J Mol Neurosci (2013) 49:211–222 DOI 10.1007/s12031-012-9913-3
Influence of Intra- and Extracellular Acidification on Free Radical Formation and Mitochondria Membrane Potential in Rat Brain Synaptosomes Tatyana G. Pekun & Valeriya V. Lemeshchenko & Tamara I. Lyskova & Tatyana V. Waseem & Sergei V. Fedorovich
Received: 20 September 2012 / Accepted: 21 October 2012 / Published online: 6 November 2012 # Springer Science+Business Media New York 2012
Abstract Brain ischemia is accompanied by lowering of intraand extracellular pH. Stroke often leads to irreversible damage of synaptic transmission by unknown mechanism. We investigated an influence of lowering of pHi and pHo on free radical formation in synaptosomes. Three models of acidosis were used: (1) pHo 6.0 corresponding to pHi decrease down to 6.04; (2) pHo 7.0 corresponding to the lowering of pHi down to 6.92: (3) 1 mM amiloride corresponding to pHi decrease down to 6.65. We have shown that both types of extracellular acidification, but not intracellular acidification, increase 2′,7′dichlorodihydrofluorescein diacetate fluorescence that reflects free radical formation. These three treatments induce the rise of the dihydroethidium fluorescence that reports synthesis of superoxide anion. However, the impact of amiloride on superoxide anion synthesis was less than that induced by moderate extracellular acidification. Superoxide anion synthesis at pHo 7.0 was almost completely eliminated by mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. Furthermore, using fluorescent dyes JC-1 and rhodamine-123, we confirmed that pHo lowering, but not intracellular acidification, led to depolarization of intrasynaptosomal mitochondria. We have shown that pHo but not pHi lowering led to oxidative stress in neuronal presynaptic endings that might underlie the long-term irreversible changing in synaptic transmission.
Abbreviations ASIC Acid-sensitive ion channel BCECF-AM 2′7′-bis(2-Carboxyethyl)-5(6)carboxyfluorescein acetoxymethyl ester CCCP Carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone DCF 2′,7′-Dichlorodihydrofluorescein DCFDA 2′,7′-Dichlorodihydrofluorescein diacetate DPI Diphenyleniodinium chloride HEPES 4-(2-Hydroxyethyl)piperazine-N’-1ethanesulfonic acid Ie-Ic, R.U. Fluorescence intensity; the control curve was extracted from the experimental curve; relative units Ifl Fluorescence intensity Ifl, R.U. Fluorescence intensity; relative units IP3 1,4,5-Triphosphate JC-1 5,5′,6,6′-Tetrachloro-1,1′3,3′tetraethylbenzimidazolo-carbocyanine iodide MES 4-Morpholineethanesulfonic acid NMDA N-Methyl-D-aspartate ROS Reactive oxygen species SOD Superoxide dismutase Tris Tris(hydroxymethyl)aminomethane
Keywords Synaptosomes . Acidosis . Ischemia . Presynaptic . Superoxide anion . Mitochondria T. G. Pekun : V. V. Lemeshchenko : T. I. Lyskova : T. V. Waseem : S. V. Fedorovich (*) Laboratory of Biophysics and Engineering of Cell, Institute of Biophysics and Cell Engineering, Akademicheskaya St., 27, Minsk 220072, Belarus e-mail:
[email protected]
Introduction Brain ischemia is accompanied by lowering of intra- and extracellular pH down to 6.5 and even down to 5.3 in case of hyperglycemia (Thorn and Heitman 1954; Crowell and Kaufmann 1961; Kraig and Chesler 1990; Tombaugh and Sapolsky 1993; Isaev et al. 2008). Acidification is mainly,
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but not exclusively, caused by shift of metabolic pathways to the prevalence of glycolysis followed by lactate accumulation. ATP hydrolysis, activation of Na+/H+ exchanger and NMDA receptors can also contribute to this phenomenon (Tombaugh and Sapolsky 1993; Isaev et al. 2008; Obara et al. 2008). Additionally, it should be noted that pH lowering is a concomitant condition not only for stroke but also for other brain diseases such as Alzheimer’s disease, Huntington’s disease, and Pick’s disease (Yates et al. 1990) and could potentially aggravate their pathogenesis. Disturbance of synaptic transmission is one of the underinvestigated stroke consequences (Eccles et al. 1966; Mittmann et al. 1998; Bolay et al. 2002; Hofmeijer and van Putten 2012). Acidification is able to induce synaptic depression and to inhibit neurotransmitter release (Drapeau and Nachshen 1988; Herschkowitz et al. 1993; Hsu et al. 2000; Fedorovich et al. 2003). However, it was established that inhibitory