temperature (20-24°C). An
Axopatch-1D amplifier with a unity gain CV-4 headstage was used for
both current- and voltage-clamp experiments. Current and voltage
protocols were controlled by a microcomputer connected to the amplifier
via a LabMaster TL-1-125 DMA interface. pClamp (versions 5.5 and 6.0, Axon Instruments, Foster City, CA) was used for data acquisition and
analysis. APs evoked by injecting rectangular depolarizing current
pulses were low-pass filtered at 2 kHz and sampled at 4 or 20 kHz. When
necessary, current was injected to keep the membrane potential at
60
mV. Voltage commands modeled on APs ("action potential
waveforms"; APWs) were used to elicit calcium currents
(ICa). An APW consisted of a depolarizing ramp
from
60 to +35 mV, followed by a biphasic repolarizing ramp to
60.
Two APWs were used, with the following parameters (times in ms): 10.5 total duration, 1.5 rise time, 6.0 80% fall time, and 3.0 remaining
fall time and 11.7 total duration, 1.5 rise time, 6.8 80% fall time,
3.4 remaining fall time. ICa elicited by APWs
were filtered at 1 kHz and sampled at 38 kHz. In combined electrophysiological and [Ca2+]i measurement
experiments, ICa was evoked by depolarizing
voltage-clamp steps. 5K cells were held at
60 mV and stepped to 0 mV
for 5-200 ms. Because of their lower Ca current densities, it was
usually necessary to hold 25K cells at
90 mV to evoke Ca currents
with charge densities similar to those of 5K neurons. However, holding potentials of
60 mV were used for 25K cells with large
ICa and no differences in Ca handling capacities
were noted. A P/4 subtraction procedure (using quarter scale
hyperpolarizing voltage commands) was used to correct for linear
leakage currents for step depolarizations and APW commands.
Ca2+ influx charge density was calculated by dividing the
time integral of ICa by the membrane
capacitance, an estimate of membrane area. Membrane capacitance was
estimated by integrating the first 1.75 ms of capacitative transients
(filtered at 10 kHz and sampled at 20 kHz) elicited by 20-mV
hyperpolarizing steps from a holding potential of
60 mV. The membrane
capacitance of the cells used for these experiments was not
significantly altered by chronic depolarization (data not shown). This
method of controlling for cell size was used instead of cell volume
measurements due to the difficulty of measuring volume in myenteric
neurons, which have irregular shapes.
Estimation of endogenous Ca2+ buffer concentration
Two methods, both of which use the properties of an exogenously
introduced Ca binding agent, fura-2, to estimate the endogenous Ca
buffer and its properties, were used to compare the endogenous Ca
binding capacity of 5K and 25K neurons (Neher and Augustine 1992). Fura-2 was introduced into cells via tight-seal whole
cell recording pipettes filled with cesium-based internal solution plus
500 µM fura-2 pentapotassium salt without any additional Ca2+ buffering agent. Cell fluorescence was imaged and
measured as described earlier except 360 nm excitation was substituted
for 340 nm and the dynamic range of the imaging setup was adjusted to
allow measurement of Rmax at saturating Ca
levels. Small pipette tips (6 M
) were used to slow fura-2 entry into
cells. The cytoplasmic concentration of fura-2 ([BT]) and
the loading time course were estimated from the loading curve of
background-corrected, emitted light intensity at the isobestic, 360-nm
excitation, wavelength, F360 (directly
proportional to BT). [Ca2+]i was
estimated from F360/F380,
after subtraction of the background fluorescence from the fura-2-filled
pipette, measured before going whole cell. F360
and F380 were recorded every 1.5-1.6 s per
wavelength pair (4 frame averages). The Ca2+ buffer
capacity of the fura-2 that had entered a cell,
B, was calculated from Eq. 31 of Neher
and Augustine (1992)
. KB, the Ca2+-binding constant of fura-2 (the inverse of
KD), was calculated using Eq. 5 of
Grynkiewicz et al. (1985)
. Values of
KB determined from 16.7, 37.6, 64.5, 100, 150, and 225 nM and 40 µM Ca2+ were averaged to yield an
estimated KB of 0.005 nM
1.
The "kinetic" method (method 1 of Neher and Augustine
1992) analyzes changes that occur in the decay time constants
(
) of [Ca2+]i transients in a population
of cells as the concentration of fura-2 ([BT]) increases.
Extrapolation back to the decay time constant in the absence of fura-2
yields an estimate for the endogenous Ca binding capacity of that
population of cells. This method of analysis also requires calculation
of recovery time constants corrected for the diffusion of buffer
between cell and patch pipette. The corrected time constant,
', was
determined from Eq. 16 of Neher and Augustine (1992)
.
