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Involvement of mitochondrial potential and calcium buffering capacity in minocycline cytoprotective actions

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Involvement of mitochondrial potential and calcium buffering capacity in minocycline cytoprotective actions
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  INVOLVEMENT OF MITOCHONDRIAL POTENTIAL AND CALCIUMBUFFERING CAPACITY IN MINOCYCLINE CYTOPROTECTIVE ACTIONS F. J. FERNANDEZ-GOMEZ, a M. F. GALINDO, a M. GOMEZ-LAZARO, a C. GONZÁLEZ-GARCÍA, b V. CEÑA, b N. AGUIRRE, c AND J. JORDÁN a * a Departamento de Ciencias Médicas, Facultad de Medicina, Univer-sidad de Castilla-La Mancha, Avenida Almansa, s/n, 02006 Albacete,Spain b Unidad Asociada Neurodeath, CSIC-Universidad de Castilla-La Man-cha, Departamento de Ciencias Médicas, Avenida Almansa, s/n,02006 Albacete, Spain c  Departamento de Farmacologia, Centro de Investigación Médica Aplicada, Universidad de Navarra Abstract—Minocycline, a semisynthetic derivative of tetracy-cline, displays beneficial activity in neuroprotective in mod-els including, Parkinson disease, spinal cord injury, amyo-trophic lateral sclerosis, Huntington disease and stroke. Themechanisms by which minocycline inhibits apoptosis remainpoorly understood. In the present report we have investi-gatedtheeffectsofminocyclineonmitochondria,duetotheir crucial role in apoptotic pathways. In mitochondria isolatedsuspensions, minocycline failed to block superoxide-induced swelling but was effective in blocking mitochondrialswelling induced by calcium. This latter effect might be me-diated through dissipation of mitochondrial transmembranepotentialandblockadeofmitochondrialcalciumuptake.Con-sistently, minocycline fails to protect SH-SY5Y cell culturesagainst reactive oxygen species-mediated cell death, includ-ing malonate and 6-hydroxydopamine treatments, but it iseffective against staurosporine-induced cytotoxicity. The ef-fects of this antibiotic on mitochondrial respiratory chaincomplex were also analyzed. Minocycline did not modifycomplex IV activity, and only at the higher concentrationtested (100   M) inhibited complex II/III activity. Other mem-bers of the minocycline antibiotic family like tetracyclinefailed to induce these mitochondrial effects. © 2005 Pub-lished by Elsevier Ltd on behalf of IBRO.Key words: apoptosis, mitochondria, Parkinson, neurode-generation, caspases, ROS. Neurodegenerative diseases, including Alzheimer (AD),Parkinson (PD) diseases, and amyotrophic lateral sclero-sis (ALS), are one of the main causes of death in Westerncountries (Murray and Lopez, 1997). In some of these pathologies a decrease in specific neuronal populationshas been described including cholinergic neurons in AD or dopaminergic neurons in PD, where apoptotic mecha-nisms seem to be involved (Jellinger, 2001). In these pro- cesses, mitochondria appear to be a key point of conver-gence of different pathways initiated by several apoptoticstimuli including receptor activation by Fas-ligand (Ka-vurma and Khachigian, 2003) or glutamate (Stout et al., 1998), or after exposure to neurotoxins such as veratridine(Jordan et al., 2002) or staurosporine (Tafani et al., 2002). Opening of a permeability transition pore (PTP) leads tomitochondrial swelling and the release of intramitochon-drial proteins to the cytoplasm including cytochrome c,apaf-1 and caspase family members, which participate inapoptosis pathways (van Gurp et al., 2003; Joza et al., 2003; Galindo et al., 2003). Indeed, drugs with the ability to block PTP formation are cytoprotective against a variety of toxic stimuli (Loeffler and Kroemer, 2000; Zamzami andKroemer, 2001; Jordan et al., 1997, 2003a). Among other  triggers, calcium uptake