action of low pH values is primarily mediated by blocking of voltage-gated calcium and sodium channels (Drapeau and Nachshen 1988; Hsu et al. 2000; Fedorovich et al. 1997, 2003). Blocking of ion channels by protons is a fully reversible process (Moody 1984), while the impairing of synaptic transmission is only partially reversible (NeumannHaefilin and Witte 2000; Bolay et al. 2002; Hofmeijer and van Putten 2012). These results allow us to hypothesize other irreversible mechanisms of acidification action on presynaptic endings. Free radical-induced damage of neuronal terminals can potentially represent one of these mechanisms (Halliwell 2006; Keating 2008). It was demonstrated that pH lowering can induce oxidative stress in brain homogenate and in brain slices (Siesjo et al. 1985; Bralet et al. 1991; Lyskova et al. 1997). It was also established that extracellular acidification increases both lipid peroxidation and free radical accumulation in synaptosomes (Lyskova et al. 1997; Pekun et al. 2012). Reactive oxygen species (ROS) synthesized in presynaptic endings are able to damage exocytotic machinery (Keating 2008; Giniatullin et al. 2006), which drives neurotransmitter release (Sudhof 2004), thus being a cause of long-term impairment of synaptic transmission after stroke. However, some questions remain to be resolved:
uptake and calcium-sensitive neurotransmitter release (Waseem et al. 2004, 2005). Synaptosomes also have NADPH oxidase which is localized on the plasma membrane (Behrens et al. 2007; Alekseenko et al. 2012) and internal polarized mitochondria (Hajos 1975; Chinopoulos et al. 1999; Alekseenko et al. 2012). It is well known that, in neurons, the primary source of ROS is superoxide anion, which is synthesized by NADPH oxidase and the electron-transport chain of mitochondria (Halliwell 2006; Keating 2008). In the present paper, we investigated an influence of strong extracellular acidification (pHo 6.0), moderate extracellular acidification (pHo 7.0), and intracellular acidification produced by 1 mM of amiloride on synaptosomal ROS accumulation monitored by fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) and superoxide anion formation monitored by fluorescent dye dihydroethidium. As mitochondria are important sources of ROS in neurons, we investigated an influence of low pH on membrane potential of intrasynaptosomal mitochondria using fluorescent dyes 5,5′,6,6′-tetrachloro-1,1′ 3,3′-tetraethylbenzimidazolo-carbocyanine iodide (JC-1) and rhodamine-123.
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Synaptosomes Preparation
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What is the primary source of ROS at acidosis? Does the mechanism of extracellular acidification action differ from that of intracellular? Do only extreme pH values possess the prooxidant effect or does acidosis have no threshold values at all?
Isolated presynaptic endings termed synaptosomes are a convenient object with which to resolve the aforesaid questions. They retain many properties of intact neuronal terminals, for instance, the same proteome (Schrimpf et al. 2005). Synaptosomes possess sodium-dependent neurotransmitter
Materials and Methods Materials Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (CCCP), dihydroethidium, diphenyleniodinium chloride (DPI), amiloride, (-)-epinephrine, rhodamine-123, and DCFDA were purchased from Sigma (St.Lois, MO, USA). 4-(2-Hydroxyethyl)piperazine-N’-1-ethanesulfonic acid (HEPES) was obtained from Merck (Darmstadt, Germany). 5,5′,6,6′-Tetrachloro-1,1′3,3′-tetraethylbenzimidazolo-carbocyanine iodide (JC-1) and 2′7′-bis(2Carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) were received from Calbiochem (La Jolla, CA, USA). 4-Morpholineethanesulfonic acid (MES) was purchased from Reanal (Budapest, Hungary). Tris (hydroxymethyl)aminomethane (Tris) was obtained from BDH (Poole, UK).
Synaptosomes were isolated from brain hemispheres of 12– 16-week-old male Wistar rats by the method of Hajos (1975). Stock suspensions of synaptosomes (10 mg/ml) were prepared in medium A (composition in millimolars, 132 NaCl; 5 KCl; 10 glucose; 1.3 MgCl2; 1.2 NaH2PO4; 2.0 CaCl2; 15 HEPES; and 5 Tris; pH7.4, 310 milliosmoles per liter (mOsm/l)) and kept on ice. Animal experiments were carried out in accordance with the European Communities Council Directive 86/609/EEC.