The average time constant for fura-2 loading was 254 ± 29 s
in 5K cells and 176 ± 23 s in 25K cells (means ± SE). To determine endogenous Ca2+ buffer capacity
(
S),
' was plotted versus
B for the 5K
and 25K populations (separately), and the points were fitted with linear regressions. The negative x intercept of such plots
is at 1 +
S and the y intercept
indicates the recovery time constant for the cell population in the
absence of fura-2.
The "binding" method (method 2 of Neher and Augustine
1992; see also Müller et al. 1993
) determines the
fraction of entering Ca that binds fura-2 by measuring the amplitudes
of fluorescence changes that occur in response to Ca entry evoked by
depolarization of fura-2-loaded cells. This was achieved by calculating
the ratio (f) of the change in F380
to the time integral of the ICa evoked by a
depolarizing voltage-clamp step (f =
F380/
Ica
dt) (Eq. 24, Neher and Augustine 1992
). Plots
of f versus
B were fitted to the equation:
f = k*
B/(1 +
B +
S) (Eq. 5, Müller et al. 1993
) to
yield k, a proportionality constant, and
S,
the endogenous Ca2+ buffer capacity. When plotted as a
double reciprocal plot and fitted with a linear regression, the
negative x intercept yields the value
1/(1 +
S).
Curve fitting
The amplitudes and decay rates of
[Ca2+]i transients were determined with
PeakFit (version 3.11; Jandel Scientific, San Rafael, CA). Data were
fit with a mono-exponential function,
A0*exp[t/
], where
A0 is the initial amplitude, t is
time, and
is the time for the transient to decay by 1/e.
Fits were accepted if r2 was
0.9. The time
integrals of [Ca2+]i transients were
determined for the period from transient onset to recovery to baseline
(estimated from fits of the recovery phase) and were calculated with
Lotus 123 (release 3.1). Linear regression fits and nonlinear curve
fits were performed in SigmaPlot (version 5.0, for DOS; Jandel
Scientific). The ranges of data to be fit with linear regressions were
selected by eye. All accepted fits had R > 0.86.
Statistics
All means are presented ±SE. The program InStat (GraphPAD Software, San Diego, CA) was used for most analyses. Large data sets were analyzed with ABstat (version 6, Anderson-Bell, Parker, CO). The significance of differences was determined by means of two-tailed parametric (paired or unpaired t-tests) or nonparametric tests (paired Wilcoxon signed rank test or unpaired Mann-Whitney 2 sample test) as appropriate.
To estimate the significance of correlations, the
t-statistic was calculated from the correlation coefficient
and the number of data points (Brady et al. 1994), and
P values were then obtained from a two-tailed
t-distribution table (Zar 1984
). The linear fits of population data from 5K and 25K neurons were tested for significance of difference using two methods. The first method compares
the slopes of the linear regressions of the two populations, testing
for difference between the two population regression coefficients. The
second method compares the two correlation coefficients (Zar 1984
).
Solutions
Standard external solution (SES), used for [Ca2+]i and AP measurements, consisted of Hank's balanced salt solution [(in mM) 137 NaCl, 5.4 KCl, 0.44 KH2PO4, and 0.34 Na2HPO4] supplemented with (in mM) 3.0 CaCl2, 10 glucose, and 5 HEPES-Na-HEPES (pH 7.35). 25K SES consisted of SES adjusted to 25 mM KCl. For ICa measurements, external solution contained (in mM) 100 TEA-Cl, 3 CaCl2, 10 glucose, 50 sucrose, 8.5 TEA-OH, and 40 HEPES plus 3 µM tetrodotoxin (pH 7.4). Recording pipettes used for whole cell current-clamp recording contained (in mM) 150 KCl, 2 MgCl2, 1 EGTA, 1 ATP, 1 GTP, and 10 HEPES (pH 7.3). Pipettes used for perforated-patch current-clamp recordings contained (in mM) 130 K-gluconate, 20 KCl, 5 KOH, 5 MgCl2, 10 glucose, and 10 HEPES (pH 7.3). Nystatin (20% wt/vol; 0.4% final DMSO concentration) was suspended in the pipette solution before use. Pipettes for perforated patch recordings of ICa contained (in mM) 55 CsCl, 75 Cs2SO4, 7 MgCl2, and 5 CsOH (pH 7.35). For whole cell ICa recordings, pipettes contained (in mM) 100 CsCl, 15 CsOH, 2 MgCl2, 22 sucrose, 1 ATP, 1 GTP, 5 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), and 40 HEPES (pH 7.35). Fura-2 pentapotassium salt (500 µM) replaced BAPTA in this cesium-based internal solution for fura-2-loading experiments. All solutions contained 28 µM phenol red for pH indication.
