Materials and methods peroxidase EC 1.11.1.9 activities were determined spec- Results

140 E 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-K and 1 mM rotenone. i 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 NADP and 0.1 ml 4 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 12 mmol kg of ammonium acetate and rats were killed 15 As shown in Table 1, acute intoxication with ammonia min later. Rats injected only with MK-801 were injected induced a significant increase in the content of calcium in E . Kosenko et al. Brain Research 880 2000 139 –146 141 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 1 mM 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, 0.5 mM 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 respiratory i 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 1 mM 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 can take up to 10666 nmol Ca mg protein. This experiment is shown. Mean values for several experiments are given in indicates that activation of NMDA receptors in vivo by Table 1. 142 E 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 internal Ca concentration [10,11], we measured Na - deviations of three different experiments. E . Kosenko et al. Brain Research 880 2000 139 –146 143 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