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Intrasynaptosomal pH Measurement Intrasynaptosomal pH was monitored by fluorescent dye BCECF-AM as described by Nachshen and Drapeau (1988) with modifications according to Waseem et al. (2007). Synaptosomes were resuspended in medium A after additional washing in 10 volumes of the same medium. The suspension was incubated in the presence of 10 μM BCECF-AM for 30 min at 37 °C. Extracellular dye was washed out three times by sedimentation, and the final pellet was resuspended in medium A. In order to estimate intrasynaptosomal pH, 200 μl of loaded synaptosomes were added to a cuvette containing 2 ml of incubation medium A, resulting in a final protein concentration 1 mg/ml. Subsequent treatments were added directly to a cuvette. Fluorescence intensity was recorded at λex/em 0505/ 530 nm at 37 °C in constant stirring. Incubation medium B (composition in millimolars, 132 NaCl; 5 KCl; 10 glucose; 1.3 MgCl2; 1.2 NaH2PO4; 2.0 CaCl2; 10 HEPES; and 10 MES; pH6.0–7.4, 310 mOsm/l) was used in experiments on investigation of extracellular acidification and incubation medium C (composition in millimolars, 132 NaCl; 5 KCl; 10 glucose; 1.3 MgCl2; 1.2 NaH2PO4; 2.0 CaCl2; 10 HEPES; and 15 NaHCO3; pH7.4, 310 mOsm/l) was used in experiments on investigation of HCO3− influence. The suspension of synaptosomes was treated with the ionophores gramicidin D and monensin in order to get a calibration curve. Aliquots of 2 μl 0.5 M HCl were added to each sample for calibration, as such an addition shifts pH of the suspension on 0.05 units. To change the extracellular pH, the aliquots of 1 mM amiloride or 60 μl of HCl solution having different acid concentrations were added to the cuvette directly. Intrasynaptosomal ROS Determination Intrasynaptosomal ROS was monitored by fluorescent dye DCFDA according to LeBel and Bondy (1990) with modifications according to Alekseenko et al. (2008). Synaptosomes purification was carried out in calciumfree medium A, and then after additional washing, the pellet was resuspended in the same medium (protein concentration 10 mg/ml). Suspension was incubated for 60 min at 37 °C in presence of 25 μM DCFDA. Extracellular dye was removed by sedimentation, and the final pellet was resuspended in 2 ml calcium-free medium B. To investigate ROS formation, 200 μl of loaded synaptosomes were added to a cuvette containing 1.8 ml of incubation medium B. Fluorescence intensity was recorded at λex/em 0501/525 nm on spectrofluorimeter Cary Eclipse (“Varian”, USA) with constant stirring and 37 °C temperature. To change the extracellular pH, the aliquots of 1 mM amiloride or 60 μl of HCl solution having different acid
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concentrations were directly added to cuvette on 50 s. The same quantity of vehicle was added in control experiments. The control curve was extracted from the experimental curve. Determination of Superoxide Anion Formation Superoxide anion level was determined with fluorescent dye dihydroethidium according to Bindokas et al. (1996). Synaptosomes purification was carried out in calciumfree medium B. An aliquot of synaptosome suspension (200 μl) was added to a cuvette containing 2 ml of incubation medium B with 2.0 mM CaCl2. Five micromolars of dihydroethidium was added to the cuvette; then, after 1 min, different additions were made. An equal amount of vehicle was added in control experiments. Fluorescence intensity was recorded at λex/em 0490/560 nm on spectrofluorimeter Cary Eclipse (“Varian”, USA) at constant stirring and 37 °C. To change the extracellular pH, the aliquots of 1 mM amiloride or 60 μl of HCl solution having different acid concentrations were added to the cuvette directly. The same quantity of vehicle was added in control experiments. The control curve was extracted from the experimental curve. Determination of Superoxide Dismutase Activity Superoxide dismutase (SOD) was measured in synaptosomal lysate by adrenaline autooxidation according to Misra and Fridovich (1972). Suspension of synaptosomes was incubated 30 min at 30 °C for lysis with following centrifugation for 10 min at 16,000×g. Supernatant was incubated 10 min at 37 ° C in different incubation media with indicated pH. SOD activity was determined by its ability to inhibit spontaneous autooxidation of adrenaline to adrenochrome. Reaction was carried out in 10 ml of 50 mM Na2CO3 (pH10.2) containing 200 μM EDTA and 300 μM adrenaline. Lysate of synaptosomes (final protein concentration 0.1–0.2 mg/ml) also was added to the reaction medium. The reaction was started by addition of acid (pH2.0) stable adrenaline solution which is auto-oxidized at pH10.2 by free radical mechanism. Absorbance was measured on spectrophotometer SPEKOL211 (“Karl Zeiss,” Germany) at 480 nm. Difference in absorbance between the third and second minutes of reactions was taken as auto-oxidation velocity. Inhibition of this process by intact lysate was taken as 100 %. Determination of Intrasynaptosomal Mitochondria Membrane Potential with Fluorescent Dye JC-1 Membrane potential of intrasynaptosomal mitochondria was detected by fluorescent dye JC-1 according to Chinopoulos et al. (1999). Synaptosomes purification was carried out in calciumfree medium B, and then, the pellet was resuspended in the same medium (protein concentration, 5 mg/ml). Suspension
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was incubated for 15 min at 37 °C in the presence of 10 μg/ ml dye. Extracellular dye was removed by sedimentation, and the final pellet was resuspended in 2.0 ml calcium-free medium B (protein concentration, 10 mg/ml). To investigate mitochondrial membrane potential, 200 μl of loaded synaptosomes were added to a cuvette containing 1.8 ml of incubation medium B. Fluorescence intensity was recorded at λex/em 0504/535 nm or 504/595 nm on spectrofluorimeter Cary Eclipse (“Varian,” USA) at constant stirring and 37 °C. To change the extracellular pH, 60 μl of HCl solutions having different acid concentrations were directly added to cuvette at 50 s. The same quantity of vehicle was added in control experiments. The control curve was extracted from the experimental curve.
depending on the tested model system. Firstly, we evaluated how lowering of pH of incubation medium affects the intrasynaptosomal pH. Commonly accepted as physiological, the value of extracellular pHo about 7.4 gives pHi 7.15 inside synaptosomes (Table 1). Decreasing of the extracellular pH to 7.0 has shifted the internal pHi only 0.25 units, while further acidifying down to 6.0 virtually equalized extra- and intrasynaptosomal pH (Table 1). We have shown that 1 mM of amiloride (which inhibits Na+/H+ exchanger) decreased pHi down to 6.65 (Table 1). We failed to find any influence of HCO3− ions on pHi regulation in synaptosomes (Table 1), therefore standard HCO3−-free incubation medium was used in further experiments.
Determination of Intrasynaptosomal Mitochondria Membrane Potential with Fluorescent Dye Rhodamine-123
Influence of Extra- and Intracellular Acidification on ROS Formation in Synaptosomes
Additionally, membrane potential of intrasynaptosomal mitochondria was detected by fluorescent dye rhodamine-123 according to Alekseenko et al. (2012). Synaptosomes purification was carried out in calcium-free medium B, and then, the pellet was resuspended in the same medium (protein concentration, 10 mg/ml). Suspension was incubated for 15 min at 37 °C in the presence of 10 μM dye. Extracellular dye was removed by sedimentation three times, and the final pellet was resuspended in 2.0 ml calcium-free medium B. To investigate mitochondrial membrane potential, 200 μl of loaded synaptosomes were added to a cuvette containing 1.8 ml of incubation medium B. Fluorescence intensity was recorded at λex/em 0505/534 nm on spectrofluorimeter Cary Eclipse (“Varian,” USA) at constant stirring and 37 °C. To change the extracellular pH, 60 μl of HCl solutions having different acid concentrations were directly added to the cuvette at 50 s. The same quantity of vehicle was added in control experiments. The control curve was extracted from the experimental curve.
Figure 1a shows that extracellular acidification leads to an increase of DCFDA fluorescence that reflects the development of oxidative stress. Therewith, lowering of pHo down to 7.0, corresponding to a drop of pHi from 7.15 to 6.92 (Table 1), was sufficient to induce statistically significant ROS accumulation (Fig. 1a, b). In contrast, amiloride known to decrease pHi down to 6.65 resulted in only slight reduction of DCFDA fluorescence (Fig. 1b), being likely a nonspecific quenching of the dye by protons (Pekun et al. 2012). The main sources of free radicals in neurons are the electrontransport chain of mitochondria and some DPI-sensitive enzymes, for instance, NADPH oxidase and xanthine oxidase (Halliwell 2006; Keating 2008; Wind et al. 2010). Figure 1c, d shows that the mitochondrial uncoupler CCCP and NADPH oxidase inhibitor DPI do not reduce ROS accumulation during acidifying of incubation medium; furthermore, both compounds unexpectedly enhance this effect at pHo 6.0.
Other Methods Protein concentration was assayed according to Lowry et al. (1951) using bovine serum albumin as a standard. Data are presented as mean±SEM where indicated; statistical significance was evaluated using one-tailed Student’s ttest.
Results Influence of Extrasynaptosomal pH on Intrasynaptosomal pH Despite the fact that the intracellular pH is tightly regulated, the spatio-temporal characteristics of this process may vary
Table 1 Influence of incubation medium on intrasynaptosomal pH Incubation medium
Intrasynaptosomal pH
Incubation medium B, pH7.4 Incubation medium B, pH7.0
7.15±0.04 6.92±0.01
Incubation medium B, pH6.0 Incubation medium B, pH7.4, 1 mM amiloride Incubation medium C (HCO3-contained), pH7.4
6.04±0.04 6.65±0.04 7.13±0.08
Measurement was made within 3 min after placing of synaptosomes in indicated medium or within 3 min after changing of extracellular medium pH. Data presented are mean values±SEM of at least four experiments
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Fig. 1 Influence of extra- and intracellular pH on DCF fluorescence in synaptosomes. a Kinetics of DCF fluorescence increase after extracellular acidification. HCl was added where indicated. To get resulted representative curves, values of fluorescence intensity (Ic) obtained in control experiments were extracted from values of fluorescence intensity (Ie) obtained in experiments with acidification. Curves represent five independent experiments. pH6.0 extracellular acidity reached 6.0. pH7.0 extracellular acidity reached 7.0. b Influence of extra- and intracellular acidification on DCF fluorescence (Ifl). pH6.0 extracellular acidity reached 6.0. pH7.0 extracellular acidity reached 7.0. Amil 1 mM amiloride was added. Bars represent DCF fluorescence increase (Ifl) within 4 min after additions. Data presented are mean values± SEM of at least five experiments. The 100 % level corresponds to fluorescence increase in response to 1 mM H2O2. **P≤0.01 versus 0. c
Inhibitor analysis of strong acidification-induced ROS formation. Con control. CCCP incubation medium contains 10 μM of CCCP. DPI synaptosomes were preincubated with 30 μM of DPI. Bars represent DCF fluorescence increase (Ifl) within 4 min after additions. Data presented are mean values±SEM of at least four experiments. The 100 % level corresponds to fluorescence increase in response to pH 6.0. *P≤0.05; **P≤0.01 versus 0. d Inhibitor analysis of moderate acidification-induced ROS formation. Con control. CCCP incubation medium contains 10 μM of CCCP. DPI synaptosomes were preincubated with 30 μM of DPI. Bars represent DCF fluorescence increase (Ifl) within 4 min after additions. Data presented are mean values± SEM of at least four experiments. The 100 % level corresponds to fluorescence increase in response to pH7.0
Influence of Extra- and Intracellular Acidification on Superoxide Anion Formation in Synaptosomes
acidification down to 6.65 by amiloride was also less pronounced (Fig. 2b). Figure 2c shows that CCCP does not influence superoxide anion synthesis induced by strong acidification (pHo 6.0), while DPI rather enhances this effect. In contrast, both compounds markedly inhibited the increase of dihydroethidium fluorescence induced by moderate extracellular acidification (pHo 7.0); CCCP almost completely eliminated this effect (Fig. 2d).
Figure 2a shows that extracellular acidification leads to an increase of dihydroethidium fluorescence that reflects superoxide anion synthesis in synaptosomes. Lowering of pHo to 7.0 was sufficient to induce this effect (Fig. 2a, b). In contrast with the relatively gradual increase of DCFDA fluorescence (Fig. 1a), the rise of dihydroethidium fluorescence reached the plateau level within 30 s after addition of HCl and remained stable thereafter (Fig. 2a). When synaptosome-free incubation medium was acidified down to 6.0, the fluorescence of dihydroethidium slightly increased, but this increase was smaller than that detected in the presence of synaptosomes and pHo of 7.0 (Fig. 2b). Similarly, the increase of fluorescence during intracellular
pH-Dependence of Synaptosomal Superoxide Dismutase Activity Superoxide dismutase is the main enzyme in the brain eliminating superoxide anion (Halliwell 2006). Therefore, we investigated the dependence of SOD activity on pH. Figure 3 shows that lowering of pH down to 6.0 leads to
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Fig. 2 Influence of extra- and intracellular pH on dihydroethidium fluorescence in synaptosomes. a Kinetics of dihydroethidium fluorescence increase after extracellular acidification. HCl was added where indicated. To get resulted representative curves, values of fluorescence intensity (Ic) obtained in control experiments was extracted from values of fluorescence intensity (Ie) obtained in experiments with acidification. Curves represent 22 independent experiments. pH6.0 extracellular acidity reached 6.0. pH 7.0 extracellular acidity reached 7.0. b Influence of extra- and intracellular acidification on dihydroethidium fluorescence (Ifl). pH6.0 extracellular acidity reached 6.0. pH7.0 extracellular acidity reached 7.0. Amil 1 mM amiloride was added. Cell-free extracellular acidity reached 6.0 in incubation medium without synaptosomes. Bars represent dihydroethidium fluorescence increase (Ifl)within 4 min after additions. Data presented are mean values±SEM of at least 17 experiments. **P≤0.01 versus 0. c
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Inhibitor analysis of strong acidification-induced superoxide anion formation Con control; CCCP incubation medium contains 10 μM of CCCP. DPI synaptosomes were preincubated with 30 μM of DPI. Bars represent dihydroethidium fluorescence increase (Ifl) within 4 min after additions. Data presented are mean values±SEM of at least five experiments. The 100 % level corresponds to fluorescence increase in response to pH6.0. **P≤0.01 versus zero. d Inhibitor analysis of moderate acidificationinduced superoxide anion formation. Con control. CCCP incubation medium contains 10 μM of CCCP. DPI synaptosomes were preincubated with 30 μM of DPI. Bars represent dihydroethidium fluorescence increase (Ifl) within 4 min after additions. Data presented are mean values±SEM of at least five experiments. The 100 % level corresponds to fluorescence increase in response to pH7.0. *P≤0.05; **P≤0.01 versus 0
20 % inhibition of enzyme activity, while moderate acidification (pH7.0) has no effect on that. Significant inhibition of this enzyme was observed only at extreme acidification down to 5.0 (Fig. 3). Influence of Extra- and Intracellular Acidification on Intrasynaptosomal Mitochondria Membrane Potential Monitored by Fluorescent Dye JC-1
Fig. 3 Influence of acidification on SOD activity in synaptosomes. Data presented are mean values±SEM of at least eight experiments. The 100 % level corresponds to SOD activity at pH7.4. **P≤0.01 versus 0
Figure 4a shows that the mitochondrial uncoupler CCCP induces fast increase of JC-1 fluorescence recorded at emission wavelength of 535 nm. It is believed that this parameter is applicable for monitoring of intrasynaptosomal mitochondria depolarization. Figure 4b shows that decreasing pHo down to 7.0 induces an increase of JC-1 fluorescence that evidences the drop in mitochondria membrane potential.
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The response on further acidification down to pHo was biphasic. Initially, a slight fluorescence decrease was observed, which seems to be mediated by nonspecific quenching of the dye in cytosol, and thereafter, during the second phase, the fluorescence increased fivefold (Fig. 4b). No rise of fluorescence intensity was seen if the acidification followed CCCP treatment (Fig. 4c, d). Figure 4e shows that CCCP decreased the signal of JC-1 recorded at emission wavelength of 595 nm. This characteristic also can be used for monitoring of mitochondria depolarization. The data presented on Fig. 4f confirm that extracellular acidification decreases the potential of intrasynaptosomal mitochondria. The intensity of the fluorescence signal at 535 nm was chosen for further statistical analysis based on two reasons. First, a decrease of pH can cause a nonspecific decline of dye signal, probably due to quenching (Fig. 4c, d), which is difficult to distinguish from depolarization-induced signal reduction. Second, it was proved that, in synaptosomes, the specificity of dye response to stimuli measured at 535 nm is higher than that at 595 nm (Chinopoulos et al. 1999). Figure 4g shows that fluorescence increase at extracellular acidification was statistically significant. In contrast, intracellular acidification induced by 1 mM amiloride caused slight non-specific decrease of fluorescence being likely a result of dye quenching (Fig. 4g). Influence of Extra- and Intracellular Acidification on Intrasynaptosomal Mitochondria Membrane Potential Monitored by Fluorescent Dye Rhodamine-123 We additionally confirmed the results obtained with dye JC1 using another fluorescent dye. Figure 5a, b shows that extracellular acidification increases rodamine-123 fluorescence that evidences intrasynaptosomal mitochondria depolarization.
Discussion We first investigated the dependence of intrasynaptosomal pH on pH of incubation medium. As was shown before (Nachshen and Drapeau 1988; Chesler 2003), pHo 7.4 corresponds to pHi 7.1, and a reduction of extracellular pH caused an appropriate shift of intracellular pH to more acidic values. We also confirmed that, unlike in neurons, an anion exchanger is not involved in pH regulation in synaptosomes (Chesler 2003) (Table 1). This enabled the use of standard HCO3−-free incubation medium. Data presented in Fig. 1 allow us to conclude that the extracellular acidification induces oxidative stress in synaptosomes, and even moderate acidifying to pHo 7.0 is sufficient to run this process, while intrasynaptosomal acidification is not able to induce ROS formation. We have
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previously shown that lowering of pH decreased fluorescence of oxidized DCF in solution (Pekun et al. 2012), therefore, the gain of fluorescence is rather underestimated but not an artifact resulted from protons binding to the dye. Obtained results suggest that ROS formation is induced by activation of a non-identified receptor on the plasma membrane. In support of this point of view, it was previously demonstrated that acidification of incubation medium induces hyperpolarization and inhibiting of ROS formation in isolated brain mitochondria (Selivanov et al. 2008). We demonstrated that lowering of extracellular pH leads to opposite effects on potential of intrasynaptosomal mitochondria and free radical formation in synaptosomes. It is thought that an acid-sensitive ion channel (ASIC) might be a suitable candidate for this role. These channels are localized on the neuronal plasma membrane and, after activation by external acidification, pass calcium ions inside (Krishtal and Pidoplichko 1981; Kellenberger and Schild 2002; Xiong et al. 2004). Despite the fact that ASICs have been found in presynaptic terminals (Voglis and Tavernarakis 2008), our previous results exclude their involvement. Firstly, pHo-induced ROS accumulation was not dependent on external calcium (Pekun et al. 2012). Secondly, lowering of pHo was not followed by calcium influx into synaptosomes (Drapeau and Nachshen 1988; Fedorovich et al. 1997). We have shown that both the mitochondrial uncoupler CCCP and the NADPH oxidase inhibitor DPI did not inhibit oxidative stress. Furthermore, they even enhanced ROS accumulation at low pHo (Fig. 1c). It has been demonstrated that both compounds are able to significantly deplete cytosolic ATP (Tretter et al. 1997; Hutchinson et al. 2007), therefore reinforcement of prooxidant effect of acidification is most likely explained by impairment of antioxidant protection, an energy-dependent process (Halliwell 2006; Keating 2008). DCFDA reacts mainly with hydroxyl radical and peroxynitrite (Setsukinai et al. 2003). In neurons, the primary reactive form of oxygen is superoxide anion (Halliwell 2006; Keating 2008). Therefore, we investigated the influence of acidification on the fluorescence of dihydroethidium, a superoxide anion-specific dye (Bindokas et al. 1996; Kalyanaraman et al. 2012). Figure 2a, b shows dihydroethidium responses to pH changing in a manner similar to that of DCFDA. However, there are some differences. First, the kinetics of both dyes is different. The fluorescence intensity of dihydroethidium demonstrates fast rising and reaches the plateau level within 30 s (Fig. 2a), while DCFDA fluorescence has no ceiling level (Fig. 1a). This is probably explainable by the fact that superoxide anion is the primary reactive form of oxygen, actively synthesizing only within the first seconds after lowering of pHo and is further engaged to development of oxidative stress monitored by the changes in DCFDA fluorescence. Second, acidification
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Fig. 4
Influence of extra- and intracellular pH on intrasynaptosomal mitochondria membrane potential monitored by fluorescent dye JC-1. a Kinetics of JC-1 fluorescence (λem 0535 nm) increase after membrane depolarization. The 10 μM of CCCP was added where indicated. To get resulted representative curves, values of fluorescence intensity (Ic) obtained in control experiments was extracted from values of fluorescence intensity (Ie) obtained in experiments with CCCP. Curves represent seven independent experiments. b Kinetics of JC-1 fluorescence (λem 0535 nm) increase after extracellular acidification. HCl was added where indicated. To get resulted representative curves, values of fluorescence intensity (Ic) obtained in control experiments were extracted from values of fluorescence intensity (Ie) obtained in experiments with acidification. Curves represent seven independent experiments. pH6.0 extracellular acidity reached 6.0. pH7.0 extracellular acidity reached 7.0. c Influence of acidifications on JC-1 fluorescence (λem 0535 nm) (Ifl) after depolarization of intrasynaptosomal mitochondria. The 10 μM of CCCP and HCl down to pH6.0 were added where indicated. Curves represent four independent experiments. d Influence of acidification on JC-1 fluorescence (λem 0535 nm) (Ifl) after depolarization of intrasynaptosomal mitochondria. The 10 μM of CCCP and HCl down to pH7.0 were added where indicated. Curves represent four independent experiments. e Kinetics of JC-1 fluorescence (λem 0595 nm) decrease after membrane depolarization. To get resulted representative curves, values of fluorescence intensity (Ic) obtained in control experiments were extracted from values of fluorescence intensity (Ie) obtained in experiments with CCCP. The 10 μM of CCCP were added where indicated. Curves represent four independent experiments. f Kinetics of JC-1 fluorescence (λem 0595 nm) decrease after extracellular acidification. To get resulted representative curves, values of fluorescence intensity (Ic) obtained in control experiments were extracted from values of fluorescence intensity (Ie) obtained in experiments with acidification. HCl down 6.0 was added where indicated. Curves represent four independent experiments. g Influence of extra- and intracellular acidification on JC-1 fluorescence (λem 0 535 nm) (Ifl). pH6.0 extracellular acidity reached 6.0. pH7.0 extracellular acidity reached 7.0. Amil 1 mM amiloride was added. Bars represent JC-1 fluorescence increase (Ifl) within 4 min after additions. Data presented are mean values±SEM of at least seven experiments. The 100 % level corresponds to fluorescence increase in response to 10 μM of CCCP. *P≤0.05; **P≤0.01 versus 0
Fig. 5 Influence of extracellular pH on intrasynaptosomal mitochondria membrane potential monitored by fluorescent dye rhodamine-123. a Kinetics of rhodamine-123 fluorescence increase after extracellular acidification. To get resulted representative curves, values of fluorescence intensity (Ic) obtained in control experiments was extracted from values of fluorescence intensity (Ie) obtained in experiments with acidification. HCl was added where indicated. Curves represent four independent experiments. pH6.0 extracellular acidity reached 6.0. pH
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of synaptosome-free incubation medium down to 6.0 induces an increase of dihydroethidium fluorescence, which is several folds smaller as compared with that in the presence of synaptosomes (Fig. 2b). Acidification in synaptosomefree incubation medium only decreases DCF fluorescence (Pekun et al. 2012). Third, intracellular acidification slightly reinforces superoxide anion formation (Fig. 2b). However, it can be noted that the effect resulting from a reduction of the internal pH to 6.65 by 1 mM amiloride was smaller when compared with that resulting from a reduction of the internal pH on 0.25 units by lowering of external pH down to 7.0 (Table 1). Furthermore, we investigated how CCCP and DPI influence dihydroethidium signal in conditions similar to those applied during the experimental series with DCFDA. In the case of strong acidification, CCCP had no influence on superoxide level; DPI even reinforced an increase of dihydroethidium fluorescence. The reinforcing effect of DPI can probably be explained by it acting on any additional target of this compound (Wind et al. 2010). In contrast, in the case of moderate acidification, CCCP almost completely inhibited superoxide anion formation while DPI reduced this effect twofold (Fig. 2d). It is difficult to estimate the involvement of NADPH oxidase in this effect because DPI also influences complex I of mitochondria (Hutchinson et al. 2007), and it seems more plausible that mitochondria are the leading, if not the only, source of superoxide anion in this case. Thereby, in contrast to glutamate-induced oxidative stress (Alekseenko et al. 2012), activation of NADPH oxidase is not the primary reason of ROS formation in neuronal presynaptic endings in case of acidification. Figure 3 shows that acidification down to 7.0 does not influence SOD activity. Further lowering of pH inhibits the activity of this
7.0 extracellular acidity reached 7.0. b Influence of extracellular acidification on rhodamine-123 fluorescence (Ifl). pH6.0 extracellular acidity reached 6.0. pH7.0 extracellular acidity reached 7.0. Bars represent rhodamine-123 fluorescence increase (Ifl) within 4 min after additions. Data presented are mean values±SEM of at least four experiments. The 100 % level corresponds to fluorescence increase in response to 10 μM of CCCP. **P≤0.01 versus 0
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enzyme and may underlie CCCP-resistant superoxide anion accumulation (Fig. 2c). The obtained results allowed us to propose two mechanisms resulting in superoxide anion concentration: synthesis of this free radical in mitochondria at moderate extracellular acidification and disturbance of its conversion due to SOD inhibition at strong acidification. To confirm that mitochondria are the main site of superoxide anion synthesis in synaptosomes, we investigated an influence of acidification on mitochondrial potential. In our early papers using steady-state distribution of [3H]tetraphenylphosphonium, we have shown that intrasynaptosomal mitochondria were depolarized by lowering of pHo, including moderate extracellular acidification down to pHo 7.0 (Fedorovich et al. 1996). However, this method required 30 min incubation prior the response to stimulus can be measured and made it impossible to correlate fast changing in membrane potential with fast changing in superoxide anion synthesis. Therefore, the rapidly reacting fluorescent probe JC-1 was used to investigate an influence of acidification on mitochondrial potential in synaptosomes. This compound is concentrated in mitochondria with the following formation of J-aggregates whose emission is measured at 595 nm (Chinopoulos et al. 1999). Depolarization of mitochondria can be detected by both a decrease of aggregate fluorescence emitted at 595 nm or an increase of monomer fluorescence emitted at 535 nm (Chinopoulos et al. 1999). Figure 4 shows that, similarly with ROS formation, the extracellular but not the intracellular acidification leads to depolarization of intrasynaptosomal mitochondria. Depolarization of mitochondria was further confirmed by fluorescent dye rhodamine-123 (Fig. 5). Our results with fluorescent probes demonstrated that mitochondria were depolarized in as early as 1 min after lowering of pHo. Taken together, these findings provide strong evidence that development of oxidative stress in our experiments resulted from damaging of mitochondria. The following scheme of ROS formation during acidosis can be proposed according to our results: After moderate extracellular acidification (pHo 7.0), binding of protons to an unknown receptor triggers transduction of a signal to mitochondria with the following synthesis of superoxide anions which further promotes the development of oxidative stress within a cell. Inhibition of antioxidant enzymes, for instance SOD, seems to be more important in case of strong acidification (pHo 6.0). Probably, the release of iron and lessening of the reduced glutathione pool also contribute to the development of oxidative stress during strong acidosis, as it was shown for brain homogenates (Siesjo et al. 1985; Bralet et al. 1992; Ying et al. 1999). This simple scheme is compromised at some extent by the lack of CCCP effect on DCFDA fluorescence increase at pHo 7.0 (Fig. 1d). This failure could be explained by dual action of CCCP. Some experimental works have demonstrated that this compound decreased free radical formation in mitochondria in different
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pathological conditions (Reynolds and Hastings 1995), while others showed that CCCP strongly reduced the ATP level (Tretter et al. 1997) and thus seriously impaired the antioxidant protection within the cell. As yet, the nature of the signal which is transduced from the plasma membrane to the mitochondria is not clear, but, due to the reasons discussed above, ASIC channels are unlikely to account for it. It was shown that extracellular acidification led to an increase of inositol 1,4,5-triphosphate (IP3) synthesis in synaptosomes (Saadoun et al. 1998), which could induce calcium release from intracellular stores followed by its mitochondrial uptake and associated ROS synthesis (Rizzuto and Pozzan 2006). Recently, a metabotropic receptor for alkaline pH coupled with activation of tyrosine-specific protein kinase was found in the kidney (Deyev et al. 2011). Taking the discovery of this report into account, it is hypothesized that, in synaptosomes, the acidification triggers a signaling cascade by acting to the still unidentified metabotropic receptor for low pH, whose activity is mediated by phospholipase C synthesizing IP3 or, alternatively, by tyrosine-specific protein kinase. We have shown that extracellular acidification led to oxidative stress in neuronal presynaptic endings which can underlie long-term impairment of synaptic transmission. However, it was demonstrated that ROS formation in mitochondria was involved in a so-called “ischemic preconditions” phenomenon (Ravati et al. 2001; Correia et al. 2010). It consists of a weak ischemia providing protective properties toward a following strong ischemia (Correia et al. 2010). Therefore, it should not be excluded that our discovered synthesis of superoxide anion after moderate acidification plays a protective role. Acknowledgments This work was supported by Committee for Aid and Education in Neurochemistry-International Society for Neurochemistry (CAEN-ISN). Foundation body had no involvement in study design, in the collection, analysis, and interpretation of data, in the writing of the report, and in the decision to submit the article for publication. We thank Mrs. Claire Roulston for improvement of English. Declaration of competing interests Authors declare no competing financial, personal, or other interests.
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