Solution changes
Solution changes were made with a linear microcapillary array controlled by a hydraulic manipulator. The time for changes in the solution superfusing a cell was ~1.5 s. Positive pressure was supplied by a syringe pump operating at low flow rates (typically, 0.4-4.0 ml/h). The array was positioned so that the cells were exposed to flowing solution throughout the experiment.
Chemicals
All reagents were purchased from Sigma (St. Louis, MO) except for the following: 2,5-di-tert-butyl-hydroquinone, anhydrous dimethylsulfoxide (Aldrich, Milwaukee, WI); ryanodine (Calbiochem, La Jolla, CA); BAPTA, calcium calibration buffer kit II, fura-2 pentapotassium salt, fura-2 AM, Pluronic F-127 (Molecular Probes); and nitrendipine (Research Biochemicals, Natick, MA).
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RESULTS |
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Action potentials, Ca currents, and [Ca2+]i transients
Franklin et al. (1992) showed that chronic
depolarization of myenteric neurons reduces the density of
pharmacologically isolated Ca channel currents evoked by voltage-clamp
steps. To determine the physiological consequences of these changes, we
compared the waveforms and sensitivity of APs to Ca channel block in
control neurons and in neurons that had been chronically depolarized
for 3-5 days. Using either whole cell or perforated-patch recordings, we observed that the APs of chronically depolarized neurons were briefer (Fig. 1) and less sensitive to
blockade of Ca currents by CdCl2. The mean widths at
half-height were 4.5 ± 0.2 and 5.0 ± 0.1 ms in 25K
(n = 55) and 5K (n = 40) neurons,
respectively (P < 0.04, 2-tailed unpaired
t-test). CdCl2 (100 µM), which completely blocks Ca currents in myenteric neurons (Franklin and Willard 1993
), caused a 9% reduction in the amplitude and a 46%
increase in the width of APs in 5K neurons but had no significant
effect on the waveforms of 25K neurons. Chronic depolarization had no significant effect on the amplitudes of evoked APs or on their thresholds (data not shown). Thus the findings indicate that chronic depolarization reduced the contribution of Ca-dependent processes to AP
waveform in myenteric neurons.
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The decreased AP width associated with chronic depolarization may result in reduced Ca entry during electrical activity. Therefore Ca entry evoked by APs in 5K and 25K neurons was compared in two ways. First, Ca currents were evoked by voltage-clamp pulses designed to approximate the waveform of action potentials (APWs). An example is shown in Fig. 2. We found that identical APWs evoked smaller and briefer Ca2+ currents in 25K neurons than in 5K neurons. The mean densities of Ca influx evoked by APWs with a width at half-height of 5.0 ms (modeled after APs of 5K neurons) were 40.2 ± 7.6 fC/pF (n = 18) and 12.8 ± 2.8 fC/pF (n = 17) in 5K and 25K neurons, respectively (2-tailed P < 0.0001, unpaired Mann-Whitney 2 sample test). Decreasing the duration of the APW to 4.5 ms decreased Ca influx by <3% in 5K neurons and by approximately 14% in 25K neurons, suggesting that the ~70% reduction of Ca influx in 25K neurons (compared with 5K neurons) is due mainly to lower Ca current density rather than to decreased AP width.
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The second test of the hypothesis that APs evoke less Ca influx in 25K
neurons was to compare the [Ca2+]i transients
evoked in 5K and 25K neurons by stimulation with extracellular field
electrodes. Field stimulation allows measurement of
[Ca2+]i transients in a greater number of
neurons per experiment and causes less disturbance of Ca homeostatic
mechanisms than intracellular microelectrode recordings or whole cell
recordings. The evoked [Ca2+]i transients
were attributed to Ca influx elicited by APs because they were blocked
by removal of extracellular Ca or by addition of tetrodotoxin (Fig.
3). The transients had reproducible
amplitudes and waveforms when sufficient time was allowed between
stimulations for the transients to recover. They also closely resembled
spontaneous transients that occasionally were observed. As expected
from the decrease in Ca influx caused by chronic depolarization, single stimuli or brief trains of stimuli evoked smaller transients in 25K
neurons than in 5K neurons (Fig. 4).
During stimulus trains, [Ca2+]i transients
initially increased approximately linearly with the number of pulses
and then increased at a slower rate. Cohen et al. (1997)
observed a similar relationship between AP number and
[Ca2+]i transient amplitude in rabbit nodose
neurons. Figure 4B shows that increasing the number of
stimulus pulses caused a steeper rate of increase in
[Ca2+]i transients in 5K neurons than in 25K
neurons. The altered relationship between AP number and transient
amplitude suggests that chronic depolarization may have altered Ca
handling by the neurons.
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Indications that chronic depolarization altered components of Ca homeostasis in myenteric neurons came from further comparisons of the waveforms of [Ca2+]i transients elicited in 5K neurons and 25K neurons. Figure 5, a comparison of the responses of populations of 5K and 25K neurons to standardized 8-pulse trains, shows that the [Ca2+]i transients evoked in 25K neurons were smaller and decayed more rapidly. The mean amplitudes of [Ca2+]i transients were 31.6 ± 1.3 nM (n = 242) in 5K neurons and 16.7 ± 0.9 nM (n = 127) in 25K neurons (2-tailed P < 0.0001). Significantly, the [Ca2+]i transient decay time constant was reduced from 15.4 ± 0.5 s (n = 195) in 5K neurons to 9.4 ± 0.7 s (n = 127) in 25K cells (2-tailed P < 0.0001, unpaired t-test), indicating that slow Ca handling mechanisms, possibly including Ca sequestration and efflux, were increased. The faster decay of [Ca2+]i transients in 25K neurons was not due to their smaller amplitudes: there was no discernible relationship between amplitude and decay time constants of [Ca2+]i transients in either 5K or 25K neurons (data not shown). A consequence of the changes in [Ca2+]i transient waveform was that the mean time integral of the [Ca2+]i transients was reduced ~3.5-fold from 375 ± 42 nM · s in 5K neurons (n = 242) to 107 ± 18 nM · s in 25K neurons (n = 127) (2-tailed P < 0.0001, unpaired t-test). Therefore the net increase in [Ca2+]i evoked by APs was decreased by chronic depolarization. This may have implications for many Ca-dependent neuronal processes.
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Chronic depolarization increases the ability of myenteric neurons to control [Ca2+]i
Multiple elements of the intracellular Ca homeostatic system,
including sequestration by organelles and efflux via plasma membrane
mechanisms (reviewed in Carafoli 1987; Pozzan et
al. 1994
), designated here as slow Ca handling mechanisms, have
been shown to reduce [Ca2+]i on the time
scale of seconds and to help regulate the waveform of
[Ca2+]i transients in a variety of neurons
including bullfrog sympathetic neurons (Friel and Tsien
1992
), rat DRG neurons (Werth et al. 1996
), and
Xenopus spinal neurons (Holliday et al.
1991
). These relatively slow Ca clearance mechanisms combine
with faster Ca binding mechanisms to shape
[Ca2+]i transients. To compare directly the
Ca homeostatic capabilities of 5K and 25K neurons, we measured the
[Ca2+]i transients evoked by voltage-clamp
steps. The time integrals of the Ca currents elicited by the
voltage-clamp steps were used to calculate influx charge densities,
which then were compared with the resultant
[Ca2+]i transients. Table
1 shows that the mean amplitude of
[Ca2+]i transients was significantly smaller
in 25K neurons over a wide range of influx charge densities, indicating
that chronic depolarization increased fast Ca handling capabilities in
the myenteric neurons.
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To test whether the increases in [Ca2+]i due
to larger Ca influx were activating additional Ca handling components
(e.g., Stuenkel 1994; Thayer and Miller
1990
), we examined the relationship of influx charge density to
[Ca2+]i transients. This relationship was
linear at the lowest levels of Ca influx and then approached a limiting
value, as illustrated in Fig. 6. Although
the slopes of the linear phases were not significantly different in 5K
(1.56 ± 0.17; n = 15) and 25K (1.29 ± 0.13;
n = 15) neurons (2-tailed P = 0.21, Mann-Whitney 2 sample test), there was a significant decrease in the
limiting values from 968 ± 127 nM · s (n = 15) in 5K neurons to 611 ± 96 nM · s (n = 15) in 25K neurons (2-tailed P < 0.034, unpaired
Mann-Whitney 2 sample test). This suggests that the Ca handling
mechanisms activated at higher levels of Ca influx may have been
altered by chronic depolarization.
