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Research report
Alteration of mitochondrial calcium homeostasis by ammonia-induced
activation of NMDA receptors in rat brain in vivo
a a a b ,
*
Elena Kosenko , Yury Kaminsky , Irina G. Stavroskaya , Vicente Felipo
a
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Russia
b
´ ´
Instituto de Investigaciones Citologicas, Fundacion Valenciana de Investigaciones Biomedicas, Valencia, Spain
Accepted 2 August 2000
Abstract
The aim of the present work was to assess the effects of activation of NMDA receptors in rat brain in vivo on calcium homeostasis in isolated non-synaptic brain mitochondria. We have shown recently that acute intoxication with large doses of ammonia leads to activation of NMDA receptors in rat brain in vivo. In the present work we injected rats with ammonium acetate to activate NMDA receptors in vivo and isolated non-synaptic mitochondria to assess calcium homeostasis. We also tested whether blocking NMDA receptors with MK-801 prevents effects on calcium homeostasis induced by ammonium injection. It is shown that activation of NMDA receptors in rat brain in vivo leads to a rapid increase in intramitochondrial calcium content followed by a reduction in the calcium capacity and calcium uptake rate in rat brain mitochondria. Activation of NMDA receptors resulted in increased spontaneous calcium efflux from rat brain mitochondria and in a strong inhibition of Na-induced and tert-butylhydroperoxide-induced calcium efflux. All these effects were prevented by previous blocking of NMDA receptors by injection of MK-801. Cyclosporin A did not affect any of the above parameters, indicating that the mitochondrial permeability transition pore does not play a role in calcium efflux under any of the conditions studied. The results reported indicate that ammonia-induced activation of NMDA receptors in rat brain in vivo alters mitochondrial calcium homeostasis at several different steps. 2000 Elsevier Science B.V. All rights reserved.
Theme: Excitable membranes and synaptic transmission
Topic: Calcium channels physiology, pharmacology and modulation
Keywords: Hyperammonemia; Excitotoxicity; Ammonia toxicity; Glutamate; Ischemia
1. Introduction degeneration is therefore of great interest. It is well known
21
that increased intracellular Ca is an essential initial step Glutamate is the main excitatory neurotransmitter in in the process of glutamate neurotoxicity [16,22,33]. mammals; however, excessive activation of glutamate Mitochondria are implicated at multiple stages of
gluta-21
receptors is neurotoxic, leading to neuronal degeneration mate neurotoxicity, including the sequestration of Ca and death. Glutamate neurotoxicity is involved in the entering via the NMDA receptor, the bioenergetic collapse origin of several neurodegenerative diseases (amyotrophic that triggers the ischemic release of glutamate, the gene-lateral sclerosis, Huntington’s disease, etc.) and in neuronal ration of reactive oxygen species and, the triggering of an damage found in cerebral ischemia. In many systems, apoptotic cascade under certain circumstances (see Ref. glutamate neurotoxicity is mainly mediated by excessive [26] for a review).
activation of the NMDA type of glutamate receptors Excessive activation of NMDA receptor leads to the
21
[16,23,35]. opening of its ion channel allowing the entry of Ca from The clarification of the molecular mechanisms by which the extracellular space into the cytoplasm of the
post-21
excessive activation of NMDA receptors leads to neuronal synaptic neuron [9]. Much of this Ca entering the cell is sequestered by mitochondria [26].
The uptake of calcium by the mitochondria is driven by *Corresponding author. Tel.: 134-96-339-1250; fax: 1
34-96-360-the mitochondrial membrane potential and will compete 1453.
E-mail address: [email protected] (V. Felipo). with the mitochondrial ATP synthase for protons. It can be
predicted that mitochondrial ATP synthesis will cease as with 2 mg / kg of MK-801 and killed 25 min later.
