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Xenon prevents cellular damage in differentiated PC-12 cells exposed to hypoxia

Xenon prevents cellular damage in differentiated PC-12 cells exposed to hypoxia Background: The neuroprotective effect of xenon has been demonstrated for glutamatergic neurons. In the present study it is investigated if dopaminergic neurons, i.e. nerve-growth-factor differentiated PC-12 cells, are protected as well against hypoxia-induced cell damage in the presence of xenon. Results: Pheochromocytoma cells differentiated by addition of nerve growth factor were placed in a N -saturated atmosphere, a treatment that induced release of dopamine, reaching a maximum after 30 min. By determining extracellular lactate dehydrogenase concentration as marker for concomitant cellular damage, a substantial increase of enzymatic activity was found for N -treated cells. Replacement of N by xenon in such a hypoxic atmosphere resulted in complete protection against cellular damage and prevention of hypoxia-induced dopamine release. Intracellular buffering 2+ of Ca using the Ca-chelator 1, 2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA) reduced the neuroprotective effect of xenon indicating the 2+ essential participation of intracellular Ca -ions in the process of xenon-induced neuroprotection. Conclusions: The results presented demonstrate the outstanding property of xenon to protect neuron-like cells in a hypoxic situation. Background toxic mechanisms may also participate in hypoxia- Originally, hypoxia/ischemia-induced alterations in neu- induced cell damage such as cortical spreading depression ronal function have been attributed to be an over-release [7,8]. Rat pheochromocytoma (PC-12) cells are catecho- of neurotransmitters, including dopamine and glutamate. laminergic, excitable cells that have been widely used as Many studies have been performed on the mechanisms of an in vitro model for neuronal cells [9] possessing both glutamate-induced neuronal damage [1,2] but relatively D1- and D2-dopamine receptors [10]. In these cells few have investigated the hypoxia-induced damage in hypoxia causes a transient release of dopamine resulting dopaminergic neurons [3-6]. In recent years several lines from a complex cellular response consisting of increased of evidence have suggested that effects other than excito- dopamine release and reduced uptake rate. Such increased Page 1 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 dopamine concentration has been shown to be associated other hand, if the release was constant but the re-uptake with cellular damage indicated by an elevated release of inhibited by hypoxia, additional inhibition of uptake by lactate dehydrogenase (LDH) from the cells [6,11]. inhibitors would have no or little effect. In the presence of 5 nM of the dopamine reuptake inhibitor GBR 1209 the Numerous approaches have been undertaken to reduce extracellular dopamine concentration did not change in a hypoxia-induced neurotoxicity [2,12]. The pathological normoxic or xenon environment. However, in nitrogen increase of extracellular neurotransmitter concentration the extracellular dopamine concentration did not reach presents probably one of the first indicators for such dam- exactly the same value as in pure nitrogen, the dopamine age although it is not clear to what extent it contributes level was slightly but significantly reduced, thus support- directly. Thus, a reduction or even complete suppression ing the view that hypoxia-induced extracellular dopamine of such an increase of neurotransmitter concentration increase was caused by an enhanced release of dopamine after the primary neuronal damage would suggest a high and – to a lesser extent – an interference with the uptake probability for protection from the hypoxic insult. mechanism (Fig. 2). Recently, we have shown that the noble gas xenon pre- Effects of the dopamine receptor antagonists SCH 23390 vents in hypoxic cortical neurons hypoxia-induced cell damage and glutamate release [13,14]. Such neuroprotec- and sulpiride tive potential has been confirmed by Ma et al. [15] and To test if indeed the hypoxia-induced increase of extracel- Wilhelm et al.