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Chem. Res. Toxicol. 2002, 15, 1242-1247
Inhibition of the r-Ketoglutarate Dehydrogenase and Pyruvate Dehydrogenase Complexes by a Putative Aberrant Metabolite of Serotonin, Tryptamine-4,5-dione Xiang-Rong Jiang and Glenn Dryhurst* Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019 Received April 9, 2002
A transient energy impairment with resultant release and subsequent reuptake of 5-hydroxytryptamine (5-HT) and NMDA receptor activation with consequent cytoplasmic superoxide (O2-•), nitric oxide (NO•), and peroxynitrite (ONOO-) generation have all been implicated in a neurotoxic cascade which ultimately leads to the degeneration of serotonergic neurons evoked by methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA). Such observations raise the possibility that the O2-•/NO•/ONOO--mediated oxidation of 5-HT, as it returns via the plasma membrane transporter to the cytoplasm of serotonergic neurons when the MA/MDMA-induced energy impairment begins to subside, may generate an endogenous neurotoxin. In vitro the O2-•/NO•/ONOO--mediated oxidation of 5-HT forms tryptamine-4,5dione (T-4,5-D). When incubated with intact rat brain mitochondria, T-4,5-D strongly inhibits state 3 respiration with pyruvate or R-ketoglutarate as substrates at concentrations which do not affect succinate-supported (complex II) respiration. Experiments with freeze-thawed rat brain mitochondria reveal that T-4,5-D inhibits the pyruvate dehydrogenase and R-ketoglutarate dehydrogenase complexes. These and other properties of T-4,5-D raise the possibility that it may be an endogenously formed intraneuronal metabolite of 5-HT that contributes to the serotonergic neurotoxicity of MA and MDMA. Methamphetamine (MA)1 can evoke neurotoxic effects on serotonergic and dopaminergic neurons in selected regions of the brain (1). Although the underlying mechanisms are incompletely understood, a number of factors have been implicated as key participants in the neurotoxic cascades evoked by MA. These include a transient MA-induced impairment of ATP production by serotonergic and dopaminergic neurons (2, 3) caused by hyperthermia (4-6), poly(ADP-ribose) polymerase (PARP) activation (7), and other metabolic perturbations (8). This MA-induced energy impairment and resultant neuronal depolarization (9), interference with the vesicular storage of 5-hydroxytryptamine (5-HT; serotonin) and dopamine (DA) (10, 11), and reversal of the plasma transporters for these neurotransmitters (12-14) together mediate a massive release of 5-HT and DA (13, 15). After attaining peak levels, the extracellular concentrations of 5-HT and DA subsequently fall but without corresponding increases of their normal metabolites (16). Thus, as the MA-induced energy impairments subside, increasing ATP production initiates repolarization of the neuronal membranes which * Address correspondence to this author. Tel: (405) 325-4811. Fax: (405) 325-6111. E-mail:
[email protected]. 1 Abbreviations: ADP, adenosine 5′-diphosphate; ATP, adenosine 5′-triphosphate; DA, dopamine; DAT, dopamine transporter; DTT, dithiothreitol; Glu, L-glutamate; GSH, glutathione; 5-HT, 5-hydroxytryptamine; 5-HTT, 5-hydroxytryptamine transporter; KGDHC, R-ketoglutarate dehydrogenase complex; MA, methamphetamine; MDMA, 3,4-methylenedioxymethamphetamine; NMDA, N-methyl-D-aspartate; NO•, nitric oxide; ONOO-, peroxynitrite; nNOS, neuronal nitric oxide synthase; O2-•, superoxide anion radical; PARP, poly(ADP-ribose)polymerase; PDHC, pyruvate dehydrogenase complex; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; T-4,5-D, tryptamine-4,5-dione; TPH, tryptophan hydroxylase; TPP, thiamin pyrophosphate.