into isolated mitochondria inducesthe collapse of the mitochondrial membrane potential(  m), the opening of the PTP and the release of proapop-totic factors (Kristal and Dubinsky, 1997; Li et al., 1997). So, mitochondrial Ca 2  overload would be a side effect of the rise in cytosolic Ca 2  concentration and inhibition of mitochondrial calcium uptake is antiapoptotic in severalmodels of cell death (Jambrina et al., 2003; Reynolds, 1999; Bae et al., 2003; Lee et al., 2002). Often, drugs used for the treatment of a specific pa-thology are later proven to be useful in a number of other diseases. Recently, a modified tetracycline antibiotic, mi-nocycline hydrochloride, commonly used in the treatmentof moderate to severe acne vulgaris (Dreno et al., 2001), appeared to display beneficial activity in various models of neurodegeneration including, PD, spinal cord injury, amyo-trophic lateral sclerosis, Huntington disease and focal isch-emic brain injury (Brundula et al., 2002; Kriz et al., 2002; Stirling et al., 2004; Wang et al., 2003). Also, its adminis-tration in mice expressing a mutant superoxide dismutase(SOD1(G37R)) at late presymptomatic stage, delayed theonset of motor neuron degeneration and muscle strengthdecline, and increased the longevity of SOD1(G37R) mice(Kriz et al., 2002). Treatment of patients with minocycline has, therefore, been proposed as a possible therapy for some neurodegenerative diseases including multiple scle-rosis (Popovic et al., 2002), ischemia ( Arvin et al., 2002), PD and Huntington’s disease (Thomas et al., 2003, 2004), because minocycline crosses the blood–brain barrier re-gardless of the dose and route of administration (Colovicand Caccia, 2003) and several clinical trials have beenperformed (Gordon et al., 2004; Thomas and Lee, 2004; *Corresponding author. Tel:  34-967-599-200; fax:  34-967-599-327(J. Jordan).E-mail addresses: Joaquin.jordan@uclm.es (J. Jordan).  Abbreviations:  AD, Alzheimer disease; FCCP, carbonyl cyanide 4-trifluoromethoxyphenlhydrazone; GST, glutathione; iNOS, induciblenitric oxide synthase; mBCl, monochlorobimane; MTT, 3-[4,5-dimethylthiazol-2-yl]-2-5-diphenyltetrazolium bromide; PD, Parkinsondisease; PTP, permeability transition pore; SOD, superoxide dis-mutase; TMRE, tetramethylrhodamine ethyl ester; 6-OHDA, 6-hydroxydopamine;  m, mitochondrial membrane potential. Neuroscience  133 (2005) 959–967 0306-4522/05$30.00  0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO.doi:10.1016/j.neuroscience.2005.03.019 959  see also Blum et al., 2004; Domercq and Matute, 2004 for reviews). However, it has been recently reported that mi-nocycline may have variable or even deleterious effects indifferent species and models depending on the mode of administration and the dose (Diguet et al., 2004). Thus minocycline presents deleterious effects in two phenotypicmodels of PD and HD (Diguet et al., 2003, 2004). Similarly, it exacerbates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopamine damage, and reverses the neuropro-tective effect of creatine in nigrostriatal dopaminergic neu-rons (Yang et al., 2003). The exact mechanisms by which minocycline playsthese neuroprotective effects remain unknown although areduced expression of cycloxygenase-2, caspase-1 andinducible nitric oxide synthase (iNOS) mRNA have beendescribed (Chen et al., 2000). It has been shown that mitochondria might be involved in minocycline-activatedpathways. Although it has been shown that minocyclineprevented mitochondrial swelling induced by different stim-uli (Zhu et al., 2002; Wang et al., 2003) in a recent report minocycline was able to induce it (Cornet et al., 2004). Theaim of the present work was to study whether minocyclinecan interfere with mitochondrial calcium uptake as a plau-sible