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The Journal of Neurophysiology Vol. 87 No. 5 May 2002, pp. 2209-2224
Copyright ©2002 by the American Physiological Society
Departments of Anatomy and Physiology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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ABSTRACT |
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Sheldon, Claire and
John Church.
Intracellular pH Response to Anoxia in Acutely Dissociated Adult
Rat Hippocampal CA1 Neurons.
J. Neurophysiol. 87: 2209-2224, 2002.
The effects of anoxia on
intracellular pH (pHi) were examined in acutely
isolated adult rat hippocampal CA1 neurons loaded with the
H+-sensitive fluorophore,
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. During
perfusion with HCO


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INTRODUCTION |
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The extra- and intracellular
ionic changes that occur during and following anoxia and ischemia have
been studied extensively (for reviews, see Erecinska and Silver
1994
; Hansen 1985
; Lipton 1999
).
While the contribution of Ca2+ ions has received
particular attention, notably within the framework of the excitotoxic
model of cell injury, Ca2+-mediated
excitotoxicity may not be a completely valid model for the direct
actions of anoxia or ischemia on neurons, and there is renewed interest
in the role of changes in intracellular pH (pHi)
in neurodegenerative phenomena. In part, this interest has been
prompted by studies in nonneuronal cell types, such as cardiac myocytes, where anoxia/ischemia-induced changes in the activities of
pHi-regulating mechanisms, notably
Na+/H+ exchange, have been
found to play an important role in reperfusion injury (for reviews, see
Herman et al. 1990
; Karmazyn et al.
1999
). The potential importance of similar events to ischemic
neuropathology is suggested by findings that pharmacological blockers
of Na+/H+ exchange exert a
protective effect in neurons in which the antiport is sensitive to such
compounds (Kuribayashi et al. 1999
; Phillis et
al. 1999
; Vornov et al. 1996
).
A number of mechanisms that act to regulate pHi
in mammalian central neurons have now been identified. In rat
hippocampal CA1 neurons,
Na+/H+ exchange (which,
unusually, is insensitive to amiloride, amiloride analogues, and
benzoylguanidinium compounds) and Na+-dependent
HCO
exchange
contribute to acid extrusion, whereas alkali extrusion is mediated by
Na+-independent
HCO
exchange
(Baxter and Church 1996
; Bevensee et al.
1996
; Raley-Susman et al. 1991
, 1993
;
Schwiening and Boron 1994
; Smith et al.
1998
). Although it has long been known that changes in neuronal
pHi occur during and following anoxia or ischemia in vivo
and in slice preparations in vitro (for reviews, see Erecinska
and Silver 1994
; Lipton 1999
; Siesjö et al. 1996
), it is difficult under these
experimental conditions to assess the contribution of intrinsic
alterations in the activities of neuronal
pHi-regulating mechanisms to the pHi changes observed. Anoxia and ischemia, for
example, lead to complex changes in the microenvironment of neurons,
many of which [e.g., changes in extracellular pH
(pHo), postsynaptic receptor activation] can
affect the activities of pHi-regulating
mechanisms and steady-state pHi. In this regard,
isolated preparations offer an important advantage, and recent studies
in cultured postnatal rat hippocampal (Diarra et al.
1999
) and fetal mouse neocortical (Jørgensen et al.
1999
) neurons point to the involvement of changes in
Na+/H+ exchange activity
and, in the case of rat hippocampal neurons, a
Zn2+-sensitive acid extrusion mechanism in the
neuronal pHi response to anoxia. However, both
the sensitivity of mammalian central neurons to the damaging effects of
anoxia (Friedman and Haddad 1993
; Isagai et al.
1999
; Kass and Lipton 1989
; Roberts and
Chih 1997
) and the mechanisms that serve to regulate neuronal
pHi (Bevensee et al. 1996
;
Douglas et al. 2001
; Raley-Susman et al.
1993
; Roberts and Chih 1997
) are developmentally
regulated, and it remains unclear whether findings made in
phenotypically immature cells in culture can be applied to more mature
neurons, especially those such as rat hippocampal CA1 neurons that are
particularly vulnerable to the damaging effects of anoxia.
In the present study, therefore we characterized the steady-state
pHi changes that occur during and following
transient periods of anoxia in hippocampal CA1 neurons acutely isolated
from adult rats and examined the mechanisms responsible for the
increases in pHi that were found to occur during
and following anoxia. Some of these results have been presented in
abstract form (Sheldon and Church 2000
).
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METHODS |
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Cell preparation
Acutely dissociated rat hippocampal CA1 neurons were prepared as
previously described (Smith et al. 1998
). In brief, male Wistar rats (200-260 g) were anesthetized with 3% halothane in air
and decapitated. Transverse hippocampal slices (450 µm) were prepared
and allowed to recover for
1 h in
HCO