returns the reversed 5-HT transporter (5-HTT) and DA transporter (DAT) to their normal function such that they mediate the reuptake of released 5-HT and DA, respectively (17). Under conditions of impaired neuronal energy metabolism, N-methyl-D-aspartate (NMDA) receptors can be activated by endogenous extracellular L-glutamate (Glu). This, in turn, mediates an influx of Ca2+ through the NMDA receptor ion channel (18) with resultant superoxide (O2-•) production (19), activation of neuronal nitric oxide synthase (nNOS) and consequent nitric oxide (NO•), and then peroxynitrite (ONOO-) generation (20, 21). Indeed, NMDA receptor stimulation (22, 23), intraneuronal O2-• production (24), nNOS activation (25), and NO• and ONOO- generation (26) have been implicated in the serotonergic and dopaminergic neurotoxic mechanisms elicited by MA. Many of these factors have also been implicated with the neurotoxicity of 3,4-methylenedioxymethamphetamine (MDMA), although in most species this drug is a relatively selective serotonergic neurotoxin (8). A single dose of MA or MDMA evokes a rapid decrease of tryptophan hydroxylase (TPH) activity that subsequently recovers in vivo or can be restored in vitro by anaerobic reduction with dithiothreitol and ferrous iron (27-30). Thus, MA and MDMA may mediate oxidation of active site cysteine residues of TPH protein by reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) forming disulfide linkages that can be reduced both in vivo and in vitro, thus restoring activity (27-30). However, following multiple or large acute doses of MA or MDMA, the activity of TPH is not restored in vivo or in vitro prior to frank neuronal damage, implying
10.1021/tx020029b CCC: $22.00 © 2002 American Chemical Society Published on Web 09/05/2002
Mitochondrial Dehydrogenases and Tryptamine-4,5-dione
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irreversible damage to this enzyme (27-30). Both NO• (31) and ONOO- (32) irreversibly damage TPH in vitro, suggesting that these RNS might be responsible for its MA- or MDMA-induced irreversible inhibition. However, other factors implicated with MA- and MDMA-induced irreversible inhibition of TPH and serotonergic neurotoxicity are difficult to reconcile with this suggestion (33). For example, 5-HTT inhibitors (34, 35) block MA- and MDMA-induced irreversible inhibition of TPH and serotonergic neurotoxicity without affecting hyperthermia evoked by these drugs, a key factor associated with this neurotoxicity (5, 35, 36). This raises the possibility that the 5-HTT-mediated reuptake of released 5-HT and its intraneuronal oxidation by ROS/RNS as a MA- or MDMAinduced serotonergic energy impairment subsides may result in an endogenously formed toxin that contributes to irreversible TPH inhibition and neurotoxicity. A product of the oxidation of 5-HT by O2-• and ONOO- (37) is tryptamine-4,5-dione (T-4,5-D). Furthermore, in vitro T-4,5-D irreversibly inactivates rat brain TPH (33) and mitochondrial NADH-coenzyme Q1 (CoQ1) reductase (complex I) and cytochrome c oxidase (complex IV) (38) by covalent modification of active site cysteine residues. In this paper, we report that T-4,5-D inhibits the pyruvate dehydrogenase complex (PDHC; the link of glycolysis to the Kreb’s cycle) and the R-ketoglutarate dehydrogenase complex (KGDHC; the link of the Kreb’s cycle to Glu metabolism).
191.0821). 1H NMR (Me2SO-d6, 300 MHz) gave δ: 12.14 (bs, 1H, N(1)-H), 7.98 (bs, 3H, NH3+), 7.35 (d, J ) 9.9 Hz, 1H, C(7)H), 6.91 (s, 1H, C(2)-H), 5.92 (d, J ) 9.9 Hz, 1H, C(6)-H), 3.02 (t, J ) 7.2 Hz, 2H, C(β)-H2), 2.89 (t, J ) 7.2 Hz, 2H, C(R)-H). Rat Brain Mitochondrial Preparations. Rat brain mitochondria were prepared using a modification of the method of Bernard and Cockrell (39). Briefly, four male Sprague-Dawley rats (Harlan Sprague-Dawley, Madison, WI) weighing approximately 300 g were decapitated; forebrains were removed and immediately placed in ice-cold medium A (300 mM mannitol, 5 mM glycylglycine, and 0.1 mM K2EDTA, pH 7.4). Brains were minced with scissors and then rinsed several times with ice-cold medium A. The tissue was then transferred to a Wheaton Dounce-type tissue homogenizer (40 mL capacity) together with 20 mL of medium A and homogenized using 10 up-and-down strokes with the glass pestle (0.0035-0.0055 in clearance). Medium A (10 mL) was added to the homogenate which was then centrifuged at 1500g for 8 min at 2 °C (Beckman J2-HS centrifuge with a JA 17 rotor). The supernatant was removed and again centrifuged (1500g, 6 min, 2 °C). The supernatant was then centrifuged at 10000g for 10 min at 2 °C to give the crude mitochondrial pellet. After the supernatant and white layer were decanted, the pellet was resuspended in 25 mL of medium B (200 mM mannitol, 8 mM glycylglycine, 40 mM KCl, 4 mM NaCl, 70 mM K2EDTA, pH 7.4) and centrifuged at 1500g for 3 min at 2 °C. The supernatant was centrifuged at 8000g for 10 min at 2 °C to sediment the mitochondria. The pellet was carefully washed with medium A without disturbing the pellet. The final mitochondrial pellet employed for oxygen consumption studies was suspended in 300-350 µL of medium C (300 mM mannitol, 15 mM Trizma hydrochloride, 15 mM glycylglycine, and 0.1 mM K2EDTA, pH 7.4). This suspension of intact mitochondria was stored on ice and used on the same day it was prepared. For measurements of PDHC and KGDHC activities, the final mitochondrial pellet was suspended in 300 µL of medium D (250 mM sucrose, 10 mM Tris-HCl, 0.5 mM K2EDTA, pH 7.4). The latter mitochondrial preparations were stored at -80 °C until needed. Mitochondrial protein was determined by the method of Lowry et al. (40). Oxygen Electrode Studies. The rate of oxygen consumption by intact rat brain mitochondria was measured with a YSI (Yellow Springs Instrument Co., Yellow Springs, OH) model 5300 biological oxygen monitor equipped with a model 5357 Clark-type micro oxygen probe assembly thermostated at 30 °C. Mitochondria at a final concentration of 200-400 µg/mL protein were suspended in 600 µL of air-saturated medium E (300 mM mannitol, 10 mM Trizma hydrochloride, 10 mM KCl, 5 mM potassium phosphate, 0.1 mM K2EDTA, and 1 mg/mL BSA, pH 7.2) which contained known concentrations of T-4,5-D (HCl salt) contained in the oxygen electrode chamber. These solutions were incubated for 5 min at 30 °C, and then 2 µL of 0.75 M pyruvate or R-ketoglutarate (to give a final concentration of 2.5 mM) was added to initiate state 2 respiration. After 3 min, state 3 respiration was initiated by addition of 2 µL of 0.135 M ADP (final concentration of 0.45 mM). The rate of state 3 respiration (nanogram atoms of oxygen per minute per milligram of protein) was determined from the linear segment of the oxygen concentration versus time trace after addition of ADP. When the added ADP was consumed, state 4 respiration was monitored. Assays for PDHC and KGDHC Activities. Procedures for PDHC and KGDHC activity assays were based on those described by Tabatabaie et al. (41) and Lai and Cooper (42), respectively. Method I. This method was employed to monitor the time course of PDHC or KGDHC inhibition by T-4,5-D. Rat brain mitochondria were exposed to six freeze-thaw cycles immediately before assay. Freeze-thawed mitochondria (480 µg of mitochondrial protein in medium D) were incubated with (sample) or without (control) T-4,5-D in a total volume of 240 µL of 50 mM potassium phosphate buffer (pH 7.4) at 30 °C for times ranging from 0 to 80 min. Then, 40 µL of the resulting solution (containing 80 µg of mitochondrial protein) was trans-