mechanism for the cytoprotective action of this anti-biotic. We have also analyzed the effect of minocycline onCa 2  - and reactive oxygen species-induced mitochondrialswelling and on mitochondrial transmembrane potential.The effect of minocycline on glutathione (GSH) and nico-tinamide adenine dinucleotide coenzyme and its deriva-tives (NAD(P)H) levels was also addressed. All these de-terminations were performed in brain mitochondria prepa-rations, since they are different from those derived from avariety of other sources (Kristian et al., 2000) and respond differently to diverse stimuli ( Andreyev and Fiskum, 1999;Berman et al., 2000). EXPERIMENTAL PROCEDURES Measurement of cell viability SH-SY5Y cultures were grown in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 2 mM  L -glutamine, penicillin(20 units/ml), streptomycin (5   g/ml), and 15% (vol/vol) heat-inactivated fetal calf serum (GIBCO, Gaithersburg, MD, USA) asreported previously by Yuste et al. (2002). Cells were grown in a humidified cell incubator at 37 °C under a 5% CO 2  atmosphere.For viability experiments, cells were plated at a density of 4  10 4 cells/cm 2 and allowed to attach overnight. Cell viability after drugsadditions was assessed by using 3-[4,5-dimethylthiazol-2-yl]-2-5-diphenyltetrazolium bromide (MTT) cell survival assays. MTT as-says were carried out in 24-well plates. For the MTT assay, 5mg/ml MTT were added to each well 24 h after treatment. Thevolume of MTT added was equal to one-tenth of the total volumeof the well. This was followed by incubation at 37 °C for 3 h. After this, culture medium was removed and 300  l DMSO were addedto each well. Fifty microliter aliquots from each well were thentransferred to a 96-well microplate and diluted with 150  l DMSOand read in duplicate at reference wavelengths of 570 and630 nm. Mitochondrial isolation Mitochondria were isolated from the brain of adult Sprague–Dawleyrats. To exclude the possibility that the observed effects were dueto contaminating synaptosomes, we isolated brain mitochondriausing a Percoll gradient as previously described (Sims, 1990). Rats were killed by decapitation, forebrains were rapidly removed,chopped and homogenized in ice cold isolation buffer (225 mMmannitol, 25 mM sucrose, 10 mM HEPES, 1 mM K 2 EDTA, pH 7.4at 4 °C). The homogenate was centrifuged at 1330  g   for 3 min,and the pellet obtained was re-suspended and re-centrifuged at1330  g   for 3 min. The pooled supernatants were centrifuged at21,300  g   for 10 min. The pellet was re-suspended in 15% Percoll(Sigma, St. Louis, MO, USA) and layered on pre-formed gradients(40 and 23%). The Percoll gradients were then centrifuged at31,700  g   for 10 min. The mitochondrial fraction located at theinterface of the lower two layers was removed, diluted with isola-tion buffer and centrifuged at 16,700  g   for 10 min. The mitocon-drial pellet resuspended in solution III (215 mM mannitol, 71 mMsucrose and 10 mM HEPES, pH 7.4). The solution was energizedwith 10 mM succinate 5 min before starting experiments. PTP activity PTP opening was assayed spectrophotometrically as previouslydescribed (Kristal et al., 2000). Specifically, mitochondria were suspended to reach a protein concentration of 1 mg ml  1 in 200  lof solution containing 125 mM KCl, 20 mM HEPES, 2 mMKH 2 PO 4 , 1   M EGTA, 1 mM MgCl 2 , 5 mM malate and 5 mmglutamate with the pH adjusted to 7.08 with KOH. Changes inabsorbance at 540 nm (A 540 ), indicating mitochondrial swellingdue to PTP opening, were followed, after addition of differentcompounds, using a microplate reader (BioRad, Hercules, CA,USA). Initial A 540  values were   0.8, and minor differences inloading of the wells were compensated by representing the dataas the fraction of the initial absorbance determination remaining ata given time. Mitochondrial protein concentrations were quantifiedspectrophotometrically (Micro BCA Protein Reagent Kit, Pierce,Rockford, IL, USA), with bovine serum albumin used as standard. Monitoring of the   m  m Was qualitatively assayed by tetramethylrhodamine ethylester (TMRE, Molecular Probes, Leiden, The Netherlands) fluo-rescence intensity measured in a Perkin-Elmer (luminescence-spectrophotometer LS50B) fluorimeter at room temperature. Theexcitation and emission wavelengths for TMRE were 549 and581 nm (slit 3 nm) for emission. Dye (250 nM) was added to amedium containing mitochondria (0.5 mg/ml) and 125 mM KCl,20 mM HEPES, 2 mM KH 2 PO 4 , 1  M EGTA, 4 mM MgCl 2 , 5 mMglutamate with the pH adjusted to 7.08 using KOH. Assay for NAD(P)H levels NAD(P)H fluorescence in isolated mitochondria (1 mg ml  1 at25 °C) were measured spectrofluorimetrically with excitation andemission wavelengths of 340 nm (slit 3 nm) and 460 nm (slit 5 nm)at room temperature. We therefore refer to NAD(P)H, indicatingthe signal derived from either NADH or NADPH, or both. Mito-chondriawereresuspendedin125mMKCl,20mMHEPES,2mMKH 2 PO 4 , 1   M EGTA, 1 mM MgCl 2 , 5 mM malate and 5 mmglutamate with the pH adjusted to 7.08 with KOH. Under theseconditions, an increase in autofluorescence signal indicates anincrease in the reduced state of the pyridine nucleotide, NAD(P)H,and a decrease in autofluorescence signal indicates an increasedoxidation to NAD(P)  . Measurement of GSH Levels of GSH were determined by using monochlorobimane(mBCl) fluorescence. GSH is specifically conjugated with mBCl toform a fluorescent bimane-GSH adduct, in a reaction catalyzed byGSH  S  -transferases (Shrieve et al., 1988). The concentration of  F. J. Fernandez-Gomez et al. / Neuroscience 133 (2005) 959–967960  the bimane-GSH adduct increases during the initial 10–12 minperiod of this reaction with first order kinetics, before leveling off (Young et al., 1994). Fluorescence levels at 15 min were used as an indication of GSH content, as has been described previously(Shrieve et al., 1988; Nakamura et al., 2000). Culture medium was removed and cells were washed three times with 1 ml PBS (37 °C)and incubated for 30 min at 37 °C in 1 ml fresh PBS containing80   M mBCl. After incubation cells were washed twice with ice-cold PBS and scraped in 500   l 0.2% Triton X-100 in PBS,centrifuged and 300  l of the extract were used for GSH determi-nation. Mitochondria were resuspended in 125 mM KCl, 20 mMHEPES, 2 mM KH 2 PO 4 , 1  M EGTA, 1 mM MgCl 2 , 5 mM malateand 5 mm glutamate with the pH adjusted to 7.08 with KOH.Fluorescence was measured at an excitation wavelength of 340 nm and emission wavelength of 460 nm. Protein content wasdetermined by the bicinchoninic acid method (Pierce). Determination enzyme activities  All enzyme activities were determined at room temperature inmitochondria isolated suspensions using a microplate reader (BioRad). The activity of complex II–III (succinate-cytochrome  c  reductase) was determined following the method of  King (1967). Complex IV (cytochrome  c   oxidase; E.C.1.9.3.1) activity was de-termined as described by Wharton and Tzagoloff (1967). Measurement of mitochondrial Ca 2  uptake Extramitochondrial free Ca 2  was monitored in the presence of isolated mitochondria using the hexapotassium salt of CalciumGreen-5N (Molecular Probes) (Rajdev and Reynolds, 1993). Iso- lated mitochondria (1 mg ml  1 ) were resuspended in media con-taining 125 mM KCl, 2 mM K 2 HPO 4 , 1 mM MgCl 2 , 20 mM HEPES,5   M EGTA, 1   M Calcium Green-5N and 20 mM succinate pH7.0. Fluorescence was continuously monitored using a Perkin-Elmer LS-50B fluorescence spectrometer with the excitation at506 nm and the emission at 531 nm (slit 2.5 nm). RESULTS Effects of minocycline on GSH and NAD(P)H levels Cells are in constant equilibrium between oxidation andreduction reactions. GSH and NAD(P)H have been pro-posed to act as direct biological antioxidant agents, for which some cytoprotector compounds act increasing their levels (Kirsch and De Groot, 2001). Therefore, we were interested in analyzing whether minocycline could modifythe cellular levels of these two agents. Treatments withminocycline (0.1–100  M for 24 h) did not modify GSH or NAD(P)H levels in SH-SY5Y cells (data not shown). Fur-ther, we analyzed plausible modification of these two agentsin isolated mitochondria. As Fig. 1 illustrates, minocycline(1–10   M) did not significantly modify GSH and NADP(H)levels. Only, at the higher concentration tested, 100   M,minocycline showed a depletion in reduced NAD(P)H lev-els but not GSH (Fig. 1), perhaps because NAD(P)H oxi- dation is very sensitive to mitochondrial membrane depo-larization (Fig. 3). Consistently, the mitochondrial uncou- pler, carbonyl cyanide 4-trifluoromethoxyphenlhydrazone(FCCP) (1  M, FCCP) depleted and rotenone, a mitochon-drial respiratory chain complex I inhibitor (10   M, Rot),increased the nucleotide signal (Fig. 1B). Minocycline blocks calcium- but not KO 2 -inducedmitochondrial swelling  A key step in cellular death programs associated tosome pathological situations is PTP formation (Hirsch etal., 1998). In the next set of experiments, we monitoredmitochondrial swelling by following 540 nm absorbance(A 540 ) decrease, using CaCl 2  and KO 2  as inductors. Mino-cycline was only able to block Ca 2  (75  M) but not KO 2 (5  M) induced mitochondrial swelling in a concentration-dependent manner. A concentration of up to 10   M, mi-nocycline did not significantly modify Ca 2  -induced mito-chondrial swelling, but at 100   M minocycline completelyblocked Ca 2  -induced swelling (Fig. 2 A). Nevertheless,minocycline, at any of the concentrations tested, did notinhibit KO 2 -induced mitochondrial swelling (Fig. 2B). Tofurther analyze whether minocycline effect was a commoneffect of this antibiotic family, we tested the effect of tetra-cycline on mitochondrial swelling. As shown in Fig. 2, Fig. 1.  Effect of minocycline on mitochondrial GSH and NAD(P)Hlevels. Effect of different minocycline concentrations on mitochondrialGSH and NAD(P)H levels. Intact mitochondria (500   g protein) wereresuspended and GSH (A) and NAD(P)H (B) levels were monitoredfluorimetrically as described in Experimental Procedures in responseto minocycline (1–100  M), rotenone (10  M) or FCCP (1  M, FCCP)additions. Data represent means  S.E.M. of four to six experiments.*  P   0.05, ***  P   0.001 versus vehicle conditions using ANOVA, Tur-key’s test. F. J. Fernandez-Gomez et al. / Neuroscience 133 (2005) 959–967 961  tetracycline, at any of the concentrations tested, did notaffect mitochondrial-induced swelling. Minocycline induces mitochondrial potential collapse  Alterations of mitochondria’s electric transmembranal po-tential (  m) have been related to cell death processes(Prehn et al., 1996; Jordan et al., 2003b). In the next set of  experiments, we studied whether minocycline was able tomodify   m in isolated mitochondria. We monitored  mchanges by measuring the release of the cationic mem-brane-permeant fluorescent probe TMRE pre-loaded intoisolated mitochondria. Under these conditions, total fluo-rescence of the mitochondrial suspension will increase if the organelles depolarize. As shown in Fig. 3 A, minocyclineinduced mitochondrial depolarization in a concentration-dependent manner. Concentrations higher than 10  M in-duced a significant   m collapse. As shown in Fig. 3,tetracycline did not induce   m collapse at any of theconcentrations tested (0.1–100   M), and the additionof FCCP (1   M) resulted in TMRE release from themitochondria. Minocycline blocks mitochondria calciumbuffering capacity  Asmitochondriaparticipateintheregulationofcytoplasmicfree Ca 2  levels (Skulachev, 1999) and PTP is triggered by intramitochondrial Ca 2  accumulation, we were inter-ested to know whether or not minocycline was able tomodify mitochondrial Ca 2  buffering capacity. The maxi-mal quantity of Ca 2  that can be sequestered by mitochon-dria can be measured by monitoring the disappearance of extramitocondrial free Ca 2  from media following the ad-dition of known pulses of CaCl 2 , using Calcium Green-5Nfluorescence tracing. A fixed amount of CaCl 2  (4 nmol)was added repeatedly every 60 s until there was no further evidence of Ca 2  uptake. Addition of Ca 2  after this pointwas associated with a further increase in fluorescenceindicating that Ca 2  is not taken up by isolated mitochon-dria and reacts with the dye. Fig. 4 shows a typical fluo-rescence tracing of Ca 2  uptake using isolated mitochon-dria. Untreated mitochondrial preparations were able toretain up to 31.4  2.8 nmol Ca 2  /mg mitochondrial protein.The presence of minocycline significantly reduced mito-chondrial Ca 2  buffering capacity in a concentration-dependent manner  (Fig. 4), while 0.1   M or 1   M mino-cycline did not modify mitochondrial buffering capacity(31.3  2.6 and 30.6  2.7 nmol Ca 2  /mg mitochondrial pro-tein respectively), at a concentration of 10   M. The anti-biotic decreased mitochondrial buffering capacity down to28.0  3.3 nmol Ca 2  /mg mitochondrial protein ( P   0.05). A higher minocycline concentration (100   M) further de-creased mitochondrial buffering capacity to 21.7  3.9 nmolCa 2  /mg mitochondrial protein (Fig. 4). Addition of tetra- cycline (100   M; Fig. 4) did not modify the mitochondrial Ca 2  buffering capacity. Effects of minocycline on mitochondrial respiratorychain complex enzymatic activity The next set of experiments was performed to analyze thepossible effects of minocycline on mitochondrial respira- Fig. 3.  Minocycline induces   m collapse.   m Was measured byusing TMRE. Minocycline (0.1–100  M) or tetracycline (100  M) wasadded. FCCP (1  M) was used to ensure that the mitochondria couldbe depolarized. Data represent means  S.E.M. of six experiments. 0 500 1000 15000.8750.9000.9250.9500.9751.000 A CaCl 2 Tetr 110RR100CsA0    A    5   4   0 Time (s)0 500 1000 15000.8250.8500.8750.9000.9250.9500.9751.000 B KO 2 Tetr 110100CsA0    A    5   4   0 Time (s) Fig.2.  MinocyclinepreventsCa 2  -butnotKO 2 -inducedmitochondrialswelling. Changes in absorbance at 540 nm (A 540 ), indicating mito-chondrial swelling, were followed, after addition of CaCl 2  (75   M,panel A) and KO 2  (5   M, panel B)-induced swelling. The effect of minocycline (0.1–100  M), tetracycline (100  M; dashed line), Ruthe-nium Red (RR, 5  M) and cyclosporin A (CsA, 10  M) was measured.Drugs were added 15 min before starting the experiments. Similar datawere found in at least five different experiments. Data representmeans  S.E.M. of nine experiments. F. J. Fernandez-Gomez et al. / Neuroscience 133 (2005) 959–967962  tory chain complex activity. Under our experimental con-ditions mitochondrial complex IV was not modified by thetreatment with minocycline for 15 min at any of the dosestested (Fig. 5 A). However, the complex II–II activity wasmodified by minocycline. Incubation of brain mitochondria(0.3   g protein) with 100   M minocycline for 15 min didresult in a significant loss in enzymatic activity comparedwith control mitochondrial suspension while lower concen-trations (1 and 10  M; 15 min) did not alter complex II–IIIactivity (Fig. 5B). Minocycline effects on cytotoxic models The effect of