21
the Ca floods from the cytoplasm to the matrix [26]. Mitochondria were isolated from forebrain in batches of The inner mitochondrial membrane possesses a unipor- two brains essentially as previously described [18], except
21
ter able to transport Ca into the matrix. The high that 1 mM EGTA was included in homogenization mitochondrial membrane potential allows accumulation of medium instead of EDTA. Mitochondria (about 1 mg large amounts of calcium into mitochondria. In the brain, protein / ml medium) were incubated in a medium con-the inner membrane possesses a separate efflux pathway taining 65 mM KCl, 125 mM sucrose, 10 mM Hepes (pH
21 1 1 1 1
which exchanges Ca for Na . A mitochondrial Na / H 7.2), 0.2 mM P , 5 mM succinate-Ki and 1 mM rotenone. transporter is also present, and the overall ion flux under
21
conditions of constant mitochondrial Ca loading consists 2.3. Calcium transport in mitochondria
21 1 1
of sequential Ca , Na and H cycling, the last driven by
21
the respiratory chain (see Ref. [26] for a review). After external Ca was accumulated by mitochondria
21
In the presence of physiological concentrations of from nominally Ca -free medium and steady state was
21 21
phosphate, an osmotically inactive Ca –phosphate com- established, a sufficient amount of exogenous Ca was
21 21
plex forms in the matrix. When there is sufficient Ca and added (75 nmol Ca / mg mitochondrial protein) to induce
21 21 1
phosphate to form this complex, mitochondria behave as the efflux of Ca from Ca -loaded mitochondria. Na
21
excellent buffers of extramitochondrial Ca , accumulat- and tert-butylhydroperoxide (tBH) were added after
initia-21
ing Ca when its concentration is higher than the set tion of the efflux. In the assays containing cyclosporin A, point at which uptake and efflux balance and releasing this was added to the medium before addition of
mito-21
Ca below this value [26]. chondria.
21
The increase in intramitochondrial Ca would decrease Calcium transport was monitored directly in the
mito-21
ATP synthesis and affect the function of different enzymes chondria incubation medium by using a Ca -selective and of the respiratory chain and lead to alterations in the electrode (Orion 93-20) with a KCl reference electrode in activities of the calcium transport systems. a thermostated cuvette at 258C.
The aim of the present work was to assess the effects of The calcium content of mitochondria (available for activation of NMDA receptors in rat brain in vivo on the release) was estimated by adding the ionophore A23187 homeostasis of calcium in isolated non-synaptic mito- (0.15 nmol / mg protein) and carbonyl cyanide-p-chloro-chondria. We have shown recently that acute intoxication phenylhydrazone (10 mM) according to Wingrove and with large doses of ammonia leads to activation of NMDA Gunter [38,39].
receptors in rat brain in vivo [16]. In the present work we Mitochondrial swelling was measured by incubating
21
injected rats with ammonium acetate to activate NMDA mitochondria (1 mg protein / ml) with 70 nmol Ca / mg receptors in vivo and isolated non-synaptic mitochondria to protein and 5 mM potassium succinate in a thermostated assess calcium homeostasis. We also tested whether block- cuvette, and absorbance at 540 nm was registered continu-ing NMDA receptors with MK-801 prevents the effects on ously as described by Chappell and Crofts [8].
calcium homeostasis induced by ammonia-induced
activa-tion of NMDA receptors. 2.4. Enzymatic activities
Glutathione reductase (EC 1.6.4.2) and glutathione
2. Materials and methods peroxidase (EC 1.11.1.9) activities were determined
spec-trophotometrically at 340 nm [13,20]. To measure the 2.1. Materials activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 10 mM glucose 6-phosphate was added to the Ficoll 400, cyclosporin A, calcium ionophore A23187, assay mixture containing 0.1 M KCl, 20 mM Tris–HCl
1
rotenone, tert-butylhydroperoxide were from Sigma. The (pH 7.5), 5 mM MgSO , 0.5 mM NADP4 and 0.1 ml other reagents were from ICN Pharmaceuticals. brain extract (1 mg of cytosolic protein) in a total volume of 2.5 ml. The reaction was followed
spectrophotometrical-1
2.2. Treatment of rats and isolation of brain ly by reduction of NADP at 340 nm [17].
mitochondria Experiments with rats were carried out in accordance with the European Communities guidelines.
Male Wistar rats were used. Control rats were injected with 12 mmol / kg of sodium acetate and killed 15 min
later. Rats injected with ammonia were injected with 12 3. Results
mmol / kg of ammonium acetate and killed 15 min later.
Rats injected with MK-801 and ammonia were injected 3.1. Endogenous calcium in mitochondria with 2 mg / kg of MK-801; 10 min later were injected with
Table 1
21 a
Endogenous calcium, calcium capacity and influx and efflux rates of Ca in non-synaptic rat brain mitochondria
21
Endogenous Ca Ca capacity Uptake Ca efflux (nmol / min mg) (nmol / mg (nmol / mg (nmol / min
Spontaneous Spontaneous NaCl NaCl tBH tBH protein) protein) mg
1CSA 1CSA 1CSA
Control 1362 75610 7064 3.260.2 3.460.5 1861 2064 660.2 5.560.4 Ammonia 2163* 45611* 4065* 5.160.6* 5.161.0* 861* 962* 360.3* 4.260.6
MK-801 4.260.7* 10868* 7167 3.660.4 1564 861
MK-8011 4.360.8* 10666* 6961 2.960.3 1661 761
Ammonia
a
Rats were injected i.p. with either 12 mmol / kg of sodium acetate (control, n515) or 12 mmol / kg of ammonium acetate (ammonia, n515), or 2 mg / kg MK-801 (n55), or 2 mg / kg MK-801 with 12 mmol / kg ammonium acetate (n55). Brain nonsynaptic mitochondria (1 mg of protein) were added to 1 ml of the incubation medium consisting of 125 mM sucrose, 65 mM KCl, 10 mM Hepes, pH 7.2, 0.2 mM potassium phosphate, 5 mM potassium succinate
21
and 1mM rotenone. Incubation was carried out at 258C. When external Ca was accumulated by mitochondria and steady state was established, 75 nmol
21 21
Ca / mg protein was added to induce the efflux from Ca -loaded mitochondria. When indicated, 0.5mM cyclosporin a (CSA) was added to the medium
21 21
before addition of mitochondria; tert-butylhydroperoxide (tBH, 50 nmol / mg protein) was added to Ca -loaded mitochondria after Ca efflux was initiated. Calcium transport was followed as indicated in Section 2. Values are the mean6standard deviations of the number of samples indicated above. *Significantly different from controls; P,0.05.
brain mitochondria. Mitochondria from control rats con- ammonia injection significantly reduced the capacity of
21
tained 1362 nmol Ca / mg protein while mitochondria mitochondria to take up calcium and that this effect was from rats injected with ammonia contained 2163 nmol completely prevented by blocking NMDA receptors with
21
Ca / mg protein, with a significant increase (62%, P,
0.001). As shown in Table 1, injection of MK-801, an antagonist of NMDA receptor, significantly reduced intra-mitochondrial calcium in control rats, decreasing it from
21
1362 to 4.260.7 nmol Ca / mg protein. This suggests that tonic activation of NMDA receptors is responsible for a large proportion of the intramitochondrial calcium under normal conditions. The increase in intramitochondrial calcium induced by ammonia is completely prevented by previous blocking of NMDA receptors by injection of MK-801, indicating that ammonia-induced increase in mitochondrial calcium is mediated by activation of NMDA receptors.
21
3.2. Ca capacity and uptake
21
The maximal amount of Ca taken up and steady
21
retained by mitochondria is considered the Ca capacity of mitochondria. A typical result of the assays on the
21
effects of acute ammonia intoxication on the Ca capacity
21
and transport of Ca in rat brain non-synaptic mito-chondria is shown in Fig. 1. Under the experimental
1
conditions used (in the absence of Na and in the presence of low P concentration and with succinate as respiratoryi
substrate), brain mitochondria from control rats were able
21
to take up and retain 75610 nmol Ca / mg protein (n512). The calcium capacity of mitochondria was
sig-nificantly reduced by 40% (P,0.01) in rats injected with 21
Fig. 1. Effects of ammonia injection on Ca movements in rat brain ammonia, which were able to take up only 45611 nmol mitochondria. Mitochondria (1 mg protein) from control rats or from rats
21
Ca / mg protein (n520). For rats treated with MK-801, injected with ammonia were incubated in 1 ml of 65 mM KCl, 125 mM sucrose, 10 mM Hepes (pH 7.2), 0.2 mM potassium phosphate, 5 mM the calcium capacity of mitochondria was significantly
21 potassium succinate and 1mM rotenone. CaCl (75 nmol / mg protein)2
higher (10868 nmol Ca / mg protein). For rats treated
and NaCl (10 mM) were added when indicated by the arrows. Calcium with MK-801 before injection of ammonia, mitochondria transport was followed as indicated in Section 2. The result of a typical
21
MK-801. Moreover, blocking NMDA receptors with MK- dependent release of calcium at different concentrations of
21 1 21
801 in control rats also significantly increases the capacity Ca . As shown in Fig. 1, the rate of Na -dependent Ca of mitochondria to take up calcium. efflux from mitochondria from rats injected with ammonia
21
The rate of the energy-dependent Ca uptake by was significantly lower than the efflux from control
21
mitochondria is shown in Table 1. Mitochondria from mitochondria at all Ca concentrations tested.
21
control rats takes up 7064 nmol of Ca / min per mg of
21
protein. In mitochondria from rats injected with ammonia, 3.4. tert-Butylhydroperoxide-induced Ca efflux
the uptake rate was reduced by 43%. Blocking NMDA
receptors with MK-801 did not affect the uptake rate in Agents affecting the redox state of mitochondrial control rats but completely prevented the decrease in the pyridine nucleotides are known to cause changes in the
21
uptake rate induced by injection of ammonia (Table 1). rate and direction of Ca movement across the inner mitochondrial membrane. tert-Butylhydroperoxide (tBH)
1 21
3.3. Spontaneous and Na -dependent Ca efflux induces redox changes in mitochondria by acting at the level of glutathione peroxidase. Addition of tBH (50 nmol /
21 21 21
When the amount of Ca ions taken up by mito- mg protein) to Ca -loaded mitochondria after Ca efflux
21
chondria in the energy-dependent way is in excess to their was initiated resulted in 2-fold stimulation of Ca efflux
21
Ca capacity, then accumulated calcium will exit the from control mitochondria (Fig.2 and Table 1). On the loaded mitochondria spontaneously. As shown in Table 1, contrary, the addition of tBH did not change the rate of
21 21
the spontaneous Ca efflux rate was higher in mito- Ca efflux in mitochondria from hyperammonemic ani-chondria from rats injected with ammonia than in mito- mals (Fig. 2 and Table 1). Cyclosporin A, an inhibitor of chondria from control rats (5.160.6 vs. 3.260.2 nmol / min the permeability transition pore did not affect tBH-induced
21
per mg protein). The increase in spontaneous release of Ca efflux and this process was not associated with calcium induced by ammonia is completely prevented by swelling of mitochondria from control or hyperam-previous blocking of NMDA receptors with MK-801 monemic rats (not shown). Blocking NMDA receptors by (Table 1). injection of MK-801 before injecting ammonia prevents
21
It should be noted that the spontaneous Ca efflux was ammonia-induced ‘loss’ of sensibility to tBH.
independent of cyclosporin A both in control mitochondria These results indicate that excessive activation of and in those from rats injected with ammonia, indicating NMDA receptors in rat brain in vivo strongly impairs the that the mitochondrial permeability transition does not play release of calcium induced by tert-butylhydroperoxide
21
a role in calcium efflux under any of the conditions (Table 1 and Fig. 3). Hydroperoxide-stimulated Ca studied. The spontaneous release of calcium is not affected release requires active glutathione peroxidase [21] and by diltiazem or clonazepam, inhibitors of the Na / Ca glutathione reductase, to regenerate glutathione. It also exchange, thus indicating that spontaneous release takes requires glucose-6-phosphate dehydrogenase (G6PDH) to place by a mechanism which does not involve Na / Ca regenerate NADPH. We assessed whether ammonia-in-exchange nor the permeability transition pore. This mecha- duced loss of response to tBH may be due to a decrease in nism remains to be clarified.
Addition of NaCl after the beginning of spontaneous
21
Ca efflux results in a rapid increase of ca. 6-fold (563%
1 21
of the efflux in the absence of Na ) in Ca efflux from control mitochondria (Table 1). In mitochondria from rats
1 21
injected with ammonia addition of Na stimulated Ca
1
release only by 57%, indicating that Na -dependent
re-21
lease of Ca was reduced by ca. 90% by injection of ammonia.
1
In the presence of Na , the total efflux of calcium from mitochondria from rats injected with ammonia was sig-nificantly reduced and occurred at a lower rate (8 vs. 18 nmol / min per mg protein in controls; Table 1).
Blocking NMDA receptors with MK-801 completely
21
prevented the inhibition of the Na-dependent Ca efflux induced by ammonia (Table 1), indicating that this effect
1 21
Fig. 2. Effects of ammonium injection on Na -stimulated Ca efflux is mediated by activation of NMDA receptors. MK-801 did
1 21 from rat brain mitochondria. Experiments were carried out as in Fig. 1,
not affect Na -dependent Ca efflux in mitochondria 21
except that, different concentrations of Ca were added, as indicated. from control animals (Table 1). When steady state was obtained again, 10 mM NaCl were added to
1 21
1 21
As Na -dependent Ca efflux is affected by the initiate Na -dependent Ca efflux. Values are the mean6standard
21 1
peroxidase activity in mitochondria by ca. 50% but did not affect the activities of GSH-reductase and G6PDH. These results are in agreement with previous reports [19]. Previ-ous injection of MK-801 to block NMDA receptors completely prevented ammonia-induced changes in gluta-thione peroxidase (Table 2). This suggests that the impair-ment of the tBH-induced release of calcium in mito-chondria from rats injected with ammonia may be due to the reduced activity of glutathione peroxidase. We have also shown recently that the GSH levels and the GSH / GSSG ratio are decreased in mitochondria from rats injected with ammonia [19]. It seems therefore that externally added tBH cannot be reduced adequately in mitochondria from rats injected with ammonia due to decreased glutathione peroxidase activity and GSH level.