Materials and Methods Chemicals. 5-HT, cysteine, glutathione (GSH), thiamin pyrophosphate (TPP), β-nicotinamide adenine dinucleotide (NAD+, sodium salt), adenosine 5′-diphosphate (ADP), Triton X-100, pyruvic acid (disodium salt), R-ketoglutaric acid (disodium salt), coenzyme A (lithium salt), dithiothreitol (DTT), glycylglycine, ethylenediaminetetraacetic acid (dipotassium salt, K2EDTA), mannitol, sucrose, Trizma hydrochloride, and bovine serum albumin (BSA, fatty acid free) were obtained from Sigma (St. Louis, MO). These reagents were of the highest purity commercially available. Synthesis and Characterization of T-4,5-D. 5-HT (creatine sulfate complex, 39 mg, 0.078 mmol) was dissolved in 20 mL of deionized water. Potassium nitrosodisulfonate (105 mg, 0.39 mmol) was then added and the solution stirred for 25 min. The resulting deep purple solution was filtered and the filtrate pumped directly into a Gilson (Middleton, WI) model 712 preparative HPLC system equipped with a reversed phase column (Bakerbond C18, 25 × 2.1 cm, 10 µm particle size, J. T. Baker, Phillipsburg, NJ) and a model HM Holochrome UV detector (set at 254 nm). Deionized water and HPLC grade methanol (MeOH) were employed as mobile phases with the following gradient: 0-40 min, linear gradient from 100% water to 50% MeOH; 42-47 min, 50% MeOH. The flow rate was 10 mL min-1. The solution eluted under the peak corresponding to T-4,5-D at a retention time (tR) ) 28.5 min and was collected, immediately frozen, and freeze-dried. The resulting solid was purified by preparative reversed phase HPLC, using the column described previously using deionized water adjusted to pH 3.5 with HCl (solvent A) and MeOH as the mobile phases. The gradient was: 0-40 min, linear gradient from 100% solvent A to 25% MeOH; 40-42 min, linear gradient to 50% MeOH; 42-47 min, 50% MeOH. The flow rate was 10 mL min-1. The solution eluted under the peak for T-4,5-D (tR ) 28 min) was collected, immediately frozen, and freeze-dried to give a hygroscopic red-brown fluffy solid. Elemental analysis gave C(48.40%), H(5.18%), N(10.85%), Cl(13.98%), corresponding to T-4,5-D‚HCl‚ 1.2H2O (calcd 48.37% C, 5.40% H, 11.29% N, and 14.30% Cl). Fast atom bombardment mass spectrometry (glycerol matrix) gave m/z ) 191.0825 (MH+, 7%, C10H11N2O2; calcd m/z )
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Jiang and Dryhurst Table 1. Effect of T-4,5-D on State 3 Oxygen Consumption by Rat Brain Mitochondria with r-Ketoglutarate as Substrate
Figure 1. Effects of T-4,5-D on ADP-stimulated (state 3) pyruvate-supported respiration by rat brain mitochondria. In all tracings, ca. 300 µg of rat brain mitochondrial protein was incubated for 5 min at 30 °C in 600 µL of medium E in the absence (A) and presence of (B) 25 µM and (C) 50 µM T-4,5-D. Pyruvate (P, 2.5 mM) was then added and, after 3 min, 0.45 mM ADP. The numbers beside each tracing represent the rate of oxygen utilization in ng atoms of oxygen min-1 (mg of protein)-1. In tracing C, 5 mM succinate(s) was added at the indicated time. ferred into the assay solution consisting of 500 µL of 50 mM phosphate buffer (pH 7.4) containing 6 mM NAD+, 2 mM MgCl2, and 0.4 mM TPP. Then, 50 µL of 200 mM pyruvate or R-ketoglutarate, 50 µL of 2% Triton X-100, and 60 µL of 5 mM DTT, all dissolved in 50 mM phosphate buffer (pH 7.4), were added followed by 200 µL of phosphate buffer (pH 7.4). This solution, contained in an optical quartz UV cell (0.5 cm optical path length), was incubated for 5 min at 30 °C, and then 100 µL of 6 mM coenzyme A (in 50 mM phosphate buffer, pH 7.4) was added to initiate the reaction. The activity of PDHC or KGDHC was determined by monitoring the increase of NADH concentration (nanomoles of NADH per minute per milligram of mitochondrial protein) at 340 nm with a Beckman DU 640 spectrophotometer. The molar absorptivity of NADH was taken as 6.22 mM-1 cm-1. Method II. This method was used to investigate the effects of various chemicals and enzymes on the inhibition of PDHC or KGDHC by T-4,5-D. Freeze-thawed rat brain mitochondria (400 µg of protein) in a total volume of 200 µL of 50 mM potassium phosphate buffer (pH 7.4) were incubated with (sample) or without (control) T-4.5-D at 30 °C in the presence of known concentrations of cysteine, GSH, ascorbic acid (AA), SOD, or catalase. The latter antioxidants or enzymes were always added to the mitochondrial membrane preparation prior to T-4,5-D. After 60 min incubation, 40 µL aliquots of the resulting solutions (containing 80 µg of mitochondrial protein) were added to the usual assay solution to determine the activity of PDHC or KGDHC as described in method I. Statistics. All results were obtained at least in triplicate and are presented as mean ( SEM. Student’s t-test was used to determine statistical significance, with p < 0.05 being taken to indicate a significant difference.
Results Effects of T-4,5-D on Pyruvate- and R-Ketoglutarate-Supported State 3 Mitochondrial Respiration. Oxidative phosphorylation using the rat brain mitochondrial preparations employed was well-coupled with pyruvate as substrate (Figure 1, tracing A), the respiratory control ratio being g8 (ratio of ADP-stimulated state 3 oxygen utilization to that measured after ADP had been converted to ATP, i.e., state 4 respiration). In experiments using R-ketoglutarate as substrate, the respiratory control ratio was g6.5. Incubation of 25 µM T-4,5-D with intact rat brain mitochondria for 5 min followed by addition of pyruvate evoked a small, but significant increase of respiration
T-4,5-D concn (µM)
rate of state 3 oxygen uptakea,b [ng atoms of oxygen min-1 (mg of protein)-1]
0 25 50 100
99.2 ( 8.8 87.9 ( 11.3 58.7 ( 8.3c 13.7 ( 0.7d
a Measured with a Clark-type oxygen electrode assembly. Rat brain mitochondria (300 µg of protein/mL) were incubated in 600 µL of medium E with the indicated concentration of T-4,5-D for 5 min at 30 °C. R-Ketoglutarate (2.5 mM) was then added and, after 3 min, ADP (0.45 mM) to stimulate state 3 respiration. b Data are mean ( SEM (n ) 6) at each T-4,5-D concentration. c p < 0.05.d p < 0.0005.
prior to the addition of ADP, (i.e., state 2 respiration) from a control value of 10.2 ( 0.5 (n ) 16) to 13.4 ( 1.0 ng atoms of oxygen min-1 (mg of protein)-1 (n ) 7; p < 0.005) (Figure 1, tracing B). The same concentration of T-4,5-D also decreased state 3 respiration from a control value of 81.1 ( 7.7 (n ) 16) to 32.3 ( 2.7 ng atoms of oxygen min-1 (mg of protein)-1 (n ) 7; p < 0.005) (Figure 1, tracing B). Using the same experimental paradigm, 50 µM (or greater concentrations) T-4,5-D also increased ADP-independent respiration to 12.7 ( 0.8 ng atoms of oxygen min-1 (mg of protein)-1 but completely blocked pyruvate-supported state 3 respiration (Figure 1, tracing C). Addition of succinate to intact rat brain mitochondria in which pyruvate supported state 3 respiration was inhibited by T-4,5-D-restored oxygen utilization (Figure 1, tracing C). Indeed, succinate-supported state 3 oxygen utilization of mitochondria exposed to 25 µM T-4,5-D [111.09 ( 11.1 ng atoms of oxygen min-1 (mg of protein)-1, n ) 7] or 50 µM T-4,5-D [86.74 ( 8.59 ng atoms of oxygen min-1 (mg of protein)-1, n ) 10] was not statistically different from that measured for control mitochondria which had not been exposed to the dione [79.84 ( 5.07 ng atoms of oxygen min-1 (mg of protein)-1]. Incubation of rat brain mitochondria for 5 min with 25 and 50 µM T-4,5-D followed by addition of R-ketoglutarate also caused an increase of ADP-independent respiration to 18.25 ( 2.4 and 20.27 ( 1.78 ng atoms of oxygen min-1 (mg of protein)-1 (n ) 6), respectively, compared to 15.84 ( 1.38 ng atoms of oxygen min-1 (mg of protein)-1 (n ) 11) measured in the absence of the dione. However, this small T-4,5-D-mediated increase in state 2 respiration did not reach statistical significance. State 3 mitochondrial respiration was significantly inhibited by T-4,5-D when R-ketoglutarate was used as the substrate (Table 1). However, >50 µM T-4,5-D was necessary to completely inhibit R-ketoglutarate-supported state 3 respiration. Addition of succinate to T-4,5-D (20-100 µM)-inhibited mitochondria in these experiments restored oxygen consumption as described previously. Effect of T-4,5-D on PDHC Activity. When incubated with freeze-thawed rat brain mitochondria (mitochondrial membranes) for 1 min, T-4,5-D (g25 µM) evoked an almost immediate and significant decrease of PDHC activity (Figure 2). With time, PDHC activity decreased further. A large molar excess of cysteine or glutathione (GSH) blocked the inhibition of PDHC by T-4,5-D, and ascorbate significantly attenuated this inhibition (Table 2). In contrast, superoxide dismutase (SOD) and catalase had
Mitochondrial Dehydrogenases and Tryptamine-4,5-dione
Figure 2. Time-dependent effects of T-4,5-D on PDHC activity. Rat brain mitochondrial membranes (2 µg of protein/µL) were incubated in 50 mM potassium phosphate buffer (pH 7.4) in the absence (control) and presence of T-4,5-D at the indicated concentrations. Aliquots of this solution were assayed at various times for PDHC as described in method I under Materials and Methods. Data are mean ( SEM (vertical bars) (n g 3). *p < 0.05. Table 2. Effects of Antioxidants and Enzymes on the Inhibition of Rat Brain Mitochondrial PDHC Activity by T-4,5-D T-4,5-D concn (µM)
added antioxidant or enzyme (µM)
PDHC act.a [% of control (concn) act.]b
0 100 100 100 100 100 100 100 100
cysteine (10 mM) GSH (10 mM) ascorbic acid (10 mM) SOD (100 units) catalase (500 units) cysteine (3 mM), ascorbic acid (3 mM) GSH (3 mM), ascorbic acid (3 mM)
100 ( 8.7c 12.8 ( 5.8 10.8 ( 11.2 75.2 ( 3.4d 58.3 ( 3.0d 29.0 ( 4.1e 35.4 ( 5.8e 86.3 ( 8.7 105.2 ( 10.1
a
Frozen and thawed rat brain mitochondria (2 µg of protein/ µL) were incubated at 30 °C in 50 mM potassium phosphate buffer (pH 7.4) with the indicated concentration of T-4,5-D and antioxidant or enzyme. After 60 min, the activity of PDHC was assayed using method I described under Materials and Methods. b Data are mean ( SEM (n ) 3). c Control activities ranged from 31.8 ( 1.1 to 70.7 ( 5.3 nmol of NADH min-1 (mg of protein)-1. d p < 0.05. e p < 0.001.
no significant influence on the inhibition of PDHC by T-4,5-D. Effect of T-4,5-D on KGDHC Activity. T-4,5-D evoked a rapid inhibition of KGDHC when incubated with rat brain mitochondrial membranes (Figure 3). At the T-4,5-D concentrations employed (50-500 µM), the inhibition of KGDHC decreased further for ca. 20 min and then remained essentially constant. Large molar excesses of cysteine and GSH blocked the inhibition of KGDHC by T-4,5-D, and ascorbate significantly attenuated this inhibition (Table 3). However, the antioxidant enzymes SOD and catalase did not significantly affect the inhibition of KGDHC by T-4,5-D (Table 3).
Discussion The initial step in the neurotoxic cascade evoked by both MA and MDMA which leads to the degeneration of serotonergic neurons appears to be an energy impairment that induces a massive release of 5-HT (8, 13) in large part because of plasma 5-HTT reversal (12-14). As the energy impairment begins to subside, increasing ATP production initiates reactivation of Na+/K+ ATPase with
Chem. Res. Toxicol., Vol. 15, No. 10, 2002 1245
Figure 3. Time-dependent effects of T-4,5-D on KGDHC activity. Rat brain mitochondrial membranes (2 µg of protein/ µL) were incubated in 50 mM potassium phosphate buffer (pH 7.4) in the absence (control) or presence of the indicated concentration of T-4,5-D. Aliquots of this solution were assayed at various times for KGDHC activity as described in method I under Materials and Methods. Data are mean ( SEM (vertical bars) (n ) 5). *p < 0.05; **p < 0.001; ***p < 0.0005. Table 3. Effects of Antioxidants and Enzymes on the Inhibition of Rat Brain Mitochondrial KGDHC Activity by T-4,5-D T-4,5-D concn (µM)
added antioxidant or enzyme (concn)
KGDHC act.a (% of control act.)b
0 100 100 100 100 100 100 100 100
cysteine (10 mM) GSH (10 mM) ascorbic acid (10 mM) SOD (100 units) catalase (500 units) cysteine (10 mM), ascorbic acid (10 mM) GSH (10 mM), ascorbic acid (10 mM)
100 ( 14.3c 12.3 ( 4.0e 89.3 ( 7.8 97.1 ( 10.1 58.9 ( 7.8d 22.0 ( 4.3f 25.0 ( 7.1d 91.3 ( 14.1 83.6 ( 8.1
a Frozen and thawed rat brain mitochondria (2 µg of protein/ µL) were incubated at 30 °C in 50 mM potassium phosphate buffer (pH 7.4) with the indicated concentration of T-4,5-D and antioxidant or enzyme. After 60 min, the activity of KGDHC was assayed using method I described under Materials and Methods. b Data are mean ( SEM. c Control activities ranged from 44.6 ( 4.8 to 56.6 ( 3.2 nmol of NADH min-1 (mg of protein)-1. d p < 0.05. e p < 0.005. f p < 0.0005.
resultant membrane repolarization which returns the reversed 5-HTT to its normal function and initiates reuptake of released 5-HT into the cytoplasm of serotonergic neurons. During this period, MA, but not MDMA, evokes elevation of extracellular Glu (43), possibly the result of reversal of glial Glu transporters (44). Nevertheless, during the period of recovering but reduced serotonergic energy metabolism, endogenous or elevated extracellular levels of Glu and NMDA receptor activation would be expected to mediate intraneuronal O2-•, NO•, and then ONOO- generation. Oxidation of 5-HT as it returns via the 5-HTT to the cytoplasm of serotonergic neurons by such ROS/RNS would be expected to form T-4,5-D (37). This putative cytoplasmic metabolite of 5-HT is extremely electrophilic and reacts particularly avidly with sulfhydryl-containing nucleophiles such as cysteine, GSH (45), and the cysteinyl residues of a number of enzymes including alcohol dehydrogenase (37), guanine nucleotide binding regulatory proteins (46), TPH (33), and mitochondrial complex I (38). The present study demonstrates that when incubated briefly with intact rat brain mitochondria, T-4,5-D partially uncouples respiration and inhibits both pyruvate- and R-ketoglutaratesupported state 3 respiration. Using rat brain mitochondrial membrane preparations, it is also demonstrated
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that T-4,5-D inhibits both PDHC (Figure 2) and KGDHC (Figure 3), both of which contain active site cysteine residues (41, 47, 48). Large molar excesses of cysteine or GSH greatly attenuate the inhibition of PDHC and KGDHC by T-4,5-D. This is presumably because T-4,5-D is rapidly scavenged by GSH and CySH, forming, initially, 7-S-substituted thioethers (45, 49). Taken together, these observations suggest that T-4,5-D probably inhibits PDHC and KGDHC and/or their dihydrolipoate cofactor by covalent modification of essential cysteine residues. Studies with intact rat brain mitochondria indicate that the inhibition of pyruvate- or R-ketoglutarate-supported state 3 respiration by g25 µM or g50 µM T-4,5-D, respectively, is significant after 5 min incubation with the latter dione. Similarly, almost immediately after exposure of mitochondrial membranes to T-4,5-D, the activities of both PDHC and KGDHC are significantly reduced (Figures 2 and 3). The proposition that the rapid inhibition of pyruvate- and R-ketoglutarate-supported state 3 mitochondrial respiration and of PDHC and KGDHC may be the result of covalent modification of active site cysteine residues by T-4,5-D is consistent with the very facile reaction of the dione with sulfhydryl nucleophiles (45, 49). The data presented in Figures 2 and 3 indicate that the initial rapid inhibition of PDHC and KGDHC is followed by a slower more minor increase in the extent of inhibition over the next 80 min. The latter effect is probably due largely to the normal activity decay over time for PDHC and KGDHC in vitro. However, it is also conceivable that T-4,5-D also reacts more slowly with less accessible cysteinyl residues or other active site nucleophilic residues of PDHC and KGDHC, thus accounting for the apparent biphasic inhibitory effect on the activity of these complexes. The proposed covalent modification of cysteine or other nucleophilic residues of PDHC or KGDHC would be expected to cause irreversible inhibition of these enzyme complexes. However, the present experimental results do not permit a definitive conclusion concerning such irreversible inhibition. Large molar excesses of GSH greatly attenuate the inhibition of PDHC and KGDHC by T-4,5-D in vitro. Thus, in order for T-4,5-D to potentially play a role as an inhibitor of PDHC, KGDHC, and, indeed, other essential enzymes containing active site cysteine residues in vivo, it would clearly be necessary for intraneuronal GSH to be severely depleted. It is of relevance, therefore, that neuronal depolarization (50) and particularly more severe energy impairments such as those evoked by mitochondrial poisons (51, 52), ischemia (53, 54), MA (2, 3), and MDMA (8) induce a massive release of intraneuronal GSH. Using microdialysis techniques, for example, it has been shown that perfusion of neurotoxic concentrations of the dopaminergic neurotoxicant 1-methyl-4phenylpyridinium, a reversible mitochondrial complex I inhibitor, into the rat striatum results in a > 50-fold rise of extracellular GSH levels (52) indicative of a massive release and hence depletion of intraneuronal GSH. Whether intraneuronal ROS/RNS-mediated oxidation of 5-HT to T-4,5-D plays a role in the serotonergic neurotoxicity of MA and MDMA remains to be established. Furthermore, efforts to detect free T-4,5-D in the brains of rats exposed to neurotoxic doses of MA or MDMA are almost certain to be unsuccessful owing to the very rapid reaction of this highly electrophilic species with protein (33) and low molecular weight nucleophiles (49). Nevertheless, it has been estimated that the con-
Jiang and Dryhurst
centration of 5-HT in serotonergic terminals is at least 7 mM, and the concentration within vesicles must be much higher (55). Thus, the massive release of 5-HT evoked by a MA- and MDMA-induced energy impairment and its subsequent 5-HTT-mediated reuptake into the cytoplasm of serotonergic terminals and oxidation by O2-•/ ONOO- could well produce T-4,5-D at concentrations necessary to inhibit PDHC, KGDHC, and other essential proteins containing active site cysteinyl residues. In the event that T-4,5-D is formed, its ability to (weakly) uncouple mitochondrial respiration, inhibit NADH-CoQ1 reductase, cytochrome c oxidase, PDHC, and KGDHC together would be expected to severely impair mitochondrial ATP production by serotonergic neurons. This, in turn, would set the stage for Glu-mediated excitotoxicity and the eventual death of serotonergic neurons (56).
Acknowledgment. This work was supported by NIH Grant GM32367.
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