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Chronic depolarization increases fast Ca buffer capacity
Fast Ca buffers, such as calcium binding proteins, act to limit
transient changes in [Ca2+]i. As such, we
determined the fast Ca buffer capacities of 5K and 25K neurons by two
methods that entail monitoring [Ca2+]i
transient waveforms during introduction of fura-2 into the neuronal
cytoplasm from a patch pipet (Neher and Augustine 1992) (see METHODS). Both methods revealed that the fast
buffering capacity of 25K neurons was increased significantly. Figures
7 and 8
illustrate raw and transformed data, respectively, from a 5K and a 25K
neuron for analysis by the "binding" method, used to estimate
endogenous Ca buffer capacity. By observing the consequences of loading
individual cells with a concentration of fura-2 sufficient to alter
evoked [Ca2+]i transients, we estimated that
the endogenous Ca buffer capacity increased from 172 ± 44 (n = 9) in 5K neurons to 608 ± 58 (n = 12) in 25K neurons (2-tailed P = 0.0005, unpaired Mann-Whitney 2 sample test). By examining the effects
of fura-2 on the recovery time constants of
[Ca2+]i transients in a group of 5K neurons
and a group of 25K neurons (Fig. 9), we
obtained a second estimate of the endogenous Ca buffer capacity, which
increased from 132 in 5K neurons to 256 in 25K neurons. (Because this
2nd method of analysis compares pooled data for the 2 populations,
there are no estimates of variance.) As was noted by Neher and
Augustine (1992)
, estimates of buffer capacity based on
recovery kinetics may be lower than those based on the binding method
because of the presence of slow organellar Ca removal mechanisms.
Analysis of the recovery time constants also allowed us to estimate
that the "native" recovery time constants were 5.6 s in 5K
neurons and 5.1 s in 25K neurons, supporting the conclusion from
measurements of field stimulated [Ca2+]i
transients that chronic depolarization speeds the decay of the
transients.
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Chronic depolarization increases caffeine-releasable Ca2+stores
Many neuronal cell types respond to caffeine application
with an elevation of [Ca2+]i, indicating that
they have caffeine-releasable Ca stores. Caffeine (10 mM) evoked
[Ca2+]i transients in the majority of 5K and
25K neurons, although the amplitudes and time courses of the responses
were quite variable among different cells. Twenty-four of 30 5K cells
and 18 of 19 25K cells had responses similar to those shown in Fig.
10. Initial responses to caffeine did
not require extracellular Ca, but subsequent applications of caffeine
in the absence of external Ca elicited little or no response (Fig.
10A). In contrast, in SES, the neurons responded to repeated
caffeine applications. Figure 10A also shows that in Ca-free
solution [Ca2+]i fell below precaffeine
levels after caffeine was washed out presumably due to depletion of
intracellular Ca stores in the absence of external Ca. Ryanodine, which
interacts with the caffeine-sensitive channels that mediate Ca-induced
Ca release (CICR) (Kimball et al. 1996; McPherson
et al. 1991
), blocked the [Ca2+]i
responses to caffeine in a use-dependent manner and caused a slowly
rising elevation of [Ca2+]i (not shown). As
summarized in Table 2, caffeine evoked
larger and slightly more rapidly decaying
[Ca2+]i responses in 25K neurons than in 5K
neurons. This appears not to be simply a consequence of the greater
influx of Ca that occurred during culture in 25K medium because
caffeine evoked similar [Ca2+]i transients
regardless of whether it was applied in SES or in SES in which the
[KCl] had been increased to 25 mM (Fig. 10, B and
C).
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Chronic depolarization changes the effects of ER-related Ca stores on electrically evoked [Ca2+]i transients
Caffeine application increases [Ca2+]i
in most types of neurons. However, only in a subset of
caffeine-responsive neurons does CICR contribute significantly to
[Ca2+]i responses evoked by moderate
electrical activity (e.g., Thayer et al. 1988). To test
the possible contribution of CICR to AP-evoked [Ca2+]i transients in myenteric neurons and
to test whether chronic depolarization alters that contribution,
electrically evoked [Ca2+]i transients were
compared in the absence and presence of caffeine. Caffeine (1-10 mM)
reduced the amplitudes of evoked [Ca2+]i
transients in 5K neurons, An example of this effect is illustrated in
Fig. 11A for application of
10 mM caffeine. It did not significantly change the amplitudes of
[Ca2+]i transients in 25K neurons, but it did
slow their decay (Table 3). Ryanodine
decreased [Ca2+]i transients in 5K neurons
but did not alter decay rate significantly in either cell type (Table
3). These results support the hypothesis that chronic depolarization
reduced AP-evoked Ca release from caffeine-sensitive stores while it
increased Ca uptake by caffeine-sensitive mechanisms.
|
|
In PC12 cells, release of Ca from caffeine-sensitive stores can be
enhanced by release of Ca from inositol triphosphate
(IP3)-sensitive stores (Reber et al.
1993). To test whether IP3-sensitive stores modulate [Ca2+]i transients in myenteric
neurons, we examined whether electrically evoked
[Ca2+]i transients were altered by
thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ),
which empty IP3-sensitive Ca stores by inhibiting ER
Ca2+-ATPases (Kass et al. 1989
;
Thastrup et al. 1990
). As illustrated in Fig. 11,
B and C, TG elicited a slow increase in
[Ca2+]i in both 5K and 25K neurons that
partially recovered during application.