21
This may explain the inhibition of tBH-stimulated Ca efflux in rats injected with ammonia.
4. Discussion
We have shown recently, by using in vivo brain microdialysis in freely moving rats, that acute intoxication with large doses of ammonia (by intraperitoneal injection) leads to activation of NMDA receptors in rat brain in vivo [16]. We have now used this experimental approach to Fig. 3. Effect of ammonium injection on tert-butylhydroperoxide-induced assess the effects of activation of NMDA receptors in rat
21
Ca efflux from rat brain mitochondria. Experiments were carried out as brain in vivo on mitochondrial calcium homeostasis. in Fig. 1, except that tert-butylhydroperoxide (tBH, 50mM) was added
The results reported indicate that activation of NMDA instead of Na at the time indicated by the arrows. The result of a typical
receptors in rat brain in vivo by intraperitoneal injection of experiment is shown. Mean values for several experiments are given in
ammonia induces significant alterations in calcium content Table 1.
and homeostasis in brain mitochondria.
the activity of any of these enzymes in brain. Table 2 As expected, injection of ammonia (which activates shows mitochondrial glutathione peroxidase, glutathione NMDA receptors) increases the calcium content of mito-reductase and cytosolic glucose-6-phosphate dehydrogen- chondria (Table 1). This increase was completely pre-ase activities in brains of control rats and of rats injected vented by blocking NMDA receptors with MK-801. More-with ammonia. Injection of ammonia reduced glutathione over, injection of MK-801 alone significantly decreased
Table 2
a
Activities of glutathione reductase and glutathione peroxidase in mitochondria and glucose-6-phosphate dehydrogenase in the cytosol of rat brain
Enzyme Control Ammonia MK-801 MK-8011
Ammonia
(n59) (n59) (n54) (n54)
Glutathione reductase
(nmol / min per mg of 4663 4161 5262 4961
mitochondrial protein) Glucose-6-phosphate dehydrogenase (nmol / min per
mg of cytosolic protein) 4963 4961 5064 5466
Glutathione peroxidase with 5762 2562* 5169 5666
tert-butylhydroperoxide
Glutathione peroxidase with 3262 1962* 3067 2763
hydrogen peroxide
a
Rats were treated with ammonia, MK-801 or MK-8011ammonia as in Table 1. Activities of the enzymes were assayed as described in Section 2. Values are given as nmol of NADPH oxidized per minute per mg of protein. Values are the mean6standard deviations of nine preparations for controls and for rats injected only with ammonia and of four preparations for the groups injected with MK-801 or MK-8011ammonia.
21 1 1 1
the intramitochondrial content of calcium in control rats, Ca for Na . A mitochondrial Na / H transporter is also present, and the overall ion flux under conditions of indicating that basal tonic activation of NMDA receptors
21
constant mitochondrial Ca loading consists of sequential play a significant role in the modulation of mitochondrial
21 1 1
Ca , Na and H cycling, the last driven by the calcium concentration in rat brain. It has been reported that
respiratory chain. The strong reduction of the Na-depen-MK-801 have effects not limited to NMDA receptor
dent release of calcium in rats injected with ammonia may antagonism. For example, it has been shown that MK-801
by due to an impairment of this cycling process due to an can also block nicotinic receptors [1]. However, the
impairment of the respiratory chain, which has been contribution of other effects of MK-801 to the results
reported for mitochondria under these conditions [18]. A reported here can be ruled out because there is large
direct effect of excessive activation of NMDA receptors on evidence that the effects of acute ammonia intoxication in
the Na-dependent transporter of calcium cannot be ruled brain are mediated by activation of NMDA receptors and
out. prevented by at least 10 different antagonists of NMDA
Abundant evidence has been recently accumulated sup-receptors acting on three different sites of the receptor
porting the role of mitochondria in calcium homeostasis [16,15].