[16], and related to its property of being an lular dopamine itself caused the cell damage measured by NMDA-receptor antagonist. In the present paper, how- the increase in extracellular LDH, dopamine receptor ever, we show that also in the dopaminergic PC-12-system antagonists were used. Since they prevent dopamine bind- xenon exhibits profound neuroprotective properties for ing they should provide protection of dopamine-induced hypoxic cells thus underlining its usefulness as a general damage. If the D1 receptor antagonist SCH 23390 was neuroprotectant. used during the incubation period, then at the highest dose of 10 nM, a reduction of nitrogen-induced external LDH-increase could be seen. However, even at this highest Results Release of dopamine under hypoxic conditions applied dose of SCH 23390, there was still only a less than Cells kept under normoxic conditions did not release 50% reduction in extracellular LDH (Fig. 3a). If, on the dopamine during the time period studied. If, however, other hand, the D2 receptor antagonist sulpiride was they were kept in an atmosphere consisting of 100% used, no reduction in the nitrogen-induced LDH-release nitrogene, considerable amounts of dopamine were was found (Fig. 3b). Both compounds did not change the found in the extracellular space reaching a maximum at xenon-induced suppression of cellular damage. 30 min of incubation, followed by a subsequent decrease. If under the same conditions nitrogen was replaced by Cellular damage induced by external addition of xenon, no such increase in dopamine concentration was dopamine found (Fig. 1a). The level of extracellular dopamine To analyze if indeed the increased external dopamine was remained as low as in cells kept under normoxic detrimental to cells, they were incubated in the presence conditions. of 100 nM dopamine, either for 30 min followed by 120 min in normal medium, or continuously for 150 min. As Hypoxia-induced cellular damage shown in fig. 4, column (c), even the 30 min incubation In order to test if such hypoxia damaged the cells, extracel- with 100 nm dopamine (the lesser of the two dopamine lular LDH was determined after a two-hour period of challenges) was sufficient to cause considerable cell dam- treatment. A low level of LDH was found in cells kept age. Such damage was further increased if dopamine was under normoxic conditions whereas cells kept under present for the whole period of time of 150 min (column nitrogen showed a significant release of LDH indicating (e). We asked then if xenon not only prevented the release severe cellular damage (Fig. 1b). If instead of nitrogen of dopamine in a hypoxic situation but could even reduce xenon was used to create such hypoxic condition, the the damage caused by external dopamine. Cells were incu- LDH level remained at the same low level as in controls. bated for 30 min in normal buffer containing 100 nM dopamine followed by 120 min in xenon-saturated Effect of the dopamine reuptake inhibitor GBR 1209 buffer, without dopamine. As shown in column (d), the Hypoxia-induced extracellular increase of dopamine dopamine-induced damage to the cells as seen in column could be caused either by elevated release of dopamine or (c) was significantly reduced. If the cells were incubated by a reduced, or even inhibited, dopamine uptake. If for 150 min in xenon-saturated buffer containing hypoxia caused faster release but did not interfere with dopamine, even under those conditions the damage was uptake, uptake-inhibitors would cause a higher concen- low compared to cells exposed to dopamine in normal tration of dopamine in the extracellular space. On the buffer (column (f)). Page 2 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 ** control xenon ** a) nitrogen ** 0 30 60 90 120 time (min) ** control nitrogen xenon b) Figure 1 A. Dopamine release from differentiated PC-12 cells under normoxic conditions, in N , or in xenon A. Dopamine release from differentiated PC-12 cells under normoxic conditions, in N , or in xenon. Whereas almost no dopamine was released from control cells during the two-hour period, a strong increase of extracellular dopamine was found when cells were kept in N . When cells were maintained in a xenon-atmosphere, no dopamine release occurred, there was virtually no difference compared to controls. B. Assessment of cellular damage in PC-12 cells after two-hour incubation. If cells were kept in normal air, or in xenon, only a small amount of LDH was released. Much higher cellular damage was found when cells were incubated in N . (n = 5; **P < 0.01 with respect to untreated controls). Page 3 of 9 (page number not for citation purposes) dopamine (nM) LDH (mU/ml) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 *** xenon+ GBR 12909 control+ GBR 12909 nitrogen+ GBR 12909 800 *** nitrogen *** *** *** *** 0 30 60 90 120 time (min) E Figure 2 ffect of the dopamine uptake inhibitor GBR 1209 on dopamine release in hypoxic cells Effect of the dopamine uptake inhibitor GBR 1209 on dopamine release in hypoxic cells. If PC-12 cells were incubated in the presence of 5 nM of the dopamine uptake inhibitor GBR 1209, no effect was observed in cells incubated under normoxic con- ditions or in a xenon atmosphere. However, a much higher extracellular dopamine concentration was found in cells incubated in N and GBR 1209 than in N alone, indicating that indeed the release was intensified under hypoxic conditions. (n = 4; P < 2 2 0.0001 with respect to untreated controls, analyzed by 2-way ANOVA and Bonferroni posttests between GBR1209-treated and untreated groups). 