Department of Physiology, University College London, London WC1E 6BT, United Kingdom
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ABSTRACT |
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Mort, Dominic,
Païkan Marcaggi,
James Grant, and
David Attwell.
Effect of Acute Exposure to Ammonia on Glutamate Transport in
Glial Cells Isolated From the Salamander Retina.
J. Neurophysiol. 86: 836-844, 2001.
A rise of brain
ammonia level, as occurs in liver failure, initially increases
glutamate accumulation in neurons and glial cells. We investigated the
effect of acute exposure to ammonia on glutamate transporter currents
in whole cell clamped glial cells from the salamander retina. Ammonia
potentiated the current evoked by a saturating concentration of
L-glutamate, and decreased the apparent affinity of the
transporter for glutamate. The potentiation had a Michaelis-Menten
dependence on ammonia concentration, with a
Km of 1.4 mM and a maximum
potentiation of 31%. Ammonia also potentiated the transporter current
produced by D-aspartate. Potentiation of the glutamate
transport current was seen even with glutamine synthetase inhibited, so
ammonia does not act by speeding glutamine synthesis, contrary to a
suggestion in the literature. The potentiation was unchanged in the
absence of Cl ions, showing that it is not an
effect on the anion current gated by the glutamate transporter.
Ammonium ions were unable to substitute for Na+
in driving glutamate transport. Although they can partially substitute for K+ at the cation counter-transport site of
the transporter, their occupancy of these sites would produce a
potentiation of <1%. Ammonium, and the weak bases methylamine and
trimethylamine, increased the intracellular pH by similar amounts, and
intracellular alkalinization is known to increase glutamate uptake.
Methylamine and trimethylamine potentiated the uptake current by the
amount expected from the known pH dependence of uptake, but ammonia
gave a potentiation that was larger than could be explained by the pH
change, and some potentiation of uptake by ammonia was still seen when
the internal pH was 8.8, at which pH further alkalinization does not increase uptake. These data suggest that ammonia speeds glutamate uptake both by increasing cytoplasmic pH and by a separate effect on
the glutamate transporter. Approximately two-thirds of the speeding is
due to the pH change.
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INTRODUCTION |
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In acute and chronic liver
disease, failure of ammonia detoxification is strongly implicated in
the etiology of hepatic encephalopathy (Mousseau and Butterworth
1994). Sudden deterioration into hepatic encephalopathy is
frequently precipitated by nitrogen loading, for example after a high
protein meal or during the digestion of blood proteins in the gut
following gastrointestinal hemorrhage. Brain levels of ammonia can then
rise to millimolar concentrations (Mousseau and Butterworth
1994
; Zieve et al. 1984
).
Ammonia can exert diverse effects on nervous tissue, including membrane
depolarization (Fan and Szerb 1993; Raabe
1990
), alteration of intracellular pH (Gillette
1983
), increased conversion of glutamate to glutamine
(Huang et al. 1994
; Waniewski 1992
), and
inhibition of glutaminase and respiratory enzymes (Cooper and
Plum 1987
). Particularly important may be a disturbance of
excitatory glutamatergic neurotransmission (Albrecht
1998
; Fan and Szerb 1993
;
Michalak and Butterworth 1997
; Raabe
1992
). The basal extracellular glutamate concentration is two
to three times normal in animal models of hepatic encephalopathy
(De Knegt et al. 1994
; Michalak et al. 1996
; Moroni et al. 1983
), although the total
brain glutamate level is reduced in such animals and in postmortem
brains of patients dying in hepatic encephalopathy (Bosman et
al. 1992
; Lavoie et al. 1987
; Record et
al. 1976
). The increased extracellular glutamate level could
reflect increased presynaptic release of glutamate or decreased uptake
of released transmitter by Na+-dependent
glutamate transporters, located predominantly in astroglial cells.
Studies that suggest an increased glutamate release (Butterworth et al. 1991
; Moroni et al. 1983
) have to be
interpreted with caution since apparent increases in release may
actually reflect reduced efficiency of the uptake mechanism.
A reduction in glutamate uptake when ammonia levels are persistently
raised has been seen in cultured astrocytes after prolonged exposure to
ammonium (Bender and Norenberg 1996), in synaptosomes from animals with experimental liver failure (Oppong et al.
1995
), and in brain slices from patients dying in hepatic
encephalopathy (Schmidt et al. 1990
). In rats with liver
failure, and in cultured astrocytes exposed to ammonia, there is
reduced expression of the glial glutamate transporters GLT-1 (the main
glutamate transporter in the brain) (Haugeto et al.
1996
) and GLAST, suggesting that the increased extracellular
glutamate level may result from decreased transporter expression
(Knecht et al. 1997
; Norenberg et al.
1997
; Zhou and Norenberg 1999
).
By contrast, acute ammonium exposure enhances glutamate uptake
(Bender and Norenberg 1996; Rao and Murthy
1991
). A better understanding of this may explain why
downregulation of uptake occurs after more prolonged ammonium exposure.
Rao and Murthy (1991)
found that
Na+-dependent uptake of both
[3H]L-glutamate and its
nonmetabolizable analogue
[3H]D-aspartate was increased in
rat cerebellar astrocyte, neuronal and synaptosome preparations exposed
to 5 mM ammonium, and also in the same preparations from rats made
acutely hyperammonaemic. The increase was due to an increase in
Vmax, with
Km remaining unchanged. However,
another study (Bender and Norenberg 1996
) reported that
5-10 mM ammonium chloride increased
[3H]L-glutamate accumulation in rat
cortical astrocytes in culture, while
[3H]D-aspartate accumulation was
unchanged. This differential response was attributed to the fact that
glutamate, but not aspartate, undergoes increased intracellular
conversion to glutamine in the presence of ammonium, catalyzed by
glutamine synthetase, which is expressed highly in astrocytes and
radial glial cells (Hertz et al. 1999
; Poitry et
al. 2000
). This is expected to increase the driving force for
glutamate uptake (although, since both L-glutamate and
D-aspartate are transported by the same carriers, a fall of intracellular glutamate concentration might increase the uptake of both
external substrates) and also to increase the fraction of labeled
glutamate (but not D-aspartate) taken up, which is retained
within the cell as glutamine rather than leaving the cell again by
exchange on the uptake carriers.
To clarify how acute ammonia exposure potentiates glutamate
accumulation, we studied L-glutamate uptake in whole cell
voltage-clamped salamander retinal glial cells, which mainly express a
glutamate transporter homologous to the mammalian GLAST/EAAT1
(Eliasof et al. 1998; Spiridon et al.
1998
). Three Na+ ions and a proton are
carried into the cell with glutamate, in exchange for one
K+ ion (Levy et al. 1998
;
Zerangue and Kavanaugh 1996
), so glutamate transport can
be monitored as a net inward current using the whole cell patch-clamp
technique (Brew and Attwell 1987
).
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METHODS |
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Salamander retinal glial cells
Tiger salamanders were killed in accordance with United Kingdom
regulations (brain concussion followed by immediate destruction of the
brain). Glial (Müller) cells were isolated from their retinae
using papain (Barbour et al. 1991), and whole cell
clamped with pipettes of series resistance (in whole cell mode) ~3
M
, giving series resistance voltage errors <2 mV.
Solutions
NH4Cl, methylamine hydrochloride, and
trimethylamine hydrochloride were prepared freshly as 1-M stock
solutions. Adding these to the experimental solutions did not alter the
pH significantly. Unless otherwise stated, the extracellular solution
contained (in mM) 105 NaCl, 2.5 KCl, 3 CaCl2, 0.5 MgCl2, 15 glucose, 5 HEPES, and 6 BaCl2 (to block inward rectifier
K+ channels), with pH adjusted to 7.4 with NaOH.
Control experiments (described in RESULTS) showed that
Ba2+ had no effect on intracellular pH changes
produced by ammonia, implying that there is no detectable flux of
NH-free
solutions, the Cl
was replaced by gluconate.
Na+-free and K+-free
extracellular solutions contained choline chloride instead of NaCl and
KCl, respectively. When replacing internal K+
with NH
). High buffering power internal solution contained (in mM) 50 KCl, 71 HEPES, 26 KOH, 5 NaCl, 5 K2EGTA, 1 CaCl2, 7 MgCl2, and 5 Na2ATP, pH
adjusted to 7.0 with KOH. For experiments with
pHi (pipette pH) of 8.8 or 6.0, 95 mM KCl and 5 mM HEPES in the standard intracellular solution were replaced with 50 mM KCl, 30 mM KOH, and either 66 mM
N-tris(hydroxymethyl) aminopropane sulfonic acid (TAPS) or
66 mM MES, respectively (the pH inside the cell was within 0.2 units of
8.8 or 6.0) (Billups and Attwell 1996
). Extracellular
solutions for reversed uptake contained (in mM) 90 NaCl, 3 CaCl2, 0.5 MgCl2, 15 glucose, 5 HEPES, 6 BaCl2, and 0.1 ouabain (to
block any current contribution from the
Na+/K+ pump), plus either
20 choline-Cl (for the K+-free and
NH
Data analysis
Once in whole cell mode, cells were left for 2 min before
applying glutamate to ensure complete dialysis with the patch pipette solution. Glutamate uptake currents in the presence of ammonium chloride (or other drugs) were "bracketed" by preceding and
following control responses. The potentiation of the uptake current by
ammonium chloride was calculated by dividing the amplitude of the
uptake current in ammonium chloride by the average of the amplitudes of
the preceding and following control responses. When comparing results
between different cells (e.g., with and without
Cl present), cells studied in the two
conditions were interleaved to reduce variability (note that specimen
traces shown for different cells cannot be compared in absolute
amplitude because of differences in cell size). Data are presented as
means ± SE and statistical P values are from
two-tailed t-tests.
Measurement of intracellular pH
Cells were whole cell clamped with the pH-sensitive fluorescent
dye BCECF [2',7'-bis-(carboxyethyl)-carboxy-fluorescein, 96 µM]
added to the normal pipette solution for forward uptake. Cell autofluorescence was negligible compared with the BCECF signal. Fluorescence was excited at 490 nm, for which an acid pH shift decreases BCECF fluorescence, and emission at 530 nm was measured with
a photomultiplier. Glutamate and amine-evoked pH changes cause no
fluorescence change using excitation at the isosbestic wavelength of
440 nm (Bouvier et al. 1992). Calibrations using high
K+ solution containing nigericin (Boyarsky
et al. 1993
) showed a fractional change in the fluorescence (F)
of BCECF (excited at 490 nm), when changing the pH from 6.76 to 7.11 of
F/FpH=7.11 = 25.9 ± 1.3% (mean ± SE) in three cells, which is not
significantly different from the 27.2% predicted by the in vitro
calibration of Rink et al. (1982)
and the 25.1%
predicted by the Boyarsky et al. (1993)
calibration in
single astrocytes (P = 0.41 and 0.6, respectively). We
therefore estimated pH changes using the calibration of Rink et
al. (1982)
assuming an initial pH of 7.0 (the pH of the pipette
solution), for which a typical ammonia-evoked fluorescence increase of
30% implies an alkalinization of 0.3 units.
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RESULTS |
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Unless otherwise stated, results are given from experiments
conducted with pHi = 7.0 and
pHo = 7.4, at a holding potential between 60
and
70 mV. We use the terms ammonia and ammonium chloride
interchangeably: at pH 7.4 most (99%) of the ammonia present is in the
form of NH
Acute exposure to ammonia potentiates L-glutamate and D-aspartate uptake
Applying L-glutamate or D-aspartate evoked
an inward transporter-mediated current in salamander retinal glial
cells, as characterized previously (Barbour et al. 1991;
Brew and Attwell 1987
). Ammonium chloride potentiated
the current associated with uptake of both glutamate (Fig.
1A) and
D-aspartate (Fig. 1B). The potentiation had a
rapid onset, apparently as fast as the kinetics of the bath solution
changes, which occur within about 3 s in our system. The offset of
the potentiating effect was somewhat slower, and varied between cells,
taking up to 60 s to occur.
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The dependence of the potentiation of L-glutamate uptake on
the dose of NH4Cl could be roughly fitted by a
Michaelis-Menten curve, with an EC50 of 1.4 mM
and a maximum potentiation of 31% (Fig. 1C). At 5 mM
concentration, ammonium chloride potentiated the current produced by a
near-saturating glutamate dose (200 µM) by 26 ± 2%
(n = 61) and that produced by a near-saturating dose of
D-aspartate (100 µM) by 21 ± 2% (n = 12, P = 0.38 compared with the potentiation for
glutamate). In subsequent experiments this concentration of ammonia was
used, unless otherwise stated. Although previous work has found ammonia
to change only the maximum rate of uptake (Bender and Norenberg
1996; Rao and Murthy 1991
), we found that it
also significantly (P = 0.0035) decreased the apparent
affinity of the transporters, increasing the
Km for glutamate from 11 to 15 µM
(Fig. 2). The combination of the
decreased affinity with the increased
Vmax results in ammonia having little
effect on uptake at low glutamate doses, but an increase at doses above 5 µM (Fig. 2).
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Potentiating effect does not depend on glutamine synthetase activity
When 2 mM methionine sulfoximine (MSO), an inhibitor of glutamine synthetase, was included in the internal solution (Fig. 3) the potentiating effect of 5 mM ammonia on the 200 µM L-glutamate uptake current was not reduced (31 ± 5%, n = 10, P = 0.75 on 2-tailed t-test comparing with interleaved cells in the absence of MSO). Thus ammonia does not act by speeding the conversion of glutamate to glutamine. We therefore investigated other possible causes of the ammonia-induced increase in glutamate- and aspartate-evoked currents.
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Ammonia does not act just by potentiating the transporter-gated anion conductance
In addition to generating an inward current by co-transporting an
excess of Na+ and H+ into
the cell with each glutamate anion, glutamate transporters also
generate some current by activating an anion conductance when the
transporter cycles (Billups et al. 1996; Eliasof
and Jahr 1996
; Wadiche et al. 1995
). In our
experiments the chloride reversal potential is around 0 mV, so an
inward current is generated by Cl
efflux at
70 mV (lowering internal [Cl
] to 21 mM
decreases the glutamate-evoked current by 26%) (Billups at al.
1996
). A potentiation of this conductance could in principle generate an increased inward current, without any associated increase in glutamate transport. To test this we replaced both internal and
external chloride ions with the large nonpermeant anion gluconate. Abolition of the anion conductance in this way did not alter the ability of ammonium chloride to potentiate the glutamate (200 µM)
uptake current (Fig. 4): in the absence
of Cl
the potentiation was 23 ± 3%
(n = 6), as compared with 23 ± 3% (n = 7, P = 0.91) in interleaved cells
in the presence of Cl
. Thus ammonia potentiates
glutamate transport and not just the anion conductance activated during
transport.
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NH
We considered the possibility that NH).
|
NH
Replacement of internal K+ ions with
NH). We
wondered, therefore, whether the inhibition by internal
NH
, raising
[K+]o evokes an outward
current at depolarized potentials that is reduced by the presence of
200 µM external glutamate (which stops the transporter losing
glutamate at the outer membrane surface). A similar but smaller
reversed uptake current was produced by raising
[NH
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Could the potentiation of glutamate uptake by external ammonia be due
to internal NH
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(1) |
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(2) |
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(3) |
Role of intracellular pH changes in the potentiating effect
The amines methylamine and trimethylamine (5 mM) also increased
the glutamate (200 µM) uptake current, but by less than bracketing applications of ammonia (Fig. 6, A and D). The
potentiation produced by 5 mM methylamine and trimethylamine was
12.9 ± 1.0% (5 cells) and 17.8 ± 3.6% (9 cells),
respectively. A possible mechanism by which the amines and ammonia
might potentiate uptake is via an intracellular alkalinization, which
is known to increase the uptake current (Billups and Attwell
1996) probably by increasing the electrochemical gradient for
co-transported H+ ions [Fig. 6B,
redrawn from Billups and Attwell (1996)
]. The neutral
form of the amine or NH3 enters (as described
above for NH3) and binds intracellular
H+ to reform charged amine or
NH
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(4) |
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(5) |
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(6) |
We used the pH-sensitive fluorescent dye BCECF, loaded via the patch
pipette, to measure the pH changes that were actually produced by
ammonia and the amines (Fig. 6C). Ammonia (5 mM) increased the BCECF fluorescence by 33 ± 2% in five cells, similar to the ~31% increase predicted (see METHODS) for a 0.31 unit pH
increase. Without Ba2+ in the external solution
to block inward rectifier K+ channels, a similar
NH; Nagaraja and Brookes 1998
) where NH
-dependent transport and produce an
acidification. Methylamine and trimethylamine gave alkaline pH shifts
that were slightly smaller than (~0.94 of) those produced by ammonia
in the same cells (Fig. 6, C and D), possibly
because the extracellular concentration of the neutral form of these
molecules is lower, due to their higher pKa, so
they enter the cell slower than NH3 and
equilibrate less fully. In contrast to the similar pH changes produced
by the amines and ammonia, the potentiation of the uptake current produced by 5 mM methylamine and trimethylamine was only 50-60% of
that produced by ammonia in the same cells (Fig. 6D). An
intracellular alkalinization of 0.31 units is expected to increase the
uptake current by 18% (from Fig. 6B), which is similar to
the potentiation produced by methylamine and trimethylamine, but only
about two-thirds of the potentiation produced by ammonia. This suggests
that intracellular alkalinization can account for all of the
potentiation produced by the amines, but for only approximately
two-thirds of that produced by ammonia.
To try to test this further, we increased the buffering power of the
cell from its normal value of around 20 mM/pH unit (Bouvier et
al. 1992) to around 50 mM/pH unit, by raising the pipette
solution [HEPES] to 71 mM. This reduced by 37% the intracellular pH
change produced by ammonia (Fig. 6E). If all of the
potentiation were due to the pH change, the nonlinearity of Fig.
6B predicts that a 37% decrease of a 0.31 unit pH change
would reduce the potentiation by 32%. However, a smaller reduction
than this (~22%) is expected if only approximately two-thirds of the
potentiation is due to the pH change. Experimentally (Fig.
6E) raising the buffering power reduced the potentiation by
24% (not significant, P = 0.23) in 11 pairs of
interleaved cells studied with high and low buffering power.
When the intracellular (pipette) pH was lowered to 6.0, the
potentiation by ammonia increased to 128 ± 8% in seven cells
(Fig. 7, A and C).
This is consistent with part of the potentiation reflecting an
ammonia-evoked alkalinization, first because this alkalinization is
predicted to be larger when the internal pH is more acid (because more
of the NH3 entering is converted to NH
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DISCUSSION |
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We observe a stimulation of glutamate uptake by acute exposure to
ammonia in voltage-clamped glial cells, which is similar in magnitude
to that reported previously in radiotracing experiments (Bender
and Norenberg 1996; Rao and Murthy 1991
). An
early potentiation of glutamate accumulation by ammonia has been
attributed to ammonia stimulating the conversion of glutamate to
glutamine by glutamine synthetase (Bender and Norenberg
1996
; Poitry et al. 2000
). This would lower the
intracellular glutamate concentration and thus promote uptake and also
increase the fraction of labeled glutamate taken up that is retained
within the cell as glutamine rather than leaving by exchange on
transporters. However, the ammonia binding site on glutamine synthetase
is close to saturated at physiological values: Waniewski
(1992)
has shown that increases of ammonia concentration above
the physiological range (200 µM) have little effect on glutamine
formation and on glutamate concentration. Since we find a potentiation
of glutamate uptake even with glutamine synthetase inhibited, we
conclude that, at least in salamander Müller cells, ammonia can
stimulate uptake without acting as a substrate for glutamine synthetase.
We considered the possibility that NH
The glutamate uptake current is strongly increased by intracellular
alkalinization (Billups and Attwell 1996), probably
because alkalinization increases the driving force for entry of
co-transported H+ into the cell. Acute exposure
to ammonia raised the intracellular pH (Fig. 6C), as seen
previously in a number of cell types. This results from
NH3 rapidly crossing the cell membrane and
binding intracellular protons to form NH
; Thomas 1974
).
Can the alkalinizing effect of ammonia explain the potentiation of the
uptake current? At 5 mM, ammonia potentiated the
L-glutamate uptake current by 26%. From the data of
Billups and Attwell (1996
; replotted in Fig.
6B) such a potentiation could be produced by an alkaline
shift of 0.5 of a pH unit. This is much larger than the shift of about
0.25 units measured in other preparations on application of 5 mM
NH
;
Thomas 1974
), and larger than the 0.31 unit shift, which
we expect theoretically (see Eq. 6) and measure
experimentally (Fig. 6C). Thus, although a large fraction (~
What are the limitations of extrapolating these data to the effects of
ammonia in liver failure? Our experiments are on GLAST transporters,
and the properties of the more abundant glial GLT-1 transporter could
differ; in particular the dependence of its uptake rate on
intracellular pH has not been reported. Nevertheless, since GLT-1 has a
homologous structure and is known to co-transport protons (Levy
et al. 1998), its uptake rate is also likely to be increased by
intracellular alkalinization. During the application of ammonia,
cultured rat astrocytes show an alkalinization (a 0.66 unit pH change
with 20 mM ammonia) (Boyarsky et al. 1993
), that slowly
reduces as NH
).] Smaller alkaline shifts will occur in prolonged liver failure, both because of
NH
. This would only produce a 7%
increase in uptake current from the pH dependence in Fig. 6B
(and this will be reduced by an astrocyte depolarization observed by
Swain et al. in the shunted animals). However, the non-pH component of
ammonia's action will produce an extra potentiation, and the alkalinization is probably significantly larger at the onset of the
liver failure and after meals.
For hepatic encephalopathy, the decrease in glutamate uptake at late
times, caused by reduced transporter expression, is more important than
the initial increase of uptake. Transporter expression can be regulated
by cAMP and several growth factors (Zelenaia et al.
2000). Conceivably modulation of the latter pathways by NH
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ACKNOWLEDGMENTS |
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This research was supported by the Wellcome Trust and by a Marie Curie Fellowship from the European Union to P. Marcaggi.
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FOOTNOTES |
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Address for reprint requests: D. Attwell, Dept. of Physiology, University College London, Gower Street, London WC1E 6BT, UK (E-mail: D.Attwell{at}ucl.ac.uk).
Received 13 June 2000; accepted in final form 20 April 2001.
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REFERENCES |
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