and signaling and in neuronal degeneration and neurologi-Activation of NMDA receptors by injecting ammonia
cal diseases [3,24,25,32,35]. In cultured neurons, exposure significantly reduced the calcium capacity of mitochondria
to NMDA induces the production of reactive oxygen and the calcium uptake rate (Table 1). Blocking NMDA
species [7,12,30] which is blocked by inhibitors of mito-receptors with MK-801 increased mitochondrial calcium
chondrial electron transport [7,12], supporting the possi-capacity. However, as shown in Table 1, MK-801 did not
bility that calcium-dependent uncoupling of neuronal affect the uptake rate. The increased calcium capacity in
mitochondria electron transport may contribute to the mitochondria from rats injected with MK-801 may be due
oxidative stress induced by glutamate and to the sub-to the reduced intramisub-tochondrial content (Table 1).
sequent neuronal death. Exposure of cultured neurons to These results suggest that physiological, basal activation
toxic glutamate stimulus also leads to accumulation of of NMDA receptors present in control rats modulates the
21
large quantities of Ca in mitochondria [36,28]. The intramitochondrial content of calcium but does not alter the
increase of calcium in mitochondria induced by NMDA transporter of calcium nor the uptake rate. In contrast,
cannot be observed in the presence of ruthenium red, an excessive pathological activation of NMDA receptors by
21
inhibitor of the mitochondrial Ca uniporter, or in the injection of ammonia leads to an increased content of
presence of compounds that breaks down the mitochon-calcium in mitochondria but also to a decrease in the
21
drial membrane potential necessary for the Ca uptake calcium capacity and calcium uptake rate (Table 1). This
21
[7,12,28]. Moreover, it has been reported that Ca suggests that excessive activation of NMDA receptors in
entering the neurons through NMDA receptors has a rat brain in vivo leads to a rapid increase in the uptake of
privileged access to mitochondria, when cultured neurons calcium by mitochondria, followed by an alteration of the
are treated with NMDA, increases in cytosolic and mito-transporter of calcium which results in a lower rate of
21
chondrial Ca show similar time courses and rate of calcium uptake and a reduced capacity to take up calcium.
changes. In contrast, when neurons are treated with other The increase in the spontaneous efflux of calcium from
21
compounds that increase cytosolic Ca (kainate, KCl, mitochondria from rats injected with ammonia may be due
21
ionomycin), mitochondrial Ca increases lagged behind to an adaptation of the transporter to reduce the initial
21
cytosolic Ca increase [29]. increase in intramitochondrial calcium in these
mitochon-21
Ca efflux from mitochondria contributes to the pro-dria (Table 1). It should be noted that the spontaneous
longed elevation of cytosolic calcium after glutamate release of calcium is the same in the presence or the
removal [36,37], and may contribute to the delayed absence of cyclosporin A, an inhibitor of the permeability
21
transition pore (Table 1), thus indicating that the release of cytoplasmic Ca deregulation [6]. Inhibitors of the mito-calcium through this pore does not play a significant role chondrial permeability transition pore only marginally
21
under normal conditions (controls) or following excessive delayed the onset of delayed cytoplasmic Ca deregula-activation of NMDA receptors in rat brain in vivo by tion [29]. A role for the mitochondrial Na / Ca exchanger in
21
injection of ammonia. The spontaneous release of calcium the modulation of the release of Ca from mitochondria is not affected by diltiazem or clonazepam, inhibitors of has been proposed [37].
21
the Na / Ca exchange, thus indicating that spontaneous Ca accumulation within mitochondria dissipates dc
release takes place by a mechanism which does not involve and leads to mitochondrial depolarization [5,31,34,36], Na / Ca exchange nor the permeability transition pore. This which is cyclosporin A-sensitive, indicating a possible role mechanism remains to be clarified. for the permeability transition pore [2,31,36]. Excessive
21
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calcium homeostasis with an initial increase in intramito- [15] C. Hermenegildo, G. Marcaida, C. Montoliu, S. Grisolıa, S.M.D.´ ˜
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Acknowledgements
Effects of acute hyperammonemia in vivo on oxidative metabolism in nonsynaptic rat brain mitochondria, Metab. Brain. Dis. 12 (1997) Supported in part by grants 98-04-48548 from the RFBR 69–82.
´
of Russia; and by grants from Ministerio de Educacion y [19] E. Kosenko, Y. Kaminski, O. Lopata, N. Muravyov, V. Felipo, Blocking NMDA receptors prevents the oxidative stress induced by ´
Cultura of Spain, Promocion General del Conocimiento
acute ammonia intoxication, Free Radic. Biol. Med. 26 (1999) ´
(SAF97-0001 and PM98-0065) and from Fundacio La
1369–1374. ´
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