Solutions and test compounds
The standard
HCO



), and we have found that the rate of acid extrusion
from acutely dissociated CA1 neurons in the complete absence of
external Na+ is not influenced by the addition of
2-4 mM Na+ (C. Brett, C. Sheldon, and J. Church, unpublished observations). In experiments in which
Na+-free media could be employed (see Fig. 6),
NaH2PO4 was omitted and
NMDG+ and/or KCl were employed as substitutes;
solutions were titrated to pH 7.35 with 10 M HCl or 2 M KOH,
respectively. For Ca2+-free media,
CaCl2 was omitted, [Mg2+]
was increased to 3.5 mM, and 200 µM EGTA was added. Neurons were
superfused at a rate of 2 ml/min for the entire duration of an
experiment, and, unless otherwise noted, all experiments were performed
at 37°C and at pHo 7.35. The pH of each
experimental solution was rechecked at the end of every experiment.
Test compounds were obtained from Sigma Chemical with the exceptions of 2',5'-dideoxyadenosine (Biomol Research Laboratories, Plymouth Meeting, PA), the Rp-isomer of adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS, Na+ salt; Biolog Life Science Institute, La Jolla, CA), and omeprazole and SCH 28080 (generous gifts from, respectively, AstraZeneca, Mississauga, Ontario, Canada, and Schering Canada, Pointe-Claire, Quebec, Canada). BCECF-AM and fura-2-AM were obtained from Molecular Probes (Eugene, OR).
Induction of anoxia
Anoxia was induced by the addition of 1 or 2 mM sodium
dithionite, an O2 scavenger, to the superfusing
medium (see Diarra et al. 1999
; Friedman and
Haddad 1993
). Solutions containing sodium dithionite were
prepared immediately prior to use and were bubbled with either 5%
CO2-95% Ar
(HCO
To assess the possibility that sodium dithionite might induce
changes in pHi via mechanisms unrelated to its
O2 scavenging property,
HCO

18 h. In samples obtained anaerobically from the
recording chamber, the Po2 in media bubbled
with Ar for 1 h was 25.3 ± 0.8 (SE) mmHg (n = 4), whereas, measured in eight different samples, the
Po2 in media bubbled with Ar for
18 h was
<1 mmHg, a value the same as that measured in media containing 1 or 2 mM sodium dithionite. When a 5-min period of anoxia was imposed by
exposing neurons to
HCO
18 h, the resultant steady-state pHi
changes were not significantly different to those observed when the
Po2 was reduced to <1 mmHg by the
addition of sodium dithionite under identical buffering conditions
(Table 1; Fig. 2B). Thus the
steady-state pHi changes evoked by exposure to
media containing sodium dithionite reflect a reduction in
Po2 and are not secondary to any additional
properties of the O2 scavenger.
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Recording techniques
Intracellular free calcium concentration
([Ca2+]i) and
pHi were measured using the dual-excitation ratio
method, employing a fluorescence ratio-imaging system (Atto
Instruments, Rockville, MD; Carl Zeiss Canada, Don Mills, Ontario,
Canada). Details of the methods employed have been presented previously
(Baxter and Church 1996
; Church et al.
1998
; Smith et al. 1998
). In brief, fluorescence emissions measured at 520 or 510 nm from neurons loaded
with BCECF or fura-2, respectively, were detected by an intensified
charge-coupled device camera (Atto Instruments) and collected from
regions of interest placed on individual neuronal somata. Raw emission
intensity data at each excitation wavelength (488 and 452 nm for BCECF;
334 and 380 nm for fura-2) were corrected for background fluorescence
prior to calculation of the ratio. Ratio pairs were acquired at 1- to
12-s intervals and analyzed off-line. Analysis was restricted to those
neurons able to retain BCECF (as judged by raw emission intensity
values recorded during excitation at 452 nm; see Fig. 2A)
throughout the course of an experiment (see Bevensee et al.
1995
). To reduce photobleaching of the fluorophores and cell
damage, the output of the 100 W mercury arc lamp was attenuated
electronically, neutral density filters were placed in the light path,
and a high-speed shutter was employed to limit UV exposure to the
periods required for data acquisition.
The one-point high-[K+]/nigericin technique was
employed to convert background-corrected BCECF emission intensity
ratios
(BI488/BI452) into pHi values as described (Baxter and
Church 1996
; Smith et al. 1998
). Parameters
employed in the calculation of pHi values were
derived from nonlinear least-squares regression fits to
background-subtracted ratio versus pH data, which, in turn, were
obtained in full calibration experiments (see Baxter and Church
1996
). For the 15 full-calibration experiments utilized in
analyzing all BCECF-derived data, the mean values for
Rn(max) (the maximum obtainable value
for the normalized ratio), Rn(min)
(the minimum obtainable value for the normalized ratio), and
pKa (the
log of the dissociation
constant of BCECF) were (means ± SE) 1.89 ± 0.04, 0.43 ± 0.02, and 7.19 ± 0.02, respectively. These values were not
dependent on the temperature at which the calibration was conducted
(data not shown). To limit potential cross-contamination by nigericin,
perfusion lines were replaced and the imaging chamber was
decontaminated after each experiment by soaking first in ethanol and
then in 20% Decon 75 (BDH, Toronto, Ontario, Canada) (see
Bevensee et al. 1999
; Richmond and Vaughan-Jones
1997
). In addition, selected experiments were repeated using an
experimental chamber that had never been exposed to the ionophore.
Although the data from these experiments were not calibrated (and,
therefore are not included in RESULTS), the BI488/BI452
ratio values obtained were typical of those recorded during the course
of equivalent experiments conducted in nigericin-decontaminated chambers. Thus we failed to reveal any evidence that nigericin contamination might have contributed to the results obtained in the
study. Calibration of the fura-2 signal was not attempted and the
effects of experimental maneuvers on
[Ca2+]i are presented as
changes in background-corrected
I334/I380 ratio values. Nevertheless, under conditions identical to those employed in the present experiments, we have found that a
BI334/BI380 ratio value of
0.5 (as was observed in quiescent neurons in the present study; Fig. 3) represents an
[Ca2+]i
80 nM (see
Church et al. 1998
).
It has been reported that BCECF inhibits the plasmalemmal
Ca2+-ATPase in erythrocytes
(IC50 100 µM) (Gatto and Milanick
1993
). Because the Ca2+-ATPase in
hippocampal neurons is a
Ca

), the possibility existed that the rises in pHi measured with BCECF under the high
[Ca2+]i conditions that
pertain during and following anoxia (see RESULTS) might
have been artifacts consequent on reduced background acid loading.
Therefore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS; Molecular
Probes), a fluorescent ratiometric H+-sensitive
indicator that is reported not to inhibit activity-dependent pHi changes in snail neurons (Willoughby
et al. 1998
), was employed in a limited number of experiments
to measure anoxia-evoked changes in pHi. Loading
of this membrane-impermeant dye was achieved by enzymatically treating
and triturating hippocampal CA1 regions in the presence of 40 mM HPTS.
The excitation wavelengths were 452 and 380 nm, and the one-point
high-[K+]/nigericin technique was employed to
convert background-corrected HPTS emission intensity ratios
(BI452/BI380)
into pHi values using the equation
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(1) |
= fn2a/fn2b,
where fn2a and
fn2b are the normalized fluorescence
intensities at the acidic and basic extremes while exciting the dye at
380 nm. The constant parameters of Eq. 1 were derived from
full calibration experiments (see preceding text). The increases in
pHi observed during and following 5 min anoxia in
HPTS-loaded neurons were not significantly different to those observed
in BCECF-loaded cells (Table 1; Fig. 2C), leading us to
conclude that BCECF is an appropriate pH indicator for use in the
present experiments. Our findings are in agreement with a recent report
in cultured rat cerebellar granule cells in which activity-dependent
changes in pHi were recorded when BCECF was employed as the H+-sensitive fluorophore
(Wu et al. 1999Experimental procedures and data analysis
The effects of transient periods of anoxia were examined on both
steady-state pHi and on rates of
pHi recovery from internal acid loads imposed by
the NH
; Smith et al. 1998
). In brief, instantaneous rates of pHi recovery under control
and test conditions were plotted against absolute
pHi values (e.g., see Fig. 1B) and compared at corresponding absolute values of pHi.
The consistency of rates of pHi recovery
following two consecutive acid loads imposed in the absence of an
anoxic insult was established in control experiments (Fig.
1B). In experiments where the composition of the external
medium was altered during the recovery of pHi from an intracellular acid load (see Figs. 5C and 6),
individual portions of the recovery were fit to a linear equation, as
described by Raley-Susman et al. (1991)
.
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Data are reported as means ± SE with the accompanying n value referring to the number of neurons from which data were obtained. Statistical analyses were performed with Student's two-tailed unpaired t-test or two-way ANOVA, as appropriate. Significance was assumed at the 5% level.
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RESULTS |
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Resting pHi values under normoxic conditions
Under
HCO


) and others
(Bevensee et al. 1996
) for acutely isolated mature rat
hippocampal CA1 neurons at 37°C.
Steady-state pHi response to anoxia
The steady-state pHi changes evoked by 5- and 10-min periods of anoxia were first examined under
HCO


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To assess the potential contribution of HCO



). Similarly, although
the magnitudes of the rises in pHi observed in
the continued absence of O2 increased under both
buffering conditions as the duration of the anoxic insult increased,
for a given duration of anoxia, no significant difference was found
between the rises in pHi under
HCO

; Pirttilä and Kauppinen
1994
), changes in the activities of
HCO

In 13 additional neurons examined under HEPES-buffered conditions, 5 min anoxia elicited a different pattern of pHi
changes to that described in the preceding text. In these neurons, the fall in pHi during anoxia was significantly
smaller (0.04 ± 0.01 pH units) than the response observed in the
majority of cells examined under identical buffering conditions, and
the small acidification gave way to a marked internal alkalinization
(0.53 ± 0.05 pH units) that started during anoxia and continued
into the postanoxic period (Fig. 2D). This atypical pattern
of pHi changes was also observed in HPTS-loaded
neurons (data not shown) and, interestingly, is reported to be the
usual response of mouse CA1 hippocampal neurons to
O2 deprivation under HEPES-buffered conditions
(Yao et al. 2001
). Although no attempt was made to
characterize the mechanism(s) underlying the atypical
pHi response to anoxia, it is noteworthy that
neurons that exhibited the response had low resting
pHi values (mean pHi prior
to the induction of anoxia was 6.92 ± 0.07). This finding is
consistent with the possibility (Bevensee et al. 1996
) that a "low pHi " population of mature rat
hippocampal CA1 neurons exists that exhibits a distinct pattern of
pHi regulation.
Because the typical steady-state pHi changes
evoked by anoxia in adult rat hippocampal CA1 neurons under
HCO

Steady-state [Ca2+]i response to anoxia
In adult CA1 neurons, anoxia leads to a disruption of internal ion
homeostasis that is associated with energy failure and an abrupt
depolarization of the plasma membrane (reviewed by Hansen 1985
; Lipton 1999
; also see Rader and
Lanthorn 1989
; Silver and Erecinska 1990
;
Tanaka et al. 1997
). In the present study, anoxia evoked
a 2.0 ± 0.4 (n = 8) unit increase in the fura-2
BI334/BI380 ratio value, which commenced at approximately 2 min after the induction
of anoxia (as did the rise in pHi; Fig.
3A) and which remained
elevated after the return to normoxia for as long as stable recordings
could be maintained (
25 min following the end of an anoxic insult)
(also see Friedman and Haddad 1993
; Kubo et al.
2001
).
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The potential contribution of changes in
[Ca2+]i to the changes in
steady-state pHi evoked by anoxia was assessed by
imposing anoxia under external Ca2+-free
conditions. As shown in Fig. 3B, exposure to
Ca2+-free medium caused a 0.30 ± 0.02 (n = 6) ratio unit decrease in resting
BI334/BI380
values, and anoxia failed to induce the rapid and marked rises in
BI334/BI380
values that were observed in the presence of Ca2+
(the increase in the
BI334/BI380
value observed under external Ca2+-free
conditions was 0.02 ± 0.01 ratio units). Interestingly, in 4/6
neurons examined under Ca2+-free conditions, a
small 0.08 ± 0.02 ratio unit increase in
BI334/BI380 values was observed in the immediate postanoxic period (Fig.
3B); although the basis for this transient increase was not
investigated, it may reflect Ca2+ release from
intracellular stores consequent on anoxia-evoked changes in
pHi (see Ou Yang et al. 1994
). In
parallel experiments in BCECF-loaded neurons, exposure to
Ca2+-free medium evoked an increase in
steady-state pHi of 0.11 ± 0.04 pH units
(n = 6), as previously reported (Smith et al.
1998
). Once a new steady-state pHi value
had been reached, a 5-min period of anoxia induced a triphasic
pHi response, the individual components of which
were not significantly different to those observed in the presence of 2 mM Ca
Mechanisms underlying the increases in pHi observed during and after anoxia
Although a fall in pHi appears to be a
universal response of mammalian central neurons to anoxia or ischemia,
the increases in pHi that occurred during and
after anoxia in the present study have been observed only relatively
infrequently in neurons in vivo or in slice preparations in vitro
(see Fujiwara et al. 1992
; Mabe et al.
1983
; Melzian et al. 1996
; Pirttilä
and Kauppinen 1992
). In subsequent experiments, therefore we
examined the mechanisms responsible for these increases in
pHi.
Under HCO
; Bevensee et al. 1996
; Raley-Susman
et al. 1991
; Schwiening and Boron 1994
). To
inhibit Na+/H+ exchange
therefore, neurons were perfused with
reduced-Na

;
Raley-Susman et al. 1991
). Under
reduced-Na
), additional
mechanism(s) must contribute to these phases of the anoxic
pHi response.
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As noted in the preceding text, the internal alkalinizations that
occurred during and after anoxia appeared to be associated temporally
with marked and persistent increases in
[Ca2+]i, raising the
possibility that they may reflect H+ efflux
through a H+-conductive pathway activated by
anoxic membrane depolarization. In all cell types studied to date,
voltage-gated H+ conductances
(gH+s) are blocked by
micromolar concentrations of Zn2+ (for reviews,
see DeCoursey and Cherny 1994a
, 2000
; Eder and DeCoursey 2001
). Therefore we examined the effects of 100-500 µM Zn2+ on the rises in
pHi observed during and following anoxia. While the application of Zn2+ did not change resting
pHi prior to anoxia, there was a significant reduction in the magnitudes of the rises in pHi
during and after anoxia (Fig. 4). When Zn2+ was
applied under
reduced-[Na+]o,
NMDG+-substituted conditions, the magnitudes of
the rises in pHi observed during and after anoxia
were reduced to values significantly less than those observed under
reduced-Na
pHi recovery from internal acid loads imposed immediately after the return to normoxia
To further investigate the possibilities that
Na+/H+ exchange and a
presumed gH+ contribute to the
internal alkalinization observed after anoxia, we compared rates of
pHi recovery from internal acid loads imposed
prior to and immediately following anoxia. Examined in 17 neurons,
instantaneous rates of pHi recovery were
increased significantly after anoxia at all absolute values of
pHi (Fig. 5,
A and B). The increases in
pHi evoked by NH

)
were similar prior to and after anoxia (0.24 ± 0.02 and 0.21 ± 0.02 pH unit increases, respectively; n = 17 in each
case; P > 0.05), suggesting that alterations in
intracellular buffering power are unlikely to underlie the changes in
the rates of pHi recovery observed after anoxia.
Next, internal acid loads were imposed under
reduced-Na






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Next, internal acid loads were applied immediately after anoxia and
pHi recovery was allowed to proceed in the
presence of 100-500 µM Zn2+ and/or under
reduced-Na


To support the possibility that the
Zn2+-sensitive component of the recovery of
pHi from acid loads imposed after anoxia might be
activated by membrane depolarization (see preceding text), internal
acid loads were applied during normoxia under
Na
) was
more than twofold faster under depolarizing (139.5 mM
K

3 pH
units/s prior to the application of Zn2+ to
0.5 ± 0.3 × 10
3 pH units/s in its
presence (n = 9 in each case; P < 0.05; Fig. 6). In contrast, the effect of
high-[K+]o to increase
rates of pHi recovery from acid loads was not
affected by the P-type
H+,K+-ATPase inhibitors
omeprazole (25-50 µM, n = 7) or SCH 28080 (500 µM,
n = 2) or by the V-type H+-ATPase
inhibitor bafilomycin A1 (2 µM,
n = 5).
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Effects of changes in pHo and temperature on the pHi response to anoxia
Anoxia and ischemia in vivo and in slice preparations in vitro
lead to reductions in pHo (e.g.,
Obrenovitch et al. 1990
; Roberts and Chih
1997
; Silver and Erecinska 1990
, 1992
). In
addition, the activities of
Na+/H+ exchangers and
gH+s are reduced by falls in
pHo (DeCoursey and Cherny 2000
;
Green et al. 1988
; Ritucci et al. 1998
;
Vaughan-Jones and Wu 1990
; Wu and Vaughan-Jones
1997
). Therefore we examined the effects of lowering
pHo on the increases in pHi observed during and after anoxia. Reducing pHo
from 7.35 to 6.60 caused a 0.49 ± 0.03 pH unit fall in
pHi (n = 19) (also see
Church et al. 1998
). Once pHi had
stabilized at a new resting level, anoxia evoked an internal
acidification followed by increases in pHi during
and after anoxia that were significantly smaller than those observed at
pHo 7.35 (Fig. 4, A and B).
Next, intracellular acid loads were imposed prior to and following
anoxia at pHo 6.60 (Fig.
7A). Prior to anoxia, rates of
pHi recovery were decreased at
pHo 6.60, compared with rates of
pHi recovery observed at the same absolute values
of pHi under control (pHo
7.35) conditions (Fig. 7B). When acid loads were imposed
immediately after anoxia, rates of pHi recovery
increased, compared with rates of recovery established prior to anoxia
also at pHo 6.60 (n = 9; Fig.
7A). Plots of the pHi dependence of
the rates of pHi recovery obtained at
pHo 6.60 (Fig. 7B) indicated that, at
all absolute values of pHi, rates of
pHi recovery after anoxia were reduced at
pHo 6.60 compared with rates established after
anoxia at pHo 7.35. Taken together, the results
are consistent with contributions from
Na+/H+ exchange and a
gH+ to the pHi
response to anoxia. The data also suggest that, even at
pHo 6.60, the mechanism(s) can continue to
participate in acid extrusion after anoxia. Qualitatively opposite
results were obtained under pHo 7.60 conditions
(data not shown).
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A reduction in temperature also inhibits
Na+/H+ exchange and
gH+s (see Baxter and Church
1996
; Eder and DeCoursey 2001
). Consistent with
the possibilities that
Na+/H+ exchange and a
gH+ contribute to acid extrusion during and/or following anoxia, the increases in
pHi observed during and after anoxia at
pHo 7.35 were significantly reduced at room
temperature, compared with corresponding changes observed at 37°C
(Figs. 4 and 8A). Similar
effects were observed when anoxia was imposed under
HCO
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Role of cAMP/cAMP-dependent protein kinase in the pHi response to anoxia
Of particular interest is the finding, detailed in the preceding
text, that activation of
Na+/H+ exchange occurred in
the immediate postanoxic period even though external pH was held at a
constant value (i.e., pH 7.35). Thus a return to normal
pHo values from an external acidification, as
would occur after anoxia in vivo, is not an absolute requirement for
the postanoxic activation of
Na+/H+ exchange in isolated
hippocampal neurons. One mechanism that could contribute to the
activation of Na+/H+
exchange after anoxia is an anoxia-induced change in the activity of
intracellular second-messenger system(s) which, in turn, act to
regulate Na+/H+ exchange.
In hippocampal neurons, the intracellular concentration of
adenosine-3',5'-cyclic monophosphate ([cAMP]i)
rises rapidly in the immediate postanoxic period (e.g.,
Domanska-Janik 1996
; Small et al. 1996
;
Whittingham et al. 1984
), and we have shown previously
that increases in [cAMP]i, acting via
cAMP-dependent protein kinase (PKA), activate
Na+/H+ exchange in acutely
isolated rat CA1 neurons under normoxic conditions (Smith et al.
1998
). Therefore we investigated the effect of modulating the
activity of the cAMP/PKA system on the rise in steady-state pHi observed after anoxia.
As previously reported (Smith et al. 1998
), the
selective PKA inhibitor Rp-cAMPS (50 µM) failed to affect
steady-state pHi under HEPES-buffered, normoxic
conditions (n = 12). However, as illustrated in Fig.
9, A and B, the
magnitude of the internal alkalinization observed after anoxia was
significantly reduced in the presence of Rp-cAMPS. In
contrast, 50 µM Rp-cAMPS failed to significantly affect
the residual increase in pHi observed after
anoxia under reduced-Na
adrenergic agonists to increase
[cAMP]i is potentiated after ischemia
(Domanska-Janik 1996
; Lin et al. 1983
).
As shown in Fig. 9, A and B, stimulation of the
cAMP/PKA pathway with the
-adrenoceptor agonist isoproterenol (10 µM) significantly increased the magnitude of the postanoxic
alkalinization, an effect that was attenuated by
Rp-cAMPS or the full
-adrenoceptor antagonist, propranolol. Furthermore, the isoproterenol-evoked increase in the magnitude of the postanoxic alkalinization was significantly attenuated under reduced-Na
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