2+ Buffering of intracellular Ca -ions using BAPTA and cell damage was investigated in rat embryonic pri- 2+ In order to test if changes in intracellular Ca were mary mesencephalic cell cultures that are known to con- required for the neuroprotective effect of xenon, cells were tain 0.5 – 1.5% of dopaminergic cells [26]. As shown in incubated with the cell-permeant Ca-chelator BAPTA-AM. fig. 6, in an hypoxic atmosphere a very similar pattern of 2+ As shown in fig. 5, chelating intracellular Ca does not dopamine and LDH release is obtained compared to PC- damage the cells per se (control + 10 µM BAPTA). Surpris- 12 cells. Xenon prevents also in these primary cells the ingly, such chelation reduces significantly the neuropro- hypoxia-induced neurotransmitter and LDH release. tective effect of xenon, indicating an essential role for 2+ intracellular Ca for this effect to occur. A slight but sig- Discussion nificant reduction in cellular damage is observed when In hypoxia/ischemia a key feature of secondary damage BAPTA-treated cells are incubated in nitrogen-saturated after the primary neuron-damaging event is the over- buffer. release of neurotransmitters [17]. Consequently, an inter- ference with the hypoxia-induced release mechanism with Comparison with another dopaminergic cell system respect to its control systems may be extremely useful to To exclude that the results obtained were limited to the reduce cellular damage. The results presented here show PC-12-system itself, hypoxia-induced dopamine release that xenon has such properties, namely to prevent cellular Page 4 of 9 (page number not for citation purposes) dopamine (nM) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 control xenon a) nitrogen ** ** 0.0 2.0 5.0 10.0 SCH 23390 (nM) control b) xenon nitrogen 025 10 sulpiride (nM) Figure 3 A. Effect of various concentrations of the D1-receptor antagonist SCH 23390 on cell survival after two-hour incubation A. Effect of various concentrations of the D1-receptor antagonist SCH 23390 on cell survival after two-hour incubation. No effect was observed on PC-12 cells maintained under normal conditions or in xenon, however, for cells kept in nitrogen, a con- centration-dependent decrease of cellular damage was found, indicating that the D1-receptor was involved to convey the cellu- lar damage. (n = 4; **P < 0.01 and *P < 0.05 with respect to untreated controls). B. Effect of various concentrations of the D2- receptor antagonist sulpiride on cell survival after two-hour incubation. No protective effect of sulpiride was found for cells incubated in nitrogen, even a slight increase of cellular damage was seen with increasing concentrations of sulpiride. Page 5 of 9 (page number not for citation purposes) LDH (mU/ml) LDH (mU/ml) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 *** *** *** *** *** 20 60 a b no dopamine 30 min dopamine 150 min dopamine control BAPTA Dopamine-indu Figure 4 ced cellular damage Dopamine-induced cellular damage. Cells were incubated for 2+ 150 min with and without 100 nM dopamine and LDH- Figure 5 Effects of intracellular Ca -buffering by BAPTA 2+ release was determined. (a) untreated control; (b) untreated Effects of intracellular Ca -buffering by BAPTA. If PC-12 cells kept for 30 min in normoxia followed by 120 min in cells were preloaded with 10 µM BAPTA, the neuroprotec- tive effect of xenon was strongly reduced. At the same time, xenon-atmosphere; (c) cells under normoxic conditions treated for 30 min with 100 nM dopamine followed by 120 however, a small reduction in cellular damage was observed min in normal medium; (d) cells under normoxic conditions in nitrogen-treated cells. (n = 5; P < 0.0001 analyzed by 2- treated for 30 min with 100 nM dopamine, followed by 120 way ANOVA and Bonferroni posttests between BAPTA- treated and untreated groups; no significant difference was min incubation in xenon-atmosphere in normal medium without dopamine; (e) cells under normoxic conditions found when the nitrogen-group was compared to the nitro- treated for 150 min with 100 nM dopamine; (f) cells under gen-BAPTA group). normoxic conditions treated for 30 min with 100 nM dopamine, followed by 120 min incubation in xenon-atmos- phere in medium containing 100 nM dopamine. No significant difference was found between (a) and (b) whereas the differ- ence between (c) and (d), and (e) and (f) was highly significant (P < 0.0001). calcineurin system that has been implicated in the regula- tion of monoamine release [24]. Alternatively, xenon might interact upstream of these regulatory systems with 2+ other Ca -dependent events required to occur in hypoxia-induced cell damage. Such a scenario is suggested damage and neurotransmitter release in a hypoxic situa- by our demonstration that the neuroprotective effect of tion thus qualifying it as an almost ideal early neuropro- xenon is strongly reduced if PC-12 cells are loaded with tectant. Concerning possible cellular targets for xenon, a BAPTA. Thus, at present all evidence obtained by us 2+ first indication for the participation of Ca -regulated [13,14,18,19] and others [15,16] establish a complex and events was obtained when it was shown that xenon composite picture of targets susceptible to xenon includ- 2+ blocked cells in metaphase and that the block could be ing NMDA receptors, Ca -regulating and -regulated sys- 2+ lifted by artificial small intracellular Ca - increases [18]. tems up to the activaton of transcription factors whereby Since the CaM KII complex is known to play a decisive such targets are probably not essentially and sequentially role in the metaphase/anaphase transition, it was tested if linked to each other. the CaMKII-inhibitor KN-93 had likewise metaphase- blocking properties. Such effects were obtained [19]. It is To summarize briefly our main findings: (1) The presence well known that in dopaminergic differentiated PC-12 of xenon blocks hypoxia-induced dopamine release in cells, the CaMKII complex is involved in the regulation of dopaminergic cells. (2) Hypoxia-induced dopamine neurotransmitter release [20-22] as well as its participa- increase is caused by an enhanced release of dopamine 2+ tion in a multitude of other Ca -dependent regulatory rather than a reduced uptake of dopamine. (3) When events [23]. Thus, it appears to be plausible that one of the measuring LDH release as a marker of cellular damage, targets for xenon might be the CaMKII complex, either xenon was found to block such release, which suggests 2+ directly or via interference with other Ca -dependent sys- that xenon reduces hypoxia-induced cellular damage. (4) 2+ tems. One of those may be the Ca /calmodulin-activated Increased extracellular dopamine can damage Page 6 of 9 (page number not for citation purposes) LDH (mU/ml) LDH (mU/ml) control xenon control+10µM BAPTA xenon+10µM BAPTA N +10µM BAPTA 2 BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 ** control a) ** nitrogen 0 30 60 90 120 time (min) ** control nitrogen xenon 30 b) A Figure 6 . Dopamine release from mesencephalic dopaminergic cells under normoxic conditions, in N , or in xenon A. Dopamine release from mesencephalic dopaminergic cells under normoxic conditions, in N , or in xenon. Mesencephalic cells containing dopaminergic neurons were exposed on day 14 after preparation for two hours either to normal atmosphere, or xenon or nitrogen. Dopamine was not released from cells maintained under normal conditions or in xenon whereas a signif- icant amount of dopamine was liberated from cells maintained in nitrogen. B. Cellular damage in mesencephalic dopaminergic cells after two-hour incubation. If cells were kept in normal air, or in xenon, only a small amount of LDH was released. Much higher cellular damage was found when cells were incubated in nitrogen. (n = 4; **P < 0.01). Page 7 of 9 (page number not for citation purposes) LDH (mU/ml) dopamine (nM) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 dopaminergic cells directly. This is mainly mediated by chased from Molecular Probes, (Leiden, The Nether- D1 receptor agonism rather than D2. (5) Such direct extra- lands), and all standard chemical products were obtained cellular dopamine-induced damage can be reduced by the from Merck (Berlin, Germany). presence of xenon, even when the increase in extracellular dopamine has not been caused by an episode of cellular Statistical analysis hypoxia. (6) The above described protective effects of All experiments were repeated at least five times, i.e. in xenon depend on the presence of calcium ions. five different plates on five different days. The data were presented as means ± SEM. The results of multiple groups Further studies will show if indeed in the hypoxic cell were analyzed using one-way ANOVA with Dunnett's multiple intracellular targets exist for xenon and how they multiple comparison post test or two-way ANOVA with are orchestrated together to result in cellular protection. Bonferroni posttests using GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego California USA. Differences with p values less than 0.05 were consid- Conclusions Based on the present results obtained with NGF-differen- ered statistically significant. tiated PC-12 cells and on the literature cited in this paper, xenon appears to be a neuroprotectant for a broad spec- Authors' contributions trum of neuronal cells; given its proven non-toxicity based CP conceived the study, participated in its design and on its long clinical use, it may come close to fulfilling the coordination, carried out the cellular studies involving the requirements for an ideal or "gold standard" various gas treatments, participated in the preparation of neuroprotectant. the cells, and drafted the manuscript. PB and WS partici- pated in the design of the experiments and the gas appli- Methods cations, JM performed neurotransmitter analysis and LDH Cells determinations, and WK participated in the design of the Rat pheochromocytoma cells (PC-12) were maintained in study and performed the statistical analysis. All authors RPMI 1640 medium containing 5% fetal calf serum, 10% read and approved the final manuscript. horse serum, at 37°C, 5% CO . For experiments, cells were seeded in 24-well plates at a density of 1 × 10 cells/ Acknowledgements The partial support of this work by Linde Gas Therapeutics is gratefully well and nerve-growth-factor (Promega, Heidelberg, Ger- acknowledged. many) was added (0.4 µg/ml) whereupon cells entered differentiation. They were used on day five after the addi- References tion of growth factor [25]. Primary dopaminergic cells 1. Lipton P: Ischemic cell death in brain neurons. Physiol Rev 1999, from rat embryonic brain were prepared as described (26) 79:431-1568. and used on day 14. 2. Dirnagl U, Iadekola C, Moskowitz MA: Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 1999, 22:391-397. 3. Akiyama Y, Ito A, Koshimura K, Ohue T, Yamagata S, Miwa S, Kikuchi Determination of dopamine and LDH H: Effects of transient forebrain ischemia and reperfusion on function of dopaminergic neurons and dopamine reuptake in Hypoxia-treatment was performed as described [13]. Sam- vivo in rat striatum. Brain Res 1991, 561:120-127. ples from individual wells were taken at the intervals indi- 4. Pastuszko A: Metabolic responses of the dopaminergic system cated and deproteinated using 5% perchloric acid (1:1 = during hypoxia in newborn brain. Biochem Med Metab Biol 1994, 51:1-15. vol/vol). Supernatants were transferred to Eppendorff 5. Gross J, Ungethum U, Andreeva N, Heldt J, Gao J, Marschhausen G, tubes and the same volume (0.5 ml) of 0.4 M perchloric Altmann T, Muller I, Husemann B, Andersson K: Hypoxia during early developmental period induces long-term changes in acid was added, mixed on vortex and centrifuged (6000 the dopamine content and release in a mesencephalic cell rpm, 3 min) to remove cell debris. Dopamine concentra- culture. Neuroscience 1999, 92:699-704. tion was determined by high-pressure liquid chromatog- 6. Kuo JS, Cheng FC, Shen CC, Ou HC, Wu TF, Huang HM: Differen- tial alteration of catecholamine release during chemical raphy (Bio-Tek, Neufahrn, Germany) using an hypoxia is correlated with cell toxicity and is blocked by pro- electrochemical detector (Biometra, Göttingen, tein kinase C inhibitors in PC12 cells. J Cell Biochem 2000, Germany). Cellular damage after the experiment was 79:191-201. 7. Obrenovitch TP, Richards DA: Extracellular neurotransmitter assessed by measuring spectrophotometrically the con- changes in cerebral ischaemia. Cerebrovasc Brain Metab Rev 1995, centration of LDH in the original supernatant, before the 7:1-54. 8. Obrenovitch TP: High extracellular glutamate and neuronal addition of perchloric acid (Roche Diagnostics, Man- death in neurological disorders. Cause, contribution or nheim, Germany). consequence? Ann N Y Acad Sci 1999, 890:273-286. 9. Greene LA, Tischler AS: PC12 pheochromocytoma cell cultures in neurobiological research. Adv Cell Neurobiol 1982, 3:373-414. Chemicals 10. Zachor DA, Moore JF, Brezausek C, Theibert A, Percy AK: Cocaine Gases were supplied by AGA-Linde (Berlin, Germany). inhibits NGF-induced PC12 cells differentiation through D(1)-type dopamine receptors. Brain Res 2000, 869:85-97. 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic 11. Kim DK, Natarajan N, Prabhakar NR, Kumar GK: Facilitation of acid tetrakis(acetoxymethyl) ester (BAPTA-AM) was pur- dopamine and acetylcholine release by intermittent hypoxia Page 8 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 in PC12 cells: involvement of calcium and reactive oxygen species. J Appl Physiol 2004, 96:1206-1215. 12. Dirnagl U, Simon RP, Hallenbeck JM: Ischemic tolerance and endogenous neuroprotection. Trends Neurosci 2003, 26:248-254. 13. Petzelt C: New concepts in neuroprotection. J Anästhes Intensivbeh 2001, 3:38. 14. Petzelt C, Blom P, Schmehl W, Müller J, Kox WJ: Prevention of neurotoxicity in hypoxic cortical neurons by the noble gas xenon. Life Sci 2003, 72:1909-1918. 15. Ma D, Wilhelm S, Maze M, Franks NP: Neuroprotective and neu- rotoxic properties of the 'inert' gas, xenon. Br J Anaesth 2002, 89:739-746. 16. Wilhelm SW, Daqing M, Maze M, Franks NP: Effects of xenon on in vitro and in vivo models of neuronal injury. Anesthesiology 2002, 9:1485-1491. 17. Choi DW, Maulucci-Gedde M, Kriegstein AR: Glutamate neuro- toxicity in cortical cell culture. J Neurosci 1987, 7:357-368. 18. Petzelt C, Taschenberger G, Schmehl W, Kox WJ: Xenon-induced 2+ inhibition of Ca -regulated transitions in the cell cycle of human endothelial cells. P Flugers Arch 1999, 437:737-744. 19. Petzelt C, Kodirov S, Taschenberger G, Kox WJ: Participation of 2+ the Ca -calmodulin-activated kinase in the control of met- aphase-anaphase transition in human cells. Cell Biol Int 2001, 25:403-409. 20. Uchikawa T, Kiuchi Y, Yura A, Nakachi N, Yamazaki Y, Yokomizo C, Oguchi K: Ca(2+)-dependent enhancement of [3H]dopamine uptake in rat striatum: possible involvement of calmodulin- dependent kinases. J Neurochem 1995, 65:2065-2071. 21. Uchida J, Kiuchi Y, Ohno M, Yura A, Oguchi K: Ca(2+)-dependent enhancement of [(3)H]noradrenaline uptake in PC12 cells through calmodulin-dependent kinases. Brain Res 1998, 809:155-164. 22. Fu XZ, Zhang QG, Meng FJ, Zhang GY: NMDA receptor-medi- ated immediate Ser831 phosphorylation of GluR1 through CaMKIIalpha in rat hippocampus during early global ischemia. Neurosci Res 2004, 48:85-91. 23. Parnas H, Segel L, Dudel J, Parnas I: Autoreceptors, membrane potential and the regulation of transmitter release. Trends Neurosci 2000, 23:60-68. 24. Erdemli G, Crunelli V: Release of monoamines and nitric oxide is involved in the modulation of hyperpolarization-activated inward current during acute thalamic hypoxia. Neuroscience 2000, 96:565-574. 25. Yao R, Yoshihara M, Osada H: Specific activation of a c-Jun NH- 2-terminal kinase isoform and induction of neurite out- growth in PC-12 cells by staurosporine. J Biol Chem 1997, 272:18261-18266. 26. Andreeva N, Ungethüm U, Heldt J, Marschhausen G, Altmann Th, Andersson K, Gross J: Elevated potassium enhances glutamate vulnerability of dopaminergic neurons developing in mesen- cephalic cell cultures. Exp Neurol 1996, 137:255-262. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Neuroscience Springer Journals

Xenon prevents cellular damage in differentiated PC-12 cells exposed to hypoxia

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Copyright © 2004 by Petzelt et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurobiology; Animal Models
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Abstract

Background: The neuroprotective effect of xenon has been demonstrated for glutamatergic neurons. In the present study it is investigated if dopaminergic neurons, i.e. nerve-growth-factor differentiated PC-12 cells, are protected as well against hypoxia-induced cell damage in the presence of xenon. Results: Pheochromocytoma cells differentiated by addition of nerve growth factor were placed in a N -saturated atmosphere, a treatment that induced release of dopamine, reaching a maximum after 30 min. By determining extracellular lactate dehydrogenase concentration as marker for concomitant cellular damage, a substantial increase of enzymatic activity was found for N -treated cells. Replacement of N by xenon in such a hypoxic atmosphere resulted in complete protection against cellular damage and prevention of hypoxia-induced dopamine release. Intracellular buffering 2+ of Ca using the Ca-chelator 1, 2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA) reduced the neuroprotective effect of xenon indicating the 2+ essential participation of intracellular Ca -ions in the process of xenon-induced neuroprotection. Conclusions: The results presented demonstrate the outstanding property of xenon to protect neuron-like cells in a hypoxic situation. Background toxic mechanisms may also participate in hypoxia- Originally, hypoxia/ischemia-induced alterations in neu- induced cell damage such as cortical spreading depression ronal function have been attributed to be an over-release [7,8]. Rat pheochromocytoma (PC-12) cells are catecho- of neurotransmitters, including dopamine and glutamate. laminergic, excitable cells that have been widely used as Many studies have been performed on the mechanisms of an in vitro model for neuronal cells [9] possessing both glutamate-induced neuronal damage [1,2] but relatively D1- and D2-dopamine receptors [10]. In these cells few have investigated the hypoxia-induced damage in hypoxia causes a transient release of dopamine resulting dopaminergic neurons [3-6]. In recent years several lines from a complex cellular response consisting of increased of evidence have suggested that effects other than excito- dopamine release and reduced uptake rate. Such increased Page 1 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 dopamine concentration has been shown to be associated other hand, if the release was constant but the re-uptake with cellular damage indicated by an elevated release of inhibited by hypoxia, additional inhibition of uptake by lactate dehydrogenase (LDH) from the cells [6,11]. inhibitors would have no or little effect. In the presence of 5 nM of the dopamine reuptake inhibitor GBR 1209 the Numerous approaches have been undertaken to reduce extracellular dopamine concentration did not change in a hypoxia-induced neurotoxicity [2,12]. The pathological normoxic or xenon environment. However, in nitrogen increase of extracellular neurotransmitter concentration the extracellular dopamine concentration did not reach presents probably one of the first indicators for such dam- exactly the same value as in pure nitrogen, the dopamine age although it is not clear to what extent it contributes level was slightly but significantly reduced, thus support- directly. Thus, a reduction or even complete suppression ing the view that hypoxia-induced extracellular dopamine of such an increase of neurotransmitter concentration increase was caused by an enhanced release of dopamine after the primary neuronal damage would suggest a high and – to a lesser extent – an interference with the uptake probability for protection from the hypoxic insult. mechanism (Fig. 2). Recently, we have shown that the noble gas xenon pre- Effects of the dopamine receptor antagonists SCH 23390 vents in hypoxic cortical neurons hypoxia-induced cell damage and glutamate release [13,14]. Such neuroprotec- and sulpiride tive potential has been confirmed by Ma et al. [15] and To test if indeed the hypoxia-induced increase of extracel- Wilhelm et al.[16], and related to its property of being an lular dopamine itself caused the cell damage measured by NMDA-receptor antagonist. In the present paper, how- the increase in extracellular LDH, dopamine receptor ever, we show that also in the dopaminergic PC-12-system antagonists were used. Since they prevent dopamine bind- xenon exhibits profound neuroprotective properties for ing they should provide protection of dopamine-induced hypoxic cells thus underlining its usefulness as a general damage. If the D1 receptor antagonist SCH 23390 was neuroprotectant. used during the incubation period, then at the highest dose of 10 nM, a reduction of nitrogen-induced external LDH-increase could be seen. However, even at this highest Results Release of dopamine under hypoxic conditions applied dose of SCH 23390, there was still only a less than Cells kept under normoxic conditions did not release 50% reduction in extracellular LDH (Fig. 3a). If, on the dopamine during the time period studied. If, however, other hand, the D2 receptor antagonist sulpiride was they were kept in an atmosphere consisting of 100% used, no reduction in the nitrogen-induced LDH-release nitrogene, considerable amounts of dopamine were was found (Fig. 3b). Both compounds did not change the found in the extracellular space reaching a maximum at xenon-induced suppression of cellular damage. 30 min of incubation, followed by a subsequent decrease. If under the same conditions nitrogen was replaced by Cellular damage induced by external addition of xenon, no such increase in dopamine concentration was dopamine found (Fig. 1a). The level of extracellular dopamine To analyze if indeed the increased external dopamine was remained as low as in cells kept under normoxic detrimental to cells, they were incubated in the presence conditions. of 100 nM dopamine, either for 30 min followed by 120 min in normal medium, or continuously for 150 min. As Hypoxia-induced cellular damage shown in fig. 4, column (c), even the 30 min incubation In order to test if such hypoxia damaged the cells, extracel- with 100 nm dopamine (the lesser of the two dopamine lular LDH was determined after a two-hour period of challenges) was sufficient to cause considerable cell dam- treatment. A low level of LDH was found in cells kept age. Such damage was further increased if dopamine was under normoxic conditions whereas cells kept under present for the whole period of time of 150 min (column nitrogen showed a significant release of LDH indicating (e). We asked then if xenon not only prevented the release severe cellular damage (Fig. 1b). If instead of nitrogen of dopamine in a hypoxic situation but could even reduce xenon was used to create such hypoxic condition, the the damage caused by external dopamine. Cells were incu- LDH level remained at the same low level as in controls. bated for 30 min in normal buffer containing 100 nM dopamine followed by 120 min in xenon-saturated Effect of the dopamine reuptake inhibitor GBR 1209 buffer, without dopamine. As shown in column (d), the Hypoxia-induced extracellular increase of dopamine dopamine-induced damage to the cells as seen in column could be caused either by elevated release of dopamine or (c) was significantly reduced. If the cells were incubated by a reduced, or even inhibited, dopamine uptake. If for 150 min in xenon-saturated buffer containing hypoxia caused faster release but did not interfere with dopamine, even under those conditions the damage was uptake, uptake-inhibitors would cause a higher concen- low compared to cells exposed to dopamine in normal tration of dopamine in the extracellular space. On the buffer (column (f)). Page 2 of 9 (page number not for citation purposes) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 ** control xenon ** a) nitrogen ** 0 30 60 90 120 time (min) ** control nitrogen xenon b) Figure 1 A. Dopamine release from differentiated PC-12 cells under normoxic conditions, in N , or in xenon A. Dopamine release from differentiated PC-12 cells under normoxic conditions, in N , or in xenon. Whereas almost no dopamine was released from control cells during the two-hour period, a strong increase of extracellular dopamine was found when cells were kept in N . When cells were maintained in a xenon-atmosphere, no dopamine release occurred, there was virtually no difference compared to controls. B. Assessment of cellular damage in PC-12 cells after two-hour incubation. If cells were kept in normal air, or in xenon, only a small amount of LDH was released. Much higher cellular damage was found when cells were incubated in N . (n = 5; **P < 0.01 with respect to untreated controls). Page 3 of 9 (page number not for citation purposes) dopamine (nM) LDH (mU/ml) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 *** xenon+ GBR 12909 control+ GBR 12909 nitrogen+ GBR 12909 800 *** nitrogen *** *** *** *** 0 30 60 90 120 time (min) E Figure 2 ffect of the dopamine uptake inhibitor GBR 1209 on dopamine release in hypoxic cells Effect of the dopamine uptake inhibitor GBR 1209 on dopamine release in hypoxic cells. If PC-12 cells were incubated in the presence of 5 nM of the dopamine uptake inhibitor GBR 1209, no effect was observed in cells incubated under normoxic con- ditions or in a xenon atmosphere. However, a much higher extracellular dopamine concentration was found in cells incubated in N and GBR 1209 than in N alone, indicating that indeed the release was intensified under hypoxic conditions. (n = 4; P < 2 2 0.0001 with respect to untreated controls, analyzed by 2-way ANOVA and Bonferroni posttests between GBR1209-treated and untreated groups). 2+ Buffering of intracellular Ca -ions using BAPTA and cell damage was investigated in rat embryonic pri- 2+ In order to test if changes in intracellular Ca were mary mesencephalic cell cultures that are known to con- required for the neuroprotective effect of xenon, cells were tain 0.5 – 1.5% of dopaminergic cells [26]. As shown in incubated with the cell-permeant Ca-chelator BAPTA-AM. fig. 6, in an hypoxic atmosphere a very similar pattern of 2+ As shown in fig. 5, chelating intracellular Ca does not dopamine and LDH release is obtained compared to PC- damage the cells per se (control + 10 µM BAPTA). Surpris- 12 cells. Xenon prevents also in these primary cells the ingly, such chelation reduces significantly the neuropro- hypoxia-induced neurotransmitter and LDH release. tective effect of xenon, indicating an essential role for 2+ intracellular Ca for this effect to occur. A slight but sig- Discussion nificant reduction in cellular damage is observed when In hypoxia/ischemia a key feature of secondary damage BAPTA-treated cells are incubated in nitrogen-saturated after the primary neuron-damaging event is the over- buffer. release of neurotransmitters [17]. Consequently, an inter- ference with the hypoxia-induced release mechanism with Comparison with another dopaminergic cell system respect to its control systems may be extremely useful to To exclude that the results obtained were limited to the reduce cellular damage. The results presented here show PC-12-system itself, hypoxia-induced dopamine release that xenon has such properties, namely to prevent cellular Page 4 of 9 (page number not for citation purposes) dopamine (nM) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 control xenon a) nitrