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Short-term effects of zinc on acetylcholine metabolism and viability of SN56 cholinergic neuroblastoma cells
Neurochemistry International 56 (2010) 143–151
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Short-term effects of zinc on acetylcholine metabolism and viability of SN56 cholinergic neuroblastoma cells Anna Ronowska a, Aleksandra Dys´ a, Agnieszka Jankowska-Kulawy a, Joanna Klimaszewska-Łata a, Hanna Bielarczyk a, Piotr Romianowski a, Tadeusz Pawełczyk b, Andrzej Szutowicz a,* a b
Department of Laboratory Medicine, Medical University of Gdan´sk, Ul. Debinki 7, 80-211 Gdansk, Poland Department of Molecular Medicine, Medical University of Gdan´sk, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 August 2009 Received in revised form 14 September 2009 Accepted 17 September 2009 Available online 23 September 2009
Excessive accumulation of zinc in the brain is one of putative factors involved in pathomechanism of cholinergic encephalopathies. The aim of this work was to investigate whether short-term increase of zinc concentration in the extracellular space may affect energy and acetylcholine metabolism in SN56 cholinergic cells of septal origin. Short 30 min exposition of SN56 cells to increasing zinc levels caused greater loss of viability of differentiated (DC, [EC0.4] 0.09 mM) than nondifferentiated cells (NC, [EC0.4] 0.14 mM). Concentration-dependent accumulation of zinc displayed exponential non-saturable kinetics. Zinc accumulation caused the decrease of calcium accumulation in mitochondria and its increase in cytoplasmic compartment of SN56 cells. Significant inverse and direct correlations were found between zinc accumulation and calcium levels in mitochondrial (r = 0.96, p = 0.028) and cytoplasmic (r = 0.97, p = 0.028) compartments of DC, respectively. Zinc exerted similar inhibition of pyruvate dehydrogenase, aconitase and isocitrate dehydrogenase both in NC and DC homogenates, at Ki values equal to about 0.07, 0.08 and 0.005 mM, respectively. On the other hand, ketoglutarate dehydrogenase activity in DC was inhibited by zinc (Ki 0.0005 mM) 8 times stronger that in NC (Ki 0.004 mM). Also zinc-evoked decreases in acetylcholine content and its release were significantly greater in DC than in NC. Same conditions caused suppression of cytoplasmic and mitochondrial content of acetyl-CoA, that positively correlated with inhibition of transmitter functions (r = 0.995, p = 005) and loss of cell viability (r = 0.990, p = 0.0006), respectively. Significant correlations were also found in zinc-challenged cells between pyruvate dehydrogenase activity and both mitochondrial acetyl-CoA content and cell viability. These data indicate that pyruvate dehydrogenase-dependent acetyl-CoA synthesis in neuronal mitochondria may be a primary target for short-term neurotoxic effects of zinc. In consequence, shortages of acetylCoA in the mitochondrial compartment would cause fast loss of functional and structural integrity of cholinergic neurons. ß 2009 Published by Elsevier Ltd.
Keywords: Acetyl-CoA Acetylcholine Cholinergic cells Pyruvate dehydrogenase Neurotoxicity Zinc
1. Introduction Zinc is an important trace element in living organisms being an essential component for over 300 enzymes, transporters, transcription factors, transmitters and hormones. The zinc concentration in the brain is the highest in comparison to other tissues. Its
Abbreviations: ACh, acetylcholine; AD, Alzheimer’s disease; AMPA, a-amino-3hydroxyl-5-methyl-4-isoxazole-propionate; ChAT, choline acetyltransferase; DC, differentiated SN56 cells; DMEM, Dulecco Modified Eagle’s Medium; FBS, fetal bovine serum; ICDH-NADP, NADP-dependent isocitrate dehydrogenase; NC, nondifferentiated SN56 cells; NMDA, N-methyl-D-aspartate; KDH, ketoglutarate dehydrogenase; PDH, pyruvate dehydrogenase; TCA, trichloroacetic acid; ZEN, zinc enriched neurons. * Corresponding author. Tel.: +48 58 349 2770; fax: +48 58 449 2784. E-mail address: [email protected] (A. Szutowicz). 0197-0186/$ – see front matter ß 2009 Published by Elsevier Ltd. doi:10.1016/j.neuint.2009.09.012
average level in whole brain was estimated to be about 0.15 mM (Zatta et al., 2003). However, in particular compartments the level of free Zn2+ is estimated to be of sub-nanomolar and in brain extracellular fluid of submicromolar range (Bertoni-Freddari et al., 2006; Frederickson et al., 2006). On the other hand, in the vesicles of presynaptic terminals of excitatory glutamatergic neurons, Zn concentration was found to be above 1 mM (Mocchegiani et al., 2005). The latter ones belong to the subgroup of zinc enriched neurons (ZEN) that are particularly abundant in olfactory nucleus, hippocampus and other forebrain regions (Airado et al., 2008). Excessive activation of ZEN neurons in course of several brain pathologies like hypoxia, hypoglycaemia, amyloid-beta overload leads to excessive release of glutamate. Prolonged activation of N-methyl-D-aspartate (NMDA) receptors by a glutamate yields over-production of NO and free radicals in receptive cells. Simultaneously massive amounts of Zn2+ are released from
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glutamatergic vesicles (Frederickson et al., 2006; Hynd et al., 2004). It may cause a build up Zn2+ concentration in the synaptic cleft to levels as high as 0.3 mM (Zatta et al., 2003). Subsequent uptake of Zn from extracellular space to postsynaptic cells, through voltage-gated Ca or NMDA channels might cause their overload with this metal (Mocchegiani et al., 2005). In cholinergic human SK-SH-SY5Y cells activation of M1 muscarinic cholinergic autoreceptors increased Zn accumulation (Zuchner et al., 2006). In turn, the Zn-overload would activate ZnT-1 and other Zn-transporting systems to excrete Zn back to extracellular space or sequestrate it in intracellular storage compartments, respectively (Lovell et al., 2005; Quin et al., 2008). In addition, Zn was reported to compete with Ca transport through voltage-gated Ca-channels (Sun et al., 2007). Through such mechanisms, excess of extracellular Zn in the brain might directly induce neurodegeneration in course of different encephalopathies (Religa et al., 2006). For instance, excess of extracellular Zn was found to facilitate formation of cytotoxic amyloid-b aggregates (Khan et al., 2005). On the other hand, Zn was reported to protect NT2 human cells against amyloidbeta toxicity (Cardoso et al., 2005). Zn also inhibited transmitterstimulated capacitative entry of extracellular Ca into the nonglutamatergic neurons and nerve terminals. Thereby, it suppressed their transmitter functions (Kresse et al., 2005). Besides chronic accumulation of Zn in neurons yielded chronic inhibition of pyruvate (pyruvate:lipoate oxidoreductase acceptor acetylating, PDH, EC 1.2.4.1) and ketoglutarate (2-oxoglutarate:lipoate oxidoreductase acceptor acylating, KDH, EC 1.2.4.2) dehydrogenases as well as aconitase (citrate(isocitrate)hydrolyase, EC 4.2.1.3), resulting in the depletion of acetyl-CoA in mitochondria. It led to extended energy deficits and cell death (Ronowska et al., 2007). By such mechanism(s) Zn could also aggravate neurotoxic actions of other compounds and interfere with various mitochondrial pathways known to be involved in the mechanisms of neurodegeneration (Castaldo et al., 2009; Gibson et al., 2000; Ronowska et al., 2007; Szutowicz et al., 2006). In addition, long-term exposure of differentiated cholinergic SN56 neuroblastoma cells to Zn caused adaptative suppression of choline acetyltransferase (acetyl-CoA:choline-O-acetyltransferase, ChAT, EC 2.3.1.6) activity, presumably through the long-lasting decrease of acetyl-CoA level in their cytoplasmic compartment (Ronowska et al., 2007). Also, sensitivity of SN56 cells to long-term NO, Al, interleukin 1b or amyloid-b burden was reported to correlate positively with their phenotypic differentiation (Bielarczyk et al., 2003a, 2005; Szutowicz et al., 2006). It is however not known whether some alterations in cell viability and ACh metabolism might occur in acute Zn-toxicity experimental paradigms. There is also no information how short-term effects of Zn on cholinergic neurons are transferred into chronic alterations in their acetyl-CoA/ACh metabolism and viability (Ronowska et al., 2007). Therefore, the aim of this study was to investigate whether short-term increase of [Zn] in extracellular space could induce instant alterations in cholinergic neurons through the interaction with pre- and postsynthetic steps of their ACh metabolism. Data presented here indicate that Zn-evoked short-term alterations in cholinergic neurons metabolism could establish the base for their modifications during their prolonged exposure to this metal (Ronowska et al., 2007). 2. Materials and methods 2.1. Materials Unless otherwise specified biochemicals were obtained from Sigma–Aldrich (Poznan´, Poland), [1-14C-acetyl]-CoA 4 mCi/mmol was from Perkin–Elmer (Boston, MA, USA), cell cultures growth media and components were provided by Gibco Life Technologies (Warsaw, Poland), cell culture disposables derived from Sarstedt (Stare Babice, Poland).
2.2. Cell cultures SN56.B5.G4 cholinergic murine neuroblastoma cells, with stable expression of PDH and ChAT between 22nd and 40th passage, were used for experiments (Hammond et al., 1990). Cells were seeded at density of 40,000 cells/cm2 on 75 cm2 Falcon vessels in Dulbecco’s modified Eagle medium containing 1 mM L-glutamine, 0.05 mg of streptomycin and 50 U of penicillin per 1 mL and 10% fetal bovine serum (DMEM-FBS) and grown at 37 8C in atmosphere 5% CO2, 95% air. Such medium was reported to contain 0.005 mM concentration of endogenous Zn (MacDonald et al., 1998). Cholinergic differentiation was obtained by combined addition of 1 mmol/L dibutyryl cAMP (cAMP) and 0.001 mmol/L all-trans-retinoic acid (RA) for 48 h. At this time media were discarded. Zn and other agents were added as indicated in DMEM-FBS, or N2 supplemented DMEM containing no differentiating agents and culture continued for 24 h. Cells that remained attached to the plate were harvested into 10 mL of ice cold 140 mmol/L NaCl containing 5 mmol/L KCl, 1.7 mmol/L NaKphosphate buffer (pH 7.4) and 5 mM glucose and collected by centrifugation at 200 g for 7 min. Supernatant was removed and cells were suspended in 320 mmol/L sucrose containing 10 mmol/L HEPES buffer (pH 7.4) and to obtain protein concentration 10.0 mg/mL. Immediately after collection the cells were used for Trypan blue exclusion assay and for metabolic studies. For ChAT and PDH assays samples were kept frozen at 20 8C for 2–7 days. Remaining enzyme activities were assessed in unfrozen samples within 24 h after harvesting cells. 2.3. Trypan blue exclusion assay Cell suspension was mixed with equal volume of 0.4% isotonic trypan blue solution. Total cell number and fraction of nonviable, dye accumulating cells were counted after 2 min in Fuchs-Rosenthal haemocytometer under light microscope (Wang et al., 2001). 2.4. Metabolic studies Depolarizing incubation medium contained in a final volume of 1.0 mL 2.5 mmol/L pyruvate, 2.5 mmol/L L-malate, 90 mmol/L NaCl, 30 mmol/L KCl, 20 mmol/L NaHEPES (pH 7.4), 1.5 mmol/L Na-phosphate, 0.01 mmol/L choline chloride, 0.015 mmol/L eserine sulfate, 32 mmol/L sucrose and 0.7–1.0 mg of cell protein. These conditions were employed to assure maximal acetylcholine release and resynthesis (Bielarczyk et al., 1998). Incubation was started by the addition of cell suspension and continued for 30 min at 37 8C with shaking at 100 cycles per min. For the determination of total acetyl-CoA content, 0.3 mL of incubation medium was centrifuged at 5000 g for 2 min. Supernatant was removed and cell pellet was deproteinized as indicated below. 2.5. Acetyl-CoA assay For assaying of whole cell acetyl-CoA content, the cell pellet was deproteinized by suspension in a small volume of 5 mmol/L HCl and incubation in a boiling bath for 1 min. To assess acetyl-CoA content in the cell mitochondria 0.5 mL of incubation medium was mixed with equal volume of lysing solution containing 1.4 mg digitonin/mL in 125 mmol/L KCl with 20 mmol/L NaHEPES buffer (pH 7.4) and 3 mmol/L EDTA. Lysate was transferred on 0.5 mL of silicone oil mixture (AR20 and AR200, 1:2) layered over 0.1 mL of buffered 320 mM sucrose. After 30 s the mitochondrial fraction was separated from the soluble one by centrifugation for 40 s at 12,000 g. After the removal of soluble fraction, silicon oils and sucrose, mitochondrial pellet was deproteinized as described above. Deproteinized extracts of whole cells and mitochondria were treated with maleic anhydride solution in ethyl ether for 2 h to remove CoA-SH. Cycling reaction was carried for 60 min in 0.1 mL of medium containing 1.9 mmol/L acetyl phosphate, 1.2 mmol/L oxaloacetate, 1.0 IU phosphotransacetylase and 0.12 IU citrate synthase. Cycling reaction was stopped by heating samples at 100 8C for 10 min and citrate formed was determined (Szutowicz and Bielarczyk, 1987). Cytoplasmic acetyl-CoA level was calculated by subtraction of mitochondrial acetyl-CoA from total acetyl-CoA content (Szutowicz and Bielarczyk, 1987). 2.6. Acetylcholine assay Samples (0.2 mL) of cells after 30 min incubation in depolarizing medium were centrifuged 3 min at 10,000 g. Supernatants were collected and used for the determination of released ACh. To calculate rates of ACh release/synthesis values obtained after 30 min incubation were subtracted from those at time zero and divided by the incubation time (Bielarczyk et al., 1998; Tucek, 1993). Cell pellets were homogenized in 0.4 mL of methanol and centrifuged. Obtained supernatants were mixed with 0.05 mL of 1.0 mol/L formic acid and centrifuged. Chloroform (0.9 mL) and water (0.4 mL) were sequentially added with ‘‘Vortex’’ mixing. After centrifugation upper methanol–water layers were collected and evaporated of Speed-Vac SC110 dessicator (Savant, Paris, France) and sediments after reconstitution with water were used for the determination of intracellular ACh content. Extra- and intracellular ACh was determined with acetylcholinesterase–choline oxidase method coupled with peroxidase–luminol detection system (Israel and Lesbats, 1982) using Junior luminometer (Berthold Technology, Bad Wild-Bad, Germany).
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Fig. 1. Effect of 30 min exposition of harvested SN56 NC and DC to increasing Zn concentrations on: (A) cell viability; (B) cell number. Data are means SEM from 3 duplicate experiments. Significantly different from respective no Zn control, *p < 0.005, from corresponding NC, +p < 0.01.
2.7. Calcium and zinc assays After separation from the incubation medium cell pellets were washed once with Ca-free incubation medium containing 1 mmol/L EDTA to remove surface-bound metals. Centrifuged and washed again with EDTA-free medium. For Ca determination, obtained pellet was deproteinized with 5% TCA and supernatants were assayed by arsenazo III spectrophotometric method (Scarpa, 1979). For Zn determinations cell pellet was deproteinized with 4% HClO4 and cation level was determined by fluorimetric method with N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) as described by Chen and Liao (2003) with emission and excitation wavelengths equal to 335 and 495 nm, respectively. To assess mitochondrial Ca, cells were lysed with digitonin followed by centrifugation through silicon oil mixture as described above (Section 2.5). 2.8. Enzyme assays Immediately before the assay samples were diluted to desired protein concentration in 0.2% (v/v) Triton X-100. ChAT activity was assessed by the radiometric method using [1-14C]acetyl-CoA as a substrate (Fonnum, 1975). PDH was assayed by the citrate synthase coupled method (Szutowicz et al., 1981). KDH activity was determined by a direct measurement of NAD reduction (Pawełczyk and Angielski, 1984). Activities of aconitase, succinate dehydrogenase (succinate:acceptor oxidoreductase, 1.3.99.1), NADP-isocitrate dehydrogenase (threo-D-isocitrate:NADP oxidoreductase (decarboxylating), ICDH-NADP, EC 1.1.1.42) and NAD Lmalate dehydrogenase (EC 1.1.1.41) were assayed by methods described elsewhere (Ochoa, 1955; Plaut, 1962; Veeger et al., 1969; Villafranca, 1974). For studies of acute Zn effects on enzyme activities the cation and/or other compounds were added directly to the assay media.
concentration (Fig. 1A). DC appeared to be more sensitive than NC to lower Zn concentrations as evidenced by [EC0.4] values equal to 0.085 and 0.14 mmol/L, respectively (Fig. 1A). No change in NC and DC number took place in these conditions (Fig. 1B). 3.2. Short-term zinc accumulation in SN56 cells The exposition of harvested DC and NC to Zn for 30 min in postculture medium (see Section 2) caused concentration-dependent metal accumulation that displayed non-saturable exponential kinetics (Fig. 2). At 0.25 mmol/L extracellular level of Zn, its intracellular accumulation reached values above 150 nmol/mg of protein level in both NC and DC (Fig. 2). 3.3. Short-term effect of Zn on Ca distribution of SN56 cells In depolarizing medium, the rise of Zn to 0.10 mmol/L caused significant alterations neither in whole SN56 cell Ca content nor in its distribution between mitochondrial and cytoplasmic compartment (Fig. 3A and B). Further increase of extracellular Zn up to
2.9. Protein assay Protein was assayed by the method of Bradford (1976) with human immunoglobulin as a standard. 2.10. Statistics Statistical analyses were carried out by one way ANOVA with Bonferroni multiple comparison test or by non-paired Student’s t-test, at p < 0.05 being considered to be statistically significant.
3. Results 3.1. Short-term cell viability Our past studies of chronic Zn-cytotoxicity in DMEM-FBS cultured SN56 cells revealed about 40% increase of nonviable cell fraction after 24 h exposition to this metal (Ronowska et al., 2007). Incubation of SN56 cells in the control depolarizing medium for 30 min caused no change in trypan blue positive fraction of NC and DC (not shown). On the other hand, Zn caused fast concentrationdependent, increase of trypan blue-retaining cell fraction that after 30 min incubation reached about 70% at 0.25 mmol/L metal
Fig. 2. Effect 30 min exposition of harvested SN56 cells to increasing extracellular Zn concentrations on its intracellular accumulation in NC and DC. Data are means SEM from 5 to 7 duplicate experiments. Significantly different from respective no Zn control, *p < 0.01.
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Fig. 3. Effect of 30 min exposition of harvested SN56 cells to increasing Zn concentrations on Ca content in their mitochondrial and cytoplasmic compartments: (A) nondifferentiated cells; (B) differentiated cells. Data are means SEM from 4 duplicate experiments. Significantly different from respective no Zn control, *p < 0.01.
0.25 mmol/L brought about concentration-dependent increases of whole cell (40–80%) and cytoplasmic Ca (128–147%), and its decrease in mitochondria (40–50%), both in NC and DC (Fig. 3A and B).
value of Zn-evoked ACh depletion in DC appeared to be 190% higher than in NC (Fig. 5A). Also, absolute value of maximal Zn-evoked inhibition of ACh release in DC was over 3 times greater than in NC (Fig. 5B).
3.4. Short-term effects of zinc on cholinergic parameters SN56 cells
3.5. Short-term effect of zinc on acetyl-CoA metabolism
Activity of ChAT in DC homogenates (0.44 0.04 nmol/min/ mg protein) was 2.5 times higher than in NC ones (Fig. 4). In DC, ChAT activity was not significantly inhibited by short-term exposure to Zn up to 0.3 mM concentration (Fig. 4). On the other hand, in NC it was inhibited at 0.3 mM Zn by about 34% (Fig. 4). ACh levels and its release from DC were 85% higher than in NC (Fig. 5A and B). The stable, steady-state level of ACh was maintained in NC and DC during entire 30 min incubation period (not shown). In such conditions amount of ACh released to the medium corresponded to its synthesis (Bielarczyk et al., 1998; Tucek, 1993). ACh release from DC was 3 times higher than in NC (Fig. 5B). Zn caused concentration-dependent suppression of ACh content both in NC and DC. Namely, at 0.15 mmol/L Zn level the relative suppression of ACh content in NC and DC was equal to 65 and 46%, respectively. ACh release from NC was almost completely inhibited by 0.05 mmol/L Zn, while in DC only 30% suppression of the transmitter release, took place (Fig. 5B). Thus, apparent [IC]50% values for Zn-evoked suppressions of ACh content and release in DC appeared to be equal to 0.15 and 0.10 mmol/L, whereas in NC to 0.10 and 0.01 mmol/L, respectively (Fig. 5A and B). However, absolute
The increase of extracellular [Zn] from 0.01 to 0.25 mmol/L caused concentration-dependent reduction of acetyl-CoA level in NC mitochondria. It fell to 50% of control values at 0.10 mmol/L [Zn] (Fig. 6A). Further increase of [Zn] resulted in slight attenuation of acetyl-CoA suppression in NC mitochondria. Acetyl-CoA level in DC mitochondria was about 30% lower than in NC (Fig. 6A). In this cell phenotype, elevation of [Zn] to 0.25 mmol/L caused a gradual depletion of mitochondrial acetyl-CoA down to 30% of control values with apparent [IC0.5] equal to 0.10 mmol/L (Fig. 6A). In NC, 0.05 mmol/L [Zn] caused twofold increase of cytoplasmic [acetylCoA]. Higher [Zn] caused a total depletion of acetyl-CoA in this compartment (Fig. 6A). On the other hand, in DC 0.1 mmol/L [Zn] brought about 80% suppression of cytoplasmic acetyl-CoA (Fig. 6B). Further increase of Zn up to 0.25 mmol/L resulted in no alteration in the level of this metabolite (Fig. 6B). 3.6. Short-term effects of zinc on selected enzyme activities The past studies revealed that diverse chronic neurotoxic conditions, cause suppression of acetyl-CoA in mitochondrial and cytoplasmic compartments of neuronal cells (Szutowicz, 2001; Szutowicz et al., 2006). These alterations could result from inhibition of brain PDH activity, acetyl-CoA utilization in TCA cycle, and its transport to cytoplasm through the mitochondrial membrane (Bubber et al., 2005; Szutowicz et al., 2006). The Zn-Ki values for PDH, aconitase and ICDH-NADP appeared to be similar for both cell phenotypes being equal to about 0.07–0.09, 0.008– 0.009 and 0.004–0.006 mmol/L, respectively (Fig. 7A–C). On the other hand, Ki for Zn-evoked inhibition of DC-KDH (0.0005 mmol/ L) appeared to be 8 times lower than for NC-KDH (0.004 mmol/L), respectively (Fig. 7D). Activities of succinate dehydrogenase, malate dehydrogenase and citrate synthase were not altered by Zn up to 0.3 mmol/L concentration (not shown). 4. Discussion
Fig. 4. Acute effect of Zn on choline acetyltransferase activity in NC and DC homogenates. Data are means SEM from 6 duplicate experiments. Significantly different from respective no Zn control, *p < 0.05; nondifferentiated cells, +p < 0.0001.
In the present work, cytotoxic effects of Zn were observed at its 0.05–0.25 mmol/L levels in extracellular space (Fig. 1B). Such
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Fig. 5. Effect of 30 min exposition of harvested SN56 NC and DC to increasing Zn concentrations on : (A) acetylcholine content; (B) depolarization-evoked Ca-dependent acetylcholine release. Data are means SEM from 3 duplicate experiments. Significantly different from respective no Zn control, *p < 0.01; corresponding NC values, +p < 0.01.
Fig. 6. Effect of 30 min exposition of harvested SN56 cells to increasing Zn concentrations on their acetyl-CoA content in: (A) mitochondrial; (B) cytoplasmic compartments. Data are means SEM from 4 to 10 duplicate experiments. Significantly different from respective no Zn control, *p < 0.01; corresponding NC values, +p < 0.01.
concentrations of Zn are compatible with those reported for the whole brain and the synaptic cleft of glutamatergic terminals in vivo (Zatta et al., 2003). Therefore, the Zn effects, reported here, are likely to be of pathophysiological significance. The hyperbolar shape of the correlation plots between load of cells with Zn and their mortality, indicates the existence of Zn-multiple target interactions leading to the cholinergic cell death (Fig. 8). It is known that several Ca-permeable channels, including aamino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), kainate, NMDA and GABA receptors participate in the Zn entry into the neuronal cells (Marin et al., 2000; Mocchegiani et al., 2005; Sun et al., 2007). Therefore, a concave up shape of concentration-dependent plots of Zn accumulation might result from consecutive activation of the multiple cation-permeable channels of decreasing affinities and increasing transport capacities to Zn (Fig. 2) (Sun et al., 2007). However, stimulation of M1 autoreceptors (Zuchner et al., 2006) did not contribute to the Zn-overload in SN56 cells, as ACh release markedly decreased with the increase of extracellular [Zn] (Fig. 5). Similar levels of Zn accumulation in NC and DC indicate that increased expression of cholinergic phenotype did not modify the overall Zn-transporting capacity in the SN56 cells (Fig. 2). Therefore, the Zn accumulation itself could not be a cause of differential susceptibility of NC and DC to this metal (Figs. 1 and 2).
The decrease of mitochondrial Ca in Zn-treated cells could result from the inhibition of Ca uniporter in their inner membrane (Fig. 3) (Malaiyandi et al., 2005; Sensi et al., 2002). The impairment of intramitochondrial Ca sequestration might contribute, at least in part, to its increased accumulation in the cytoplasmic compartment (Fig. 3). Such conclusion is justified by the existence of direct and inverse correlations between level of intracellular Zn and Ca content in cytoplasmic and mitochondrial compartments, respectively (Fig. 9). However, presented findings remain in conflict with data demonstrating that Zn inhibited NMDA and AMPA channels (Mocchegiani et al., 2005; Sun et al., 2007). Hence, Zn should yield rather decrease not the increase of whole cell and cytoplasmic Ca (Fig. 3). Such phenomenon was observed in SN56 cells treated with aluminum, that inhibited verapamil-sensitive Ca-channels in plasma membranes (Jankowska et al., 2000). However, in present experiments Zn caused rapid impairment of membrane integrity as evidenced by a concentration-dependent, short-term increase of trypan blue positive cells fraction (Figs. 1–3) (Marin et al., 2000; Sun et al., 2007). Such conclusion is also supported by the existence of significant direct correlation between acute Zn-induced increases of Ca accumulation in cytoplasmic compartment and rise of trypan blue positive fraction of SN56 cells (Figs. 1, 3 and 10B). Therefore, prolonged hiper-zincaemia could be responsible for chronic detrimental effects of this metal on cholinergic cells
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Fig. 7. Dixon plots of acute concentration-dependent effects of Zn on pyruvate dehydrogenase, aconitase and NADP-isocitrate dehydrogenase activities in NC and DC homogenates. Data are means from 4 to 6 duplicate experiments. (A) pyruvate dehydrogenase; (B) aconitase; (C) isocitrate-NADPdehydrogenase; (D)ketoglutaratedehydrogenase.
(Ronowska et al., 2007). Similar patterns of Zn-evoked alterations in subcellular distributions of Ca in NC and DC indicate that they may not directly contribute to differential, phenotype-dependent sensitivity of these cells to Zn (Figs. 1B, 2 and 3). It is known, that the size of intracellular ACh pool is resultant of ChAT activity and concentrations of transmitter’s precursors in cytoplasmic compartment (Szutowicz et al., 1996, 1998; Tucek, 1993). In this respect, proportionality between ChAT activities and ACh contents or releases in DC and NC is in accord with past reports (Figs. 4–6) (Bielarczyk et al., 2003b; Szutowicz et al., 1998; Tucek, 1993). However, the mechanism of tight correlations between ChAT activity and size of ACh pool, still remains unsolved. Therefore, fast reduction of intracellular ACh pool by Zn, at the absence of significant alterations of ChAT activity, indicates that the former could be due to reduction in cytoplasmic levels of acetyl-CoA (Figs. 4–6 and 11). Significant correlations between cytoplasmic acetyl-CoA and ACh content/release from Zn-challenged DC may be explained on the basis of mass action law mechanism at equilibrium constant for ChAT reaction equal to 11.4 (Fig. 11) (Tucek, 1993). Similar dependences were also observed between level of choline and ACh release in brain cortex (Napoli et al., 2008). Thus, the inhibition of transmitter functions in cholinergic cells acutely exposed to Zn resulted mainly from the decrease of cytoplasmic acetyl-CoA level (Figs. 6B and 11). Inhibition of acetyl-CoA production by PDH and its transport
Fig. 8. Nonlinear correlation plots of dependence between intracellular Zn accumulation and nonviable cell fraction after 30 min exposition of harvested NC and DC to increasing Zn concentrations in the incubation medium. Data were calculated from Figs. 1A and 2.
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Fig. 11. Linear correlations between acetyl-CoA content in cytoplasmic compartment and acetylcholine level and release from DC exposed for 30 min to increasing concentrations of Zn. Data were calculated from Figs. 5A, B and 6B. Fig. 9. Linear correlations between intracellular Zn accumulation and Ca levels in mitochondrial and cytoplasmic compartments of Zn-challenged SN56 DC. Data were calculated from Figs. 2 and 3B.
from mitochondria, could be main causes of this deficit (Figs. 5, 6 and 7A), as the ChAT activity itself was not affected by these conditions (Fig. 4). Also, the decrease of mitochondrial Ca by Zn could inhibit permeability transition-dependent direct transport of acetyl-CoA from neuronal mitochondria (Figs. 4 and 7) (Bielarczyk et al., 1998; Szutowicz et al., 1998). In consequence, ACh pool in SN56 cells available for release has been reduced (Fig. 5A and B). This conclusion is supported by the existence of significant correlation between cytoplasmic acetyl-CoA content and ACh level/release in Zn-challenged DC (Fig. 11). It also explains why, the Zn-evoked increase of cytoplasmic Ca did not activate ACh release (Figs. 3 and 5) (Tucek, 1993). In addition, when such conditions were extended in time, adaptative alterations in the expression of cholinergic phenotype might appear (Ronowska et al., 2007). The acute inhibition of PDH activity by Zn occurred through its reversible interaction with lipoamide binding sites of lipoamide dehydrogenase subunit (Fig. 7A) (Gazaryan et al., 2002; Ronowska et al., 2007). This inhibition caused the decrease of acetyl-CoA synthesis from pyruvate, yielding reduction of its level in the
mitochondria (Figs. 2, 6A and 7A). The existence of significant correlation between PDH activity and mitochondrial level of this metabolite supports this thesis (Fig. 12B). Thus, the key mechanism of Zn-toxicity could be due to a shortage of acetylCoA both for energy production in TCA cycle as well as for transport through the mitochondrial membrane (Fig. 6A and B) (Bielarczyk et al., 2003a; Szutowicz, 2001). Significant, multiple correlations between Zn-evoked depletion of Ca from mitochondria, inhibition of PDH activity, shortage of mitochondrial acetylCoA and increase of nonviable SN56 cell fraction remain in accord with this hypothesis (Figs. 9, 10A, 12A and B). Therefore, we postulate that maintenance of mitochondrial acetyl-CoA availability plays a pivotal role in survival of cholinergic neurons in neurodegenerative conditions. Values of [Zn]-Ki for PDH appeared to be similar in DC and NC. It suggests that limitation of acetyl-CoA production in Zn-overloaded DC and NC was similar both at high and low rates of ACh metabolism, respectively (Figs. 2, 5 and 7A). Therefore, deeper shortage of mitochondrial acetyl-CoA in Zn-treated DC can be explained by its greater than in NC utilization for ACh synthesis (Figs. 5 and 6) (Szutowicz et al., 2006).
Fig. 10. (A) Inverse linear correlations between Ca accumulation in mitochondria; (B) Direct linear correlations between Ca accumulation in cytoplasm and nonviable SN56 NC and DC fractions after 30 min. incubation in the presence of increasing concentrations of extracellular Zn. Data were calculated from Figs. 1A, 3A and B.
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Fig. 12. (A) Direct linear correlation between PDH activity and acetyl-CoA content in DC mitochondria and inverse correlations between PDH activity and nonviable DC fraction after 30 min exposition to increasing concentrations of extracellular Zn. Data were calculated from Figs. 1A, 6A and 7A. (B) Inverse correlation between mitochondrial acetyl-CoA and nonviable DC fraction after 30 incubation with increasing concentrations of Zn. Data were calculated from Figs. 1A and 6A.
The [Zn]-Ki values for aconitase and ICDH-NADP were similar both in DC and NC homogenates (Fig. 7B and C). Therefore, these inhibitory influences themselves do not explain greater, short- and long-term, susceptibility of DC than NC to Zn and other neurotoxic factors (Fig. 1) (Jankowska et al., 2000; Szutowicz et al., 2006). One should stress that Zn inhibited these enzymes about 10 times stronger than PDH, as demonstrated by respective values of [Zn]-Ki (Fig. 7A–C). However, their inhibition by Zn presumably did not limit metabolic flow of pyruvate-derived acetyl-CoA throughout aconitase-ICDH-NADP steps, as their Vmax values were from 4 to 6 times higher than those of PDH (Fig. 7A–C). On the other hand, low Vmax for KDH and its high sensitivity to Zn, could make this step of TCA cycle the key target for this metal neurotoxicity (Fig. 7D) (Brown et al., 2000; Gibson et al., 2000). Moreover, we report here for the first time, that inhibition of KDH by Zn in DC was 8 times stronger than in NC (Fig. 7D). It could, at least in part, contribute to greater sensitivity of DC to acute excitotoxic insults, by blocking metabolic flow through the second part of TCA cycle (Figs. 1 and 7D). This differential inhibition of KDH by Zn is also compatible with earlier data demonstrating preferential sensitivity of DC to various AD neurotoxins (Fig. 7D) (Bielarczyk et al., 2003a; Jankowska et al., 2000; Szutowicz et al., 2005, 2006). However, neither aconitase nor KDH were likely to be involved in the backward regulation of acetyl-CoA level in cholinergic cell mitochondria. The activities of these enzymes were strongly inhibited at Zn concentrations that were one order of magnitude lower than those that significantly inhibited PDH, suppressed acetyl-CoA level and decreased cell viability (Figs. 1A, 6 and 7A–D). These observations rise the supposition that functional competence of second half of TCA cycle and KDH itself played less important role in supporting survival of clonal SN56 cells. These findings indicate that inhibition of PDH by Zn may be a main factor leading to functional and structural injury of cholinergic cells through the decrease of acetyl-CoA availability in their mitochondria (Figs. 1, 5–7, 10 and 12) (Ronowska et al., 2007). In addition, the pattern of Zn-evoked alterations of TCA enzymes in these septal cholinergic cells fits well to the profile of enzymatic changes seen in brains of AD patients (Bubber et al., 2005). Presented data suggest, that prolonged accumulation of Zn in glutamatergic synaptic clefts might play an important role in the excitotoxic, acetyl-CoA-mediated mechanisms of cholinergic deficits in AD (Hynd et al., 2004; Mocchegiani et al., 2005; Szutowicz et al., 2006). Taken together our present and past findings consolidate the hypothesis that differential short-term
susceptibility of brain cholinergic neurons correlates directly with expression of their cholinergic phenotype and increasing utilization of mitochondrial acetyl-CoA for ACh synthesis in cytoplasm (Bielarczyk et al., 2003a,b; Szutowicz, 2001; Szutowicz et al., 2006). Long-lasting hyper-zincaemia could yield suppressive-adaptative alterations in expression of the cholinergic phenotype in surviving cholinergic neurons. Thereby, the acute limitation of acetyl-CoA provision for ACh synthesis, could preserve greater fraction of this metabolite for energy production. Such mechanism could paradoxically increase chances of cholinergic neurons for longer survival in neurodegenerative conditions (Figs. 1 and 5) (Ronowska et al., 2007). Acknowledgements Work was supported by the Ministry of Research and Higher Education projects 2P05A 110 30 and NN401 2333 33 and Medical University of Gdan´sk projects St-57 and W-20. We are grateful to Dr J.K. Blusztajn (Boston, MA, USA) for SN56 cells. References Airado, C.C., Gomez, J.S., Recio, F., Balantas, E., Weruaga, Alfonso, J.R., 2008. Zincergic innervation from the anterior olfactory nucleus to olfactory bulb displays plastic responses after mitral cell loss. J. Chem. Neuroanat. 36, 197–208. Bertoni-Freddari, C., Mocchegiani, E., Malavolta, M., Casoli, T., Di Stefano, G., Fattoretti, P., 2006. Synaptic and mitochondrial physiopathologic changes in the aging nervous system and the role of zinc homeostasis. Mech. Aging Dev. 127, 590–596. Bielarczyk, H., Tomaszewicz, M., Szutowicz, A., 1998. Effect of aluminum on acetylCoA and acetylcholine metabolism in nerve terminals. J. Neurochem. 70, 1175– 1181. Bielarczyk, H., Jankowska, A., Madziar, B., Matecki, A., Michno, A., Szutowicz, A., 2003a. Differential toxicity of nitric oxide, aluminum, and amyloid b-peptide in SN56 cholinergic cells from mouse septum. Neurochem. Int. 42, 323–331. Bielarczyk, H., Tomaszewicz, M., Madziar, B., C´wikowska, J., Pawełczyk, T., Szutowicz, A., 2003b. Relationships between cholinergic phenotype and acetyl-CoA level in hybrid murine neuroblastoma cells of septal origin. J. Neurosci. Res. 73, 717–721. Bielarczyk, H., Jankowska-Kulawy, A., Gul, S., Pawełczyk, T., Szutowicz, A., 2005. Phenotype dependent differential effects of interleukin-1b and amyloid-b on viability and cholinergic phenotype of T17 neuroblastoma cells. Neurochem. Int. 47, 466–473. Bradford, M., 1976. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brown, A.M., Kristal, B.S., Effron, M.S., Shestopalov, A.I.M., Ulucci, P.A., Sheu, K.F.R., Blass, J.P., Cooper, A.J.L., 2000. Zn2+ inhibits a-ketoglutarate-stimulated mitochondrial respiration and the isolated a-ketoglutarate dehydrogenase complex. J. Biol. Chem. 275, 13441–13447.
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Short-term effects of zinc on acetylcholine metabolism and viability of SN56 cholinergic neuroblastoma cells Anna Ronowska a, Aleksandra Dys´ a, Agnieszka Jankowska-Kulawy a, Joanna Klimaszewska-Łata a, Hanna Bielarczyk a, Piotr Romianowski a, Tadeusz Pawełczyk b, Andrzej Szutowicz a,* a b
Department of Laboratory Medicine, Medical University of Gdan´sk, Ul. Debinki 7, 80-211 Gdansk, Poland Department of Molecular Medicine, Medical University of Gdan´sk, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 August 2009 Received in revised form 14 September 2009 Accepted 17 September 2009 Available online 23 September 2009
Excessive accumulation of zinc in the brain is one of putative factors involved in pathomechanism of cholinergic encephalopathies. The aim of this work was to investigate whether short-term increase of zinc concentration in the extracellular space may affect energy and acetylcholine metabolism in SN56 cholinergic cells of septal origin. Short 30 min exposition of SN56 cells to increasing zinc levels caused greater loss of viability of differentiated (DC, [EC0.4] 0.09 mM) than nondifferentiated cells (NC, [EC0.4] 0.14 mM). Concentration-dependent accumulation of zinc displayed exponential non-saturable kinetics. Zinc accumulation caused the decrease of calcium accumulation in mitochondria and its increase in cytoplasmic compartment of SN56 cells. Significant inverse and direct correlations were found between zinc accumulation and calcium levels in mitochondrial (r = 0.96, p = 0.028) and cytoplasmic (r = 0.97, p = 0.028) compartments of DC, respectively. Zinc exerted similar inhibition of pyruvate dehydrogenase, aconitase and isocitrate dehydrogenase both in NC and DC homogenates, at Ki values equal to about 0.07, 0.08 and 0.005 mM, respectively. On the other hand, ketoglutarate dehydrogenase activity in DC was inhibited by zinc (Ki 0.0005 mM) 8 times stronger that in NC (Ki 0.004 mM). Also zinc-evoked decreases in acetylcholine content and its release were significantly greater in DC than in NC. Same conditions caused suppression of cytoplasmic and mitochondrial content of acetyl-CoA, that positively correlated with inhibition of transmitter functions (r = 0.995, p = 005) and loss of cell viability (r = 0.990, p = 0.0006), respectively. Significant correlations were also found in zinc-challenged cells between pyruvate dehydrogenase activity and both mitochondrial acetyl-CoA content and cell viability. These data indicate that pyruvate dehydrogenase-dependent acetyl-CoA synthesis in neuronal mitochondria may be a primary target for short-term neurotoxic effects of zinc. In consequence, shortages of acetylCoA in the mitochondrial compartment would cause fast loss of functional and structural integrity of cholinergic neurons. ß 2009 Published by Elsevier Ltd.
Keywords: Acetyl-CoA Acetylcholine Cholinergic cells Pyruvate dehydrogenase Neurotoxicity Zinc
1. Introduction Zinc is an important trace element in living organisms being an essential component for over 300 enzymes, transporters, transcription factors, transmitters and hormones. The zinc concentration in the brain is the highest in comparison to other tissues. Its
Abbreviations: ACh, acetylcholine; AD, Alzheimer’s disease; AMPA, a-amino-3hydroxyl-5-methyl-4-isoxazole-propionate; ChAT, choline acetyltransferase; DC, differentiated SN56 cells; DMEM, Dulecco Modified Eagle’s Medium; FBS, fetal bovine serum; ICDH-NADP, NADP-dependent isocitrate dehydrogenase; NC, nondifferentiated SN56 cells; NMDA, N-methyl-D-aspartate; KDH, ketoglutarate dehydrogenase; PDH, pyruvate dehydrogenase; TCA, trichloroacetic acid; ZEN, zinc enriched neurons. * Corresponding author. Tel.: +48 58 349 2770; fax: +48 58 449 2784. E-mail address: [email protected] (A. Szutowicz). 0197-0186/$ – see front matter ß 2009 Published by Elsevier Ltd. doi:10.1016/j.neuint.2009.09.012
average level in whole brain was estimated to be about 0.15 mM (Zatta et al., 2003). However, in particular compartments the level of free Zn2+ is estimated to be of sub-nanomolar and in brain extracellular fluid of submicromolar range (Bertoni-Freddari et al., 2006; Frederickson et al., 2006). On the other hand, in the vesicles of presynaptic terminals of excitatory glutamatergic neurons, Zn concentration was found to be above 1 mM (Mocchegiani et al., 2005). The latter ones belong to the subgroup of zinc enriched neurons (ZEN) that are particularly abundant in olfactory nucleus, hippocampus and other forebrain regions (Airado et al., 2008). Excessive activation of ZEN neurons in course of several brain pathologies like hypoxia, hypoglycaemia, amyloid-beta overload leads to excessive release of glutamate. Prolonged activation of N-methyl-D-aspartate (NMDA) receptors by a glutamate yields over-production of NO and free radicals in receptive cells. Simultaneously massive amounts of Zn2+ are released from
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glutamatergic vesicles (Frederickson et al., 2006; Hynd et al., 2004). It may cause a build up Zn2+ concentration in the synaptic cleft to levels as high as 0.3 mM (Zatta et al., 2003). Subsequent uptake of Zn from extracellular space to postsynaptic cells, through voltage-gated Ca or NMDA channels might cause their overload with this metal (Mocchegiani et al., 2005). In cholinergic human SK-SH-SY5Y cells activation of M1 muscarinic cholinergic autoreceptors increased Zn accumulation (Zuchner et al., 2006). In turn, the Zn-overload would activate ZnT-1 and other Zn-transporting systems to excrete Zn back to extracellular space or sequestrate it in intracellular storage compartments, respectively (Lovell et al., 2005; Quin et al., 2008). In addition, Zn was reported to compete with Ca transport through voltage-gated Ca-channels (Sun et al., 2007). Through such mechanisms, excess of extracellular Zn in the brain might directly induce neurodegeneration in course of different encephalopathies (Religa et al., 2006). For instance, excess of extracellular Zn was found to facilitate formation of cytotoxic amyloid-b aggregates (Khan et al., 2005). On the other hand, Zn was reported to protect NT2 human cells against amyloidbeta toxicity (Cardoso et al., 2005). Zn also inhibited transmitterstimulated capacitative entry of extracellular Ca into the nonglutamatergic neurons and nerve terminals. Thereby, it suppressed their transmitter functions (Kresse et al., 2005). Besides chronic accumulation of Zn in neurons yielded chronic inhibition of pyruvate (pyruvate:lipoate oxidoreductase acceptor acetylating, PDH, EC 1.2.4.1) and ketoglutarate (2-oxoglutarate:lipoate oxidoreductase acceptor acylating, KDH, EC 1.2.4.2) dehydrogenases as well as aconitase (citrate(isocitrate)hydrolyase, EC 4.2.1.3), resulting in the depletion of acetyl-CoA in mitochondria. It led to extended energy deficits and cell death (Ronowska et al., 2007). By such mechanism(s) Zn could also aggravate neurotoxic actions of other compounds and interfere with various mitochondrial pathways known to be involved in the mechanisms of neurodegeneration (Castaldo et al., 2009; Gibson et al., 2000; Ronowska et al., 2007; Szutowicz et al., 2006). In addition, long-term exposure of differentiated cholinergic SN56 neuroblastoma cells to Zn caused adaptative suppression of choline acetyltransferase (acetyl-CoA:choline-O-acetyltransferase, ChAT, EC 2.3.1.6) activity, presumably through the long-lasting decrease of acetyl-CoA level in their cytoplasmic compartment (Ronowska et al., 2007). Also, sensitivity of SN56 cells to long-term NO, Al, interleukin 1b or amyloid-b burden was reported to correlate positively with their phenotypic differentiation (Bielarczyk et al., 2003a, 2005; Szutowicz et al., 2006). It is however not known whether some alterations in cell viability and ACh metabolism might occur in acute Zn-toxicity experimental paradigms. There is also no information how short-term effects of Zn on cholinergic neurons are transferred into chronic alterations in their acetyl-CoA/ACh metabolism and viability (Ronowska et al., 2007). Therefore, the aim of this study was to investigate whether short-term increase of [Zn] in extracellular space could induce instant alterations in cholinergic neurons through the interaction with pre- and postsynthetic steps of their ACh metabolism. Data presented here indicate that Zn-evoked short-term alterations in cholinergic neurons metabolism could establish the base for their modifications during their prolonged exposure to this metal (Ronowska et al., 2007). 2. Materials and methods 2.1. Materials Unless otherwise specified biochemicals were obtained from Sigma–Aldrich (Poznan´, Poland), [1-14C-acetyl]-CoA 4 mCi/mmol was from Perkin–Elmer (Boston, MA, USA), cell cultures growth media and components were provided by Gibco Life Technologies (Warsaw, Poland), cell culture disposables derived from Sarstedt (Stare Babice, Poland).
2.2. Cell cultures SN56.B5.G4 cholinergic murine neuroblastoma cells, with stable expression of PDH and ChAT between 22nd and 40th passage, were used for experiments (Hammond et al., 1990). Cells were seeded at density of 40,000 cells/cm2 on 75 cm2 Falcon vessels in Dulbecco’s modified Eagle medium containing 1 mM L-glutamine, 0.05 mg of streptomycin and 50 U of penicillin per 1 mL and 10% fetal bovine serum (DMEM-FBS) and grown at 37 8C in atmosphere 5% CO2, 95% air. Such medium was reported to contain 0.005 mM concentration of endogenous Zn (MacDonald et al., 1998). Cholinergic differentiation was obtained by combined addition of 1 mmol/L dibutyryl cAMP (cAMP) and 0.001 mmol/L all-trans-retinoic acid (RA) for 48 h. At this time media were discarded. Zn and other agents were added as indicated in DMEM-FBS, or N2 supplemented DMEM containing no differentiating agents and culture continued for 24 h. Cells that remained attached to the plate were harvested into 10 mL of ice cold 140 mmol/L NaCl containing 5 mmol/L KCl, 1.7 mmol/L NaKphosphate buffer (pH 7.4) and 5 mM glucose and collected by centrifugation at 200 g for 7 min. Supernatant was removed and cells were suspended in 320 mmol/L sucrose containing 10 mmol/L HEPES buffer (pH 7.4) and to obtain protein concentration 10.0 mg/mL. Immediately after collection the cells were used for Trypan blue exclusion assay and for metabolic studies. For ChAT and PDH assays samples were kept frozen at 20 8C for 2–7 days. Remaining enzyme activities were assessed in unfrozen samples within 24 h after harvesting cells. 2.3. Trypan blue exclusion assay Cell suspension was mixed with equal volume of 0.4% isotonic trypan blue solution. Total cell number and fraction of nonviable, dye accumulating cells were counted after 2 min in Fuchs-Rosenthal haemocytometer under light microscope (Wang et al., 2001). 2.4. Metabolic studies Depolarizing incubation medium contained in a final volume of 1.0 mL 2.5 mmol/L pyruvate, 2.5 mmol/L L-malate, 90 mmol/L NaCl, 30 mmol/L KCl, 20 mmol/L NaHEPES (pH 7.4), 1.5 mmol/L Na-phosphate, 0.01 mmol/L choline chloride, 0.015 mmol/L eserine sulfate, 32 mmol/L sucrose and 0.7–1.0 mg of cell protein. These conditions were employed to assure maximal acetylcholine release and resynthesis (Bielarczyk et al., 1998). Incubation was started by the addition of cell suspension and continued for 30 min at 37 8C with shaking at 100 cycles per min. For the determination of total acetyl-CoA content, 0.3 mL of incubation medium was centrifuged at 5000 g for 2 min. Supernatant was removed and cell pellet was deproteinized as indicated below. 2.5. Acetyl-CoA assay For assaying of whole cell acetyl-CoA content, the cell pellet was deproteinized by suspension in a small volume of 5 mmol/L HCl and incubation in a boiling bath for 1 min. To assess acetyl-CoA content in the cell mitochondria 0.5 mL of incubation medium was mixed with equal volume of lysing solution containing 1.4 mg digitonin/mL in 125 mmol/L KCl with 20 mmol/L NaHEPES buffer (pH 7.4) and 3 mmol/L EDTA. Lysate was transferred on 0.5 mL of silicone oil mixture (AR20 and AR200, 1:2) layered over 0.1 mL of buffered 320 mM sucrose. After 30 s the mitochondrial fraction was separated from the soluble one by centrifugation for 40 s at 12,000 g. After the removal of soluble fraction, silicon oils and sucrose, mitochondrial pellet was deproteinized as described above. Deproteinized extracts of whole cells and mitochondria were treated with maleic anhydride solution in ethyl ether for 2 h to remove CoA-SH. Cycling reaction was carried for 60 min in 0.1 mL of medium containing 1.9 mmol/L acetyl phosphate, 1.2 mmol/L oxaloacetate, 1.0 IU phosphotransacetylase and 0.12 IU citrate synthase. Cycling reaction was stopped by heating samples at 100 8C for 10 min and citrate formed was determined (Szutowicz and Bielarczyk, 1987). Cytoplasmic acetyl-CoA level was calculated by subtraction of mitochondrial acetyl-CoA from total acetyl-CoA content (Szutowicz and Bielarczyk, 1987). 2.6. Acetylcholine assay Samples (0.2 mL) of cells after 30 min incubation in depolarizing medium were centrifuged 3 min at 10,000 g. Supernatants were collected and used for the determination of released ACh. To calculate rates of ACh release/synthesis values obtained after 30 min incubation were subtracted from those at time zero and divided by the incubation time (Bielarczyk et al., 1998; Tucek, 1993). Cell pellets were homogenized in 0.4 mL of methanol and centrifuged. Obtained supernatants were mixed with 0.05 mL of 1.0 mol/L formic acid and centrifuged. Chloroform (0.9 mL) and water (0.4 mL) were sequentially added with ‘‘Vortex’’ mixing. After centrifugation upper methanol–water layers were collected and evaporated of Speed-Vac SC110 dessicator (Savant, Paris, France) and sediments after reconstitution with water were used for the determination of intracellular ACh content. Extra- and intracellular ACh was determined with acetylcholinesterase–choline oxidase method coupled with peroxidase–luminol detection system (Israel and Lesbats, 1982) using Junior luminometer (Berthold Technology, Bad Wild-Bad, Germany).
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Fig. 1. Effect of 30 min exposition of harvested SN56 NC and DC to increasing Zn concentrations on: (A) cell viability; (B) cell number. Data are means SEM from 3 duplicate experiments. Significantly different from respective no Zn control, *p < 0.005, from corresponding NC, +p < 0.01.
2.7. Calcium and zinc assays After separation from the incubation medium cell pellets were washed once with Ca-free incubation medium containing 1 mmol/L EDTA to remove surface-bound metals. Centrifuged and washed again with EDTA-free medium. For Ca determination, obtained pellet was deproteinized with 5% TCA and supernatants were assayed by arsenazo III spectrophotometric method (Scarpa, 1979). For Zn determinations cell pellet was deproteinized with 4% HClO4 and cation level was determined by fluorimetric method with N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) as described by Chen and Liao (2003) with emission and excitation wavelengths equal to 335 and 495 nm, respectively. To assess mitochondrial Ca, cells were lysed with digitonin followed by centrifugation through silicon oil mixture as described above (Section 2.5). 2.8. Enzyme assays Immediately before the assay samples were diluted to desired protein concentration in 0.2% (v/v) Triton X-100. ChAT activity was assessed by the radiometric method using [1-14C]acetyl-CoA as a substrate (Fonnum, 1975). PDH was assayed by the citrate synthase coupled method (Szutowicz et al., 1981). KDH activity was determined by a direct measurement of NAD reduction (Pawełczyk and Angielski, 1984). Activities of aconitase, succinate dehydrogenase (succinate:acceptor oxidoreductase, 1.3.99.1), NADP-isocitrate dehydrogenase (threo-D-isocitrate:NADP oxidoreductase (decarboxylating), ICDH-NADP, EC 1.1.1.42) and NAD Lmalate dehydrogenase (EC 1.1.1.41) were assayed by methods described elsewhere (Ochoa, 1955; Plaut, 1962; Veeger et al., 1969; Villafranca, 1974). For studies of acute Zn effects on enzyme activities the cation and/or other compounds were added directly to the assay media.
concentration (Fig. 1A). DC appeared to be more sensitive than NC to lower Zn concentrations as evidenced by [EC0.4] values equal to 0.085 and 0.14 mmol/L, respectively (Fig. 1A). No change in NC and DC number took place in these conditions (Fig. 1B). 3.2. Short-term zinc accumulation in SN56 cells The exposition of harvested DC and NC to Zn for 30 min in postculture medium (see Section 2) caused concentration-dependent metal accumulation that displayed non-saturable exponential kinetics (Fig. 2). At 0.25 mmol/L extracellular level of Zn, its intracellular accumulation reached values above 150 nmol/mg of protein level in both NC and DC (Fig. 2). 3.3. Short-term effect of Zn on Ca distribution of SN56 cells In depolarizing medium, the rise of Zn to 0.10 mmol/L caused significant alterations neither in whole SN56 cell Ca content nor in its distribution between mitochondrial and cytoplasmic compartment (Fig. 3A and B). Further increase of extracellular Zn up to
2.9. Protein assay Protein was assayed by the method of Bradford (1976) with human immunoglobulin as a standard. 2.10. Statistics Statistical analyses were carried out by one way ANOVA with Bonferroni multiple comparison test or by non-paired Student’s t-test, at p < 0.05 being considered to be statistically significant.
3. Results 3.1. Short-term cell viability Our past studies of chronic Zn-cytotoxicity in DMEM-FBS cultured SN56 cells revealed about 40% increase of nonviable cell fraction after 24 h exposition to this metal (Ronowska et al., 2007). Incubation of SN56 cells in the control depolarizing medium for 30 min caused no change in trypan blue positive fraction of NC and DC (not shown). On the other hand, Zn caused fast concentrationdependent, increase of trypan blue-retaining cell fraction that after 30 min incubation reached about 70% at 0.25 mmol/L metal
Fig. 2. Effect 30 min exposition of harvested SN56 cells to increasing extracellular Zn concentrations on its intracellular accumulation in NC and DC. Data are means SEM from 5 to 7 duplicate experiments. Significantly different from respective no Zn control, *p < 0.01.
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Fig. 3. Effect of 30 min exposition of harvested SN56 cells to increasing Zn concentrations on Ca content in their mitochondrial and cytoplasmic compartments: (A) nondifferentiated cells; (B) differentiated cells. Data are means SEM from 4 duplicate experiments. Significantly different from respective no Zn control, *p < 0.01.
0.25 mmol/L brought about concentration-dependent increases of whole cell (40–80%) and cytoplasmic Ca (128–147%), and its decrease in mitochondria (40–50%), both in NC and DC (Fig. 3A and B).
value of Zn-evoked ACh depletion in DC appeared to be 190% higher than in NC (Fig. 5A). Also, absolute value of maximal Zn-evoked inhibition of ACh release in DC was over 3 times greater than in NC (Fig. 5B).
3.4. Short-term effects of zinc on cholinergic parameters SN56 cells
3.5. Short-term effect of zinc on acetyl-CoA metabolism
Activity of ChAT in DC homogenates (0.44 0.04 nmol/min/ mg protein) was 2.5 times higher than in NC ones (Fig. 4). In DC, ChAT activity was not significantly inhibited by short-term exposure to Zn up to 0.3 mM concentration (Fig. 4). On the other hand, in NC it was inhibited at 0.3 mM Zn by about 34% (Fig. 4). ACh levels and its release from DC were 85% higher than in NC (Fig. 5A and B). The stable, steady-state level of ACh was maintained in NC and DC during entire 30 min incubation period (not shown). In such conditions amount of ACh released to the medium corresponded to its synthesis (Bielarczyk et al., 1998; Tucek, 1993). ACh release from DC was 3 times higher than in NC (Fig. 5B). Zn caused concentration-dependent suppression of ACh content both in NC and DC. Namely, at 0.15 mmol/L Zn level the relative suppression of ACh content in NC and DC was equal to 65 and 46%, respectively. ACh release from NC was almost completely inhibited by 0.05 mmol/L Zn, while in DC only 30% suppression of the transmitter release, took place (Fig. 5B). Thus, apparent [IC]50% values for Zn-evoked suppressions of ACh content and release in DC appeared to be equal to 0.15 and 0.10 mmol/L, whereas in NC to 0.10 and 0.01 mmol/L, respectively (Fig. 5A and B). However, absolute
The increase of extracellular [Zn] from 0.01 to 0.25 mmol/L caused concentration-dependent reduction of acetyl-CoA level in NC mitochondria. It fell to 50% of control values at 0.10 mmol/L [Zn] (Fig. 6A). Further increase of [Zn] resulted in slight attenuation of acetyl-CoA suppression in NC mitochondria. Acetyl-CoA level in DC mitochondria was about 30% lower than in NC (Fig. 6A). In this cell phenotype, elevation of [Zn] to 0.25 mmol/L caused a gradual depletion of mitochondrial acetyl-CoA down to 30% of control values with apparent [IC0.5] equal to 0.10 mmol/L (Fig. 6A). In NC, 0.05 mmol/L [Zn] caused twofold increase of cytoplasmic [acetylCoA]. Higher [Zn] caused a total depletion of acetyl-CoA in this compartment (Fig. 6A). On the other hand, in DC 0.1 mmol/L [Zn] brought about 80% suppression of cytoplasmic acetyl-CoA (Fig. 6B). Further increase of Zn up to 0.25 mmol/L resulted in no alteration in the level of this metabolite (Fig. 6B). 3.6. Short-term effects of zinc on selected enzyme activities The past studies revealed that diverse chronic neurotoxic conditions, cause suppression of acetyl-CoA in mitochondrial and cytoplasmic compartments of neuronal cells (Szutowicz, 2001; Szutowicz et al., 2006). These alterations could result from inhibition of brain PDH activity, acetyl-CoA utilization in TCA cycle, and its transport to cytoplasm through the mitochondrial membrane (Bubber et al., 2005; Szutowicz et al., 2006). The Zn-Ki values for PDH, aconitase and ICDH-NADP appeared to be similar for both cell phenotypes being equal to about 0.07–0.09, 0.008– 0.009 and 0.004–0.006 mmol/L, respectively (Fig. 7A–C). On the other hand, Ki for Zn-evoked inhibition of DC-KDH (0.0005 mmol/ L) appeared to be 8 times lower than for NC-KDH (0.004 mmol/L), respectively (Fig. 7D). Activities of succinate dehydrogenase, malate dehydrogenase and citrate synthase were not altered by Zn up to 0.3 mmol/L concentration (not shown). 4. Discussion
Fig. 4. Acute effect of Zn on choline acetyltransferase activity in NC and DC homogenates. Data are means SEM from 6 duplicate experiments. Significantly different from respective no Zn control, *p < 0.05; nondifferentiated cells, +p < 0.0001.
In the present work, cytotoxic effects of Zn were observed at its 0.05–0.25 mmol/L levels in extracellular space (Fig. 1B). Such
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Fig. 5. Effect of 30 min exposition of harvested SN56 NC and DC to increasing Zn concentrations on : (A) acetylcholine content; (B) depolarization-evoked Ca-dependent acetylcholine release. Data are means SEM from 3 duplicate experiments. Significantly different from respective no Zn control, *p < 0.01; corresponding NC values, +p < 0.01.
Fig. 6. Effect of 30 min exposition of harvested SN56 cells to increasing Zn concentrations on their acetyl-CoA content in: (A) mitochondrial; (B) cytoplasmic compartments. Data are means SEM from 4 to 10 duplicate experiments. Significantly different from respective no Zn control, *p < 0.01; corresponding NC values, +p < 0.01.
concentrations of Zn are compatible with those reported for the whole brain and the synaptic cleft of glutamatergic terminals in vivo (Zatta et al., 2003). Therefore, the Zn effects, reported here, are likely to be of pathophysiological significance. The hyperbolar shape of the correlation plots between load of cells with Zn and their mortality, indicates the existence of Zn-multiple target interactions leading to the cholinergic cell death (Fig. 8). It is known that several Ca-permeable channels, including aamino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), kainate, NMDA and GABA receptors participate in the Zn entry into the neuronal cells (Marin et al., 2000; Mocchegiani et al., 2005; Sun et al., 2007). Therefore, a concave up shape of concentration-dependent plots of Zn accumulation might result from consecutive activation of the multiple cation-permeable channels of decreasing affinities and increasing transport capacities to Zn (Fig. 2) (Sun et al., 2007). However, stimulation of M1 autoreceptors (Zuchner et al., 2006) did not contribute to the Zn-overload in SN56 cells, as ACh release markedly decreased with the increase of extracellular [Zn] (Fig. 5). Similar levels of Zn accumulation in NC and DC indicate that increased expression of cholinergic phenotype did not modify the overall Zn-transporting capacity in the SN56 cells (Fig. 2). Therefore, the Zn accumulation itself could not be a cause of differential susceptibility of NC and DC to this metal (Figs. 1 and 2).
The decrease of mitochondrial Ca in Zn-treated cells could result from the inhibition of Ca uniporter in their inner membrane (Fig. 3) (Malaiyandi et al., 2005; Sensi et al., 2002). The impairment of intramitochondrial Ca sequestration might contribute, at least in part, to its increased accumulation in the cytoplasmic compartment (Fig. 3). Such conclusion is justified by the existence of direct and inverse correlations between level of intracellular Zn and Ca content in cytoplasmic and mitochondrial compartments, respectively (Fig. 9). However, presented findings remain in conflict with data demonstrating that Zn inhibited NMDA and AMPA channels (Mocchegiani et al., 2005; Sun et al., 2007). Hence, Zn should yield rather decrease not the increase of whole cell and cytoplasmic Ca (Fig. 3). Such phenomenon was observed in SN56 cells treated with aluminum, that inhibited verapamil-sensitive Ca-channels in plasma membranes (Jankowska et al., 2000). However, in present experiments Zn caused rapid impairment of membrane integrity as evidenced by a concentration-dependent, short-term increase of trypan blue positive cells fraction (Figs. 1–3) (Marin et al., 2000; Sun et al., 2007). Such conclusion is also supported by the existence of significant direct correlation between acute Zn-induced increases of Ca accumulation in cytoplasmic compartment and rise of trypan blue positive fraction of SN56 cells (Figs. 1, 3 and 10B). Therefore, prolonged hiper-zincaemia could be responsible for chronic detrimental effects of this metal on cholinergic cells
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Fig. 7. Dixon plots of acute concentration-dependent effects of Zn on pyruvate dehydrogenase, aconitase and NADP-isocitrate dehydrogenase activities in NC and DC homogenates. Data are means from 4 to 6 duplicate experiments. (A) pyruvate dehydrogenase; (B) aconitase; (C) isocitrate-NADPdehydrogenase; (D)ketoglutaratedehydrogenase.
(Ronowska et al., 2007). Similar patterns of Zn-evoked alterations in subcellular distributions of Ca in NC and DC indicate that they may not directly contribute to differential, phenotype-dependent sensitivity of these cells to Zn (Figs. 1B, 2 and 3). It is known, that the size of intracellular ACh pool is resultant of ChAT activity and concentrations of transmitter’s precursors in cytoplasmic compartment (Szutowicz et al., 1996, 1998; Tucek, 1993). In this respect, proportionality between ChAT activities and ACh contents or releases in DC and NC is in accord with past reports (Figs. 4–6) (Bielarczyk et al., 2003b; Szutowicz et al., 1998; Tucek, 1993). However, the mechanism of tight correlations between ChAT activity and size of ACh pool, still remains unsolved. Therefore, fast reduction of intracellular ACh pool by Zn, at the absence of significant alterations of ChAT activity, indicates that the former could be due to reduction in cytoplasmic levels of acetyl-CoA (Figs. 4–6 and 11). Significant correlations between cytoplasmic acetyl-CoA and ACh content/release from Zn-challenged DC may be explained on the basis of mass action law mechanism at equilibrium constant for ChAT reaction equal to 11.4 (Fig. 11) (Tucek, 1993). Similar dependences were also observed between level of choline and ACh release in brain cortex (Napoli et al., 2008). Thus, the inhibition of transmitter functions in cholinergic cells acutely exposed to Zn resulted mainly from the decrease of cytoplasmic acetyl-CoA level (Figs. 6B and 11). Inhibition of acetyl-CoA production by PDH and its transport
Fig. 8. Nonlinear correlation plots of dependence between intracellular Zn accumulation and nonviable cell fraction after 30 min exposition of harvested NC and DC to increasing Zn concentrations in the incubation medium. Data were calculated from Figs. 1A and 2.
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Fig. 11. Linear correlations between acetyl-CoA content in cytoplasmic compartment and acetylcholine level and release from DC exposed for 30 min to increasing concentrations of Zn. Data were calculated from Figs. 5A, B and 6B. Fig. 9. Linear correlations between intracellular Zn accumulation and Ca levels in mitochondrial and cytoplasmic compartments of Zn-challenged SN56 DC. Data were calculated from Figs. 2 and 3B.
from mitochondria, could be main causes of this deficit (Figs. 5, 6 and 7A), as the ChAT activity itself was not affected by these conditions (Fig. 4). Also, the decrease of mitochondrial Ca by Zn could inhibit permeability transition-dependent direct transport of acetyl-CoA from neuronal mitochondria (Figs. 4 and 7) (Bielarczyk et al., 1998; Szutowicz et al., 1998). In consequence, ACh pool in SN56 cells available for release has been reduced (Fig. 5A and B). This conclusion is supported by the existence of significant correlation between cytoplasmic acetyl-CoA content and ACh level/release in Zn-challenged DC (Fig. 11). It also explains why, the Zn-evoked increase of cytoplasmic Ca did not activate ACh release (Figs. 3 and 5) (Tucek, 1993). In addition, when such conditions were extended in time, adaptative alterations in the expression of cholinergic phenotype might appear (Ronowska et al., 2007). The acute inhibition of PDH activity by Zn occurred through its reversible interaction with lipoamide binding sites of lipoamide dehydrogenase subunit (Fig. 7A) (Gazaryan et al., 2002; Ronowska et al., 2007). This inhibition caused the decrease of acetyl-CoA synthesis from pyruvate, yielding reduction of its level in the
mitochondria (Figs. 2, 6A and 7A). The existence of significant correlation between PDH activity and mitochondrial level of this metabolite supports this thesis (Fig. 12B). Thus, the key mechanism of Zn-toxicity could be due to a shortage of acetylCoA both for energy production in TCA cycle as well as for transport through the mitochondrial membrane (Fig. 6A and B) (Bielarczyk et al., 2003a; Szutowicz, 2001). Significant, multiple correlations between Zn-evoked depletion of Ca from mitochondria, inhibition of PDH activity, shortage of mitochondrial acetylCoA and increase of nonviable SN56 cell fraction remain in accord with this hypothesis (Figs. 9, 10A, 12A and B). Therefore, we postulate that maintenance of mitochondrial acetyl-CoA availability plays a pivotal role in survival of cholinergic neurons in neurodegenerative conditions. Values of [Zn]-Ki for PDH appeared to be similar in DC and NC. It suggests that limitation of acetyl-CoA production in Zn-overloaded DC and NC was similar both at high and low rates of ACh metabolism, respectively (Figs. 2, 5 and 7A). Therefore, deeper shortage of mitochondrial acetyl-CoA in Zn-treated DC can be explained by its greater than in NC utilization for ACh synthesis (Figs. 5 and 6) (Szutowicz et al., 2006).
Fig. 10. (A) Inverse linear correlations between Ca accumulation in mitochondria; (B) Direct linear correlations between Ca accumulation in cytoplasm and nonviable SN56 NC and DC fractions after 30 min. incubation in the presence of increasing concentrations of extracellular Zn. Data were calculated from Figs. 1A, 3A and B.
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Fig. 12. (A) Direct linear correlation between PDH activity and acetyl-CoA content in DC mitochondria and inverse correlations between PDH activity and nonviable DC fraction after 30 min exposition to increasing concentrations of extracellular Zn. Data were calculated from Figs. 1A, 6A and 7A. (B) Inverse correlation between mitochondrial acetyl-CoA and nonviable DC fraction after 30 incubation with increasing concentrations of Zn. Data were calculated from Figs. 1A and 6A.
The [Zn]-Ki values for aconitase and ICDH-NADP were similar both in DC and NC homogenates (Fig. 7B and C). Therefore, these inhibitory influences themselves do not explain greater, short- and long-term, susceptibility of DC than NC to Zn and other neurotoxic factors (Fig. 1) (Jankowska et al., 2000; Szutowicz et al., 2006). One should stress that Zn inhibited these enzymes about 10 times stronger than PDH, as demonstrated by respective values of [Zn]-Ki (Fig. 7A–C). However, their inhibition by Zn presumably did not limit metabolic flow of pyruvate-derived acetyl-CoA throughout aconitase-ICDH-NADP steps, as their Vmax values were from 4 to 6 times higher than those of PDH (Fig. 7A–C). On the other hand, low Vmax for KDH and its high sensitivity to Zn, could make this step of TCA cycle the key target for this metal neurotoxicity (Fig. 7D) (Brown et al., 2000; Gibson et al., 2000). Moreover, we report here for the first time, that inhibition of KDH by Zn in DC was 8 times stronger than in NC (Fig. 7D). It could, at least in part, contribute to greater sensitivity of DC to acute excitotoxic insults, by blocking metabolic flow through the second part of TCA cycle (Figs. 1 and 7D). This differential inhibition of KDH by Zn is also compatible with earlier data demonstrating preferential sensitivity of DC to various AD neurotoxins (Fig. 7D) (Bielarczyk et al., 2003a; Jankowska et al., 2000; Szutowicz et al., 2005, 2006). However, neither aconitase nor KDH were likely to be involved in the backward regulation of acetyl-CoA level in cholinergic cell mitochondria. The activities of these enzymes were strongly inhibited at Zn concentrations that were one order of magnitude lower than those that significantly inhibited PDH, suppressed acetyl-CoA level and decreased cell viability (Figs. 1A, 6 and 7A–D). These observations rise the supposition that functional competence of second half of TCA cycle and KDH itself played less important role in supporting survival of clonal SN56 cells. These findings indicate that inhibition of PDH by Zn may be a main factor leading to functional and structural injury of cholinergic cells through the decrease of acetyl-CoA availability in their mitochondria (Figs. 1, 5–7, 10 and 12) (Ronowska et al., 2007). In addition, the pattern of Zn-evoked alterations of TCA enzymes in these septal cholinergic cells fits well to the profile of enzymatic changes seen in brains of AD patients (Bubber et al., 2005). Presented data suggest, that prolonged accumulation of Zn in glutamatergic synaptic clefts might play an important role in the excitotoxic, acetyl-CoA-mediated mechanisms of cholinergic deficits in AD (Hynd et al., 2004; Mocchegiani et al., 2005; Szutowicz et al., 2006). Taken together our present and past findings consolidate the hypothesis that differential short-term
susceptibility of brain cholinergic neurons correlates directly with expression of their cholinergic phenotype and increasing utilization of mitochondrial acetyl-CoA for ACh synthesis in cytoplasm (Bielarczyk et al., 2003a,b; Szutowicz, 2001; Szutowicz et al., 2006). Long-lasting hyper-zincaemia could yield suppressive-adaptative alterations in expression of the cholinergic phenotype in surviving cholinergic neurons. Thereby, the acute limitation of acetyl-CoA provision for ACh synthesis, could preserve greater fraction of this metabolite for energy production. Such mechanism could paradoxically increase chances of cholinergic neurons for longer survival in neurodegenerative conditions (Figs. 1 and 5) (Ronowska et al., 2007). Acknowledgements Work was supported by the Ministry of Research and Higher Education projects 2P05A 110 30 and NN401 2333 33 and Medical University of Gdan´sk projects St-57 and W-20. We are grateful to Dr J.K. Blusztajn (Boston, MA, USA) for SN56 cells. References Airado, C.C., Gomez, J.S., Recio, F., Balantas, E., Weruaga, Alfonso, J.R., 2008. Zincergic innervation from the anterior olfactory nucleus to olfactory bulb displays plastic responses after mitral cell loss. J. Chem. Neuroanat. 36, 197–208. Bertoni-Freddari, C., Mocchegiani, E., Malavolta, M., Casoli, T., Di Stefano, G., Fattoretti, P., 2006. Synaptic and mitochondrial physiopathologic changes in the aging nervous system and the role of zinc homeostasis. Mech. Aging Dev. 127, 590–596. Bielarczyk, H., Tomaszewicz, M., Szutowicz, A., 1998. Effect of aluminum on acetylCoA and acetylcholine metabolism in nerve terminals. J. Neurochem. 70, 1175– 1181. Bielarczyk, H., Jankowska, A., Madziar, B., Matecki, A., Michno, A., Szutowicz, A., 2003a. Differential toxicity of nitric oxide, aluminum, and amyloid b-peptide in SN56 cholinergic cells from mouse septum. Neurochem. Int. 42, 323–331. Bielarczyk, H., Tomaszewicz, M., Madziar, B., C´wikowska, J., Pawełczyk, T., Szutowicz, A., 2003b. Relationships between cholinergic phenotype and acetyl-CoA level in hybrid murine neuroblastoma cells of septal origin. J. Neurosci. Res. 73, 717–721. Bielarczyk, H., Jankowska-Kulawy, A., Gul, S., Pawełczyk, T., Szutowicz, A., 2005. Phenotype dependent differential effects of interleukin-1b and amyloid-b on viability and cholinergic phenotype of T17 neuroblastoma cells. Neurochem. Int. 47, 466–473. Bradford, M., 1976. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brown, A.M., Kristal, B.S., Effron, M.S., Shestopalov, A.I.M., Ulucci, P.A., Sheu, K.F.R., Blass, J.P., Cooper, A.J.L., 2000. Zn2+ inhibits a-ketoglutarate-stimulated mitochondrial respiration and the isolated a-ketoglutarate dehydrogenase complex. J. Biol. Chem. 275, 13441–13447.
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