[Ca2+]i responses to TG did not differ
significantly in 5K and 25K neurons (data not shown). TG decreased the
amplitudes of electrically evoked [Ca2+]i
transients in 5K neurons, and it slowed the decay of
[Ca2+]i transients in both cell types (Table
3). BHQ (10 µM) had effects similar to those of TG, although it
usually elevated [Ca2+]i for longer than did
TG. Although the actions of TG and BHQ were similar to those of
caffeine and ryanodine, the caffeine- and thapsigargin-sensitive Ca
stores could be separated functionally because 10 mM caffeine evoked
[Ca2+]i responses in neurons that had already
undergone an increase in [Ca2+]i in response
to 1 µM TG (not shown). Thus chronic depolarization may alter the
possible role of IP3-sensitive Ca stores in Ca handling by
myenteric neurons.
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DISCUSSION |
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By using chronic depolarization as a tool to elevate [Ca2+]i, we have found that myenteric neurons marshal several responses, including decreased Ca influx during APs, reduced contribution of Ca from internal stores, increased fast Ca buffering, and increased ER clearance of cytoplasmic Ca, to restore [Ca2+]i to an optimal level. These adaptive changes, incorporated in a schematic model in Fig. 12, demonstrate that myenteric neurons can adjust Ca homeostatic mechanisms to maintain [Ca2+]i within a range optimal for normal functioning of Ca-dependent processes and to resist potentially lethal increases in [Ca2+]i.
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Decreased Ca influx during APs
As would be predicted from the decreased Ca channel current,
increased transient K current, and unchanged Na current observed by
Franklin et al. (1992), 25K neurons had briefer APs and
had lower densities of Ca influx when stimulated with voltage-clamp pulses that approximated APs. The decreased AP duration in 25K neurons
appeared primarily to be due to increased K current density because
total Ca channel blockade with Cd actually prolonged APs in 5K neurons
and did not alter the duration of APs in 25K neurons. Chronic
depolarization also decreases AP duration in chick sensory neurons, due
to the appearance of a Ca-dependent, aminopyridine-sensitive, transient
K current and to faster activation of a delayed rectifier K current
(Yang and Zorumsky 1990
). Comparison of the Ca influx evoked by voltage-clamp pulses that mimicked APs with durations characteristic of 5K and 25K neurons showed that the reduced Ca influx
was attributable to reduced Ca current density rather than to briefer
AP duration. The briefer Ca currents evoked by APWs in 25K neurons may
be due to an effect of chronic depolarization on the deactivation
kinetics of Ca currents in myenteric neurons. Because our previous
experiments used pharmacological conditions designed to maximize Ca
channel currents, e.g., the substitution of Ba ions for Ca ions
(Franklin et al. 1992
), the present findings are
important in that they demonstrate that the changes induced by chronic
depolarization cause significant changes in the physiological properties of neurons exposed to normal concentrations of Ca and under
conditions in which ionized Ca is the charge carrier.
Altered contributions of organellar Ca release and uptake to [Ca2+]i transients
The relationship between Ca influx and the amplitudes of
[Ca2+]i transients was analyzed to assess the
contributions of Ca release from intracellular stores, putatively
labeled CICR, to [Ca2+]i transients evoked by
electrical activity in myenteric neurons. At low levels of Ca influx,
the amplitudes of [Ca2+]i transients
increased linearly in both 5K and 25K neurons. Such linearity, which
also is observed in rat DRG neurons (Thayer and Miller
1990), in bovine chromaffin cells (Neher and Augustine 1992
), and in isolated rat neurohypophysial nerve endings
(Stuenkel 1994
), suggests lack of a significant
contribution to the transients by CICR under these conditions in
myenteric neurons. In contrast, in cells in which CICR does make a
significant contribution to [Ca2+]i
transients, such as rat cerebellar Purkinje neurons (Llano et
al. 1994
) and rat DRG neurons (at higher levels of Ca influx) (Shmigol et al. 1995
), a supralinear relationship has been reported. At
higher levels of influx, the amplitudes of
[Ca2+]i transients in myenteric neurons
approached a limiting value (i.e., exhibit an infralinear
relationship), as do rat sensory neurons in which Thayer and
Miller (1990)
attributed the infralinear relationship to Ca
buffering actions of mitochondria. The lower limiting values for
[Ca2+]i transient amplitude in 25K
neurons suggest that the cellular mechanisms that handle large Ca loads
have an increased capacity and/or that they are activated at lower
[Ca2+]i.
Analysis of the effects of caffeine, ryanodine, TG, and BHQ on
basal [Ca2+]i and on the amplitude and time
course of [Ca2+]i transients (as summarized
in Table 3) permits us to draw conclusions about organellar stores and
their contributions to [Ca2+]i transient
waveform in myenteric neurons. The ability of these compounds to
elevate [Ca2+]i in both 5K and 25K myenteric
neurons indicates ER Ca stores the pharmacological properties of which
are similar to those of many other cell types (Henzi and
MacDermott 1992; Pozzan et al. 1994
;
Reber et al. 1993
; Thayer and Miller
1990
; Thayer et al. 1988
). In addition, the lack
of effects of caffeine, ryanodine, BHQ, and TG on the amplitude of
AP-evoked [Ca2+]i transients in 25K neurons
(Table 3) suggests that release of Ca from intracellular stores does
not contribute significantly to [Ca2+]i
transients in 25K neurons, whereas the ability of these compounds to
reduce transients in 5K neurons suggests that Ca release does contribute to [Ca2+]i transients in 5K
neurons. This latter conclusion seems to be in contradiction to the
finding that the relationship of Ca influx to
[Ca2+]i transient amplitude is infralinear in
5K neurons. However, similar findings have been described for
largely CICR-mediated [Ca2+]i transients in
rabbit vagal afferent neurons (Cohen et al. 1997
). AP-induced [Ca2+]i transients in hippocampal
neurons have a large contribution from Ca release from intracellular
stores triggered by Ca influx as long as the extracellular Ca
concentration is >50 µM (Jacobs and Meyer 1997
). It
is possible that the lack of effect of the pharmacological agents on
[Ca2+]i transient amplitude in 25K neurons
indicates a change in the sensitivity of the Ca release mechanism for
Ca entry. This has yet to be tested.
The lack of effect of caffeine and ryanodine on [Ca2+]i transient amplitude in 25K neurons, in combination with the increase in caffeine-releasable Ca in these neurons, suggests that chronic depolarization changes the role of caffeine-sensitive stores in 25K neurons, causing them to become more important for Ca sequestration than for Ca mobilization. It is possible that the sustained elevation of [Ca2+]i caused by chronic depolarization may trigger a compensatory increase in the size of caffeine-sensitive stores, thereby enhancing the ability of myenteric neurons to control [Ca2+]i. Thus the factor (or factors) limiting the [Ca2+]i transient amplitude in myenteric neurons remains to be described.
Increased fast Ca buffer capacity
Both methods of analysis revealed large (2- to 5-fold) increases
in the endogenous Ca buffer capacity of 25K neurons. The estimates fall
within the range previously estimated for other cell types (see
Neher 1995 for review). However, as discussed by
Zhou and Neher (1993)
, estimates of Ca binding capacity
entail multiple assumptions and can be changed significantly by
relatively small variation in measurements. In particular, it should be
noted that estimates of binding capacity based on the analysis of
recovery kinetics are less reliable (than estimates based on the
binding method) because they attribute
[Ca2+]i transient decay kinetics exclusively
to cytoplasmic Ca buffers, and we have evidence that ER
[Ca2+]i stores also contribute to these decay rates.
One plausible mechanism for increased fast Ca buffering capacity of 25K
neurons would be increased expression of Ca binding proteins such as
calbindin D28k. Vyas et al. (1994) reported
that depolarization with 50 mM KCl or treatment with low concentrations of Ca ionophore induces calbindin D28k expression in PC12
cells. Transfection of GH3 cells with plasmids encoding
calbindin D28k more than doubles their Ca buffering
capacity (Lledo et al. 1992
) and loading rat sensory
neurons with calbindin D28k significantly reduces evoked
[Ca2+]i transients (Chard et al.
1993
). Conversely, there is a significantly larger fast
component in [Ca2+]i transients in cerebellar
Purkinje neurons from mice lacking calbindin D28k
(Airaksinen et al. 1997
). Calbindin D28k is
found in about half of rat myenteric neurons in vivo (Buchan and
Baimbridge 1988
; Resibois et al. 1988
), but the
effect of [Ca2+]i perturbations in myenteric
neurons on the expression of calbindin D28k or other Ca
binding proteins has not yet been elucidated.
Increased [Ca2+]i transient recovery
The decay of [Ca2+]i transients is
faster in myenteric neurons that have undergone chronic depolarization,
as measured during AP-induced transients and extrapolated from
transients evoked by step depolarization. The extrapolated
"native" recovery time constants, which represent the recovery in
the absence of fura-2, were lower than recovery time constants measured
in cells loaded with fura-2 AM, indicating that loading with the
exogenous buffer affected the transients. Nevertheless, the lower
recovery time constant in 25K neurons cannot be due to simple
differences in dye loading in 5K and 25K neurons. Increasing the
concentration of fura-2 should slow recovery rates (i.e., increase the
recovery time constant) while decreasing
[Ca2+]i transient amplitude (e.g.,
Blumenfeld et al. 1992; Lledo et al.
1992
). Also the reduced amplitudes of
[Ca2+]i transient in 25K neurons is not
responsible for the faster recovery because transient amplitude did not
affect the recovery rates in either 5K or 25K neurons. We therefore
surmise that the faster decay of transients in 25K neurons is due to a
combination of increased Ca sequestration by internal stores (as
supported by the effects of thapsigargin and caffeine on time
constants) and increased Ca efflux by, as yet, unidentified mechanisms.
The determination of the mechanisms responsible for the faster
[Ca2+]i transient decay will be of great
importance for understanding the adaptation of Ca homeostatic
mechanisms to Ca perturbations.
Ca-dependent changes in electrical properties
The changes in Ca handling capabilities caused by chronic
depolarization of myenteric neurons may be a consequence of the long-term elevation of [Ca2+]i through a
dihydropyridine-sensitive mechanism as has been determined for the
sustained component of myenteric neuronal Ca current (Franklin et al. 1992). Activity-dependent increases in
[Ca2+]i that last hours to days can cause
changes in the ionic conductances of other neurons, including cultured
lobster stomatogastric ganglion (STG) neurons (Turrigiano et al.
1994
) and crayfish motoneurons (Hong and Lnenicka
1995
). Importantly, in crayfish motoneurons, the
activity-dependent decrease of Ca current density is accompanied by an
increase in Ca clearance capability, resulting in faster decay of
[Ca2+]i transients (Lnenicka et al.
1998
). Modeling studies have shown that activity-dependent
changes in maximal membrane conductances, regulated through
Ca-dependent pathways, can alter dramatically the ability of STG
neurons to generate patterned electrical activity (LeMasson et
al. 1993
; Liu et al. 1998
). In guinea pig
myenteric neurons, Ca-dependent K conductances can exert strong
influence on firing patterns (reviewed in Wood 1989
).
Thus in addition to altering Ca homeostasis, changes in
[Ca2+]i induced by chronic depolarization
also could alter the activity and/or expression of Ca-dependent
conductances of myenteric neurons and thereby alter the patterns of APs
they fire. It will be interesting to test whether altered patterns of
action potential activity can induce Ca-dependent changes in Ca homeostasis.
Implications of the upregulation of Ca homeostasis by chronic depolarization
Nishi and Willard (1985) originally added
depolarizing concentrations of KCl to culture medium to enhance
long-term growth and survival of rat myenteric neurons in culture. The
findings reported here support the hypothesis that chronic
depolarization enhances neuronal survival by increasing their ability
to control [Ca2+]i. The hypothesis that
increased Ca handling capabilities will enhance neuronal survival under
adverse conditions is consistent with several previous
findings. Chronic depolarization decreases N-methyl-D-aspartate (NMDA)induced
[Ca2+]i responses of mouse spinal neurons
(Tymianski et al. 1994
) and rat cerebellar granule cells
(Pearson et al. 1992
) and thereby decreases the
excitotoxic effects of NMDA receptor stimulation. Chronically
depolarized cerebellar granule cells are also resistant to apoptosis
induced by a transforming growth factor, TGF-
(De Luca et al. 1996
). Furthermore the ability of several
neurotrophic factors and cytokines to reduce the elevation of
[Ca2+]i caused by metabolic insults suggests
that stabilization of Ca homeostasis is a mechanism by which these
agents reduce neuronal vulnerability (Cheng and Mattson
1991
; Cheng et al. 1994
). The relationship
between Ca homeostasis and neuronal survival also is suggested by
recent findings that Ca homeostatic mechanisms are subject to change
with age in vivo in rat basal forebrain neurons and rat hippocampal
neurons (Hartmann et al. 1996
; Murchison and
Griffith 1998
) and may be correlated with the survival of cultured fetal rat hippocampal neurons (Porter et al.
1997
). Future experiments should be directed toward discovering
mechanisms by which elevation of [Ca2+]i
causes alterations in neuronal Ca homeostasis.
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ACKNOWLEDGMENTS |
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We thank L. Fleck for technical assistance.
This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-24362.
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FOOTNOTES |
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Address for reprint requests: D. J. Fickbohm, Biology Dept., Georgia State University, P.O. Box 4010, Atlanta, GA 30302-4010.
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 23 September 1998; accepted in final form 10 February 1999.
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REFERENCES |
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