minocycline on cell viability was examined inSH-SY5Y cells exposed to different mitochondrial toxins,including malonate and 6-hydroxydopamine (6-OHDA).We have previously shown that both drugs are able toinduce cell death in SH-SY5Y cell cultures by mechanismsinvolving reactive oxygen species formation (Jordan et al.,2004; Fernandez-Gomez et al., 2005). As shown in Fig. 6, minocycline (100  M) failed to afford cytoprotection to thecell cultures challenged with mitochondrial toxins. By con-trast and in line with previous observations, 100  M mino-cycline treatment resulted in a cytoprotective effect againststaurosporine-induced cell death (Fig. 6). DISCUSSION In the present work we have focused our attention on brainmitochondrion as a plausible pharmacological target toexplain minocycline’s neuroprotective actions. Minocy-cline, by inducing collapse of the mitochondrial potentialdecreased Ca 2  influx into the organelle and preventedmitochondrial swelling. On the other hand, our data ex-clude the possibility of a non-specific pathway involvingincreases in cellular antioxidant capacity, since minocy-cline did not affect NAD(P)H or GSH levels.Mitochondrial swelling might be a consequence of PTPformation, which leads to a massive water influx into mito-chondriaandreleaseofintramitochondrialproteinsthatmightactivate downstream apoptotic pathways (Hirsch et al.,1998). Thus, the cytoprotective effects of minocycline mayrely on its ability to block mitochondrial swelling, as mito-chondrial swelling is involved in several death pathwaysincluding those induced by 1-methyl-4-phenylpyridinium,staurosporine, veratridine, or   N  -methyl- D -aspartate (Jordanet al., 2002; Boada et al., 2000; Duan et al., 2003). In fact,drugs able to block PTP formation afford complete or partial neuroprotection against a broad type of insults C   o  n  t   r   o  l     405060708090100 1   0    M    M   i   n  o  1   0   0    M    M   i   n  o  1    M    M   i   n  o  0   . 1    M    M   i   n  o        C  o  m  p   l  e  x   I   V  a  c   t   i  v   i   t  y   (   %   o   f  c  o  n   t  r  o   l   )   l   ) AB C   o  n  t   r   o  l     405060708090100 1   0   0    M    M   i   n  o  1   0    M    M   i   n  o  1    M    M   i   n  o  0   . 1    M    M   i   n  o   **    C  o  m  p   l  e  x   I   I   /   I   I   I  a  c   t   i  v   i   t  y   (   %   o   f  c  o  n   t  r  o Fig. 5.  Effect of minocycline on mitochondrial respiratory chain com-plex activities. Brain mitochondria suspensions were incubated in theabsence (control) or presence of minocycline for 15 min at roomtemperature. Enzyme activities of complex IV (A) and complex II/III (B)were determined in the mitochondrial suspensions as described inExperimental Procedures. Values are expressed as percentages of control conditions. The mean  S.E.M. values from three different mi-tochondria preparations. **  P   0.01 versus vehicle conditions using ANOVA, Turkey’s test. 0 120 240 360 480 60002505007501000CaCl 2 110010Minocycline    C  a   l  c   i  u  m   g  r  e  e  n  -   5   N   f   l  u  o  r  e  s  c  e  n  c  e Time (s) Fig. 4.  Minocycline inhibits mitochondrial Ca 2  uptake capacity. Max-imal mitochondrial Ca 2  uptake by isolated mitochondria (1 mg/ml in0.5 ml) was measured as described using Calcium Green-5N as anindicator of the free Ca 2  concentration in the medium. Minocycline(1–100   M) or tetracycline (100   M, dashed line) was added at thearrow. Pulses of 4 nmol CaCl 2  (100  M) were added every 60 s. Dataare expressed as mean values obtained from one experiment per-formed in triplicate. Similar data were found in at least four differentexperiments. F. J. Fernandez-Gomez et al. / Neuroscience 133 (2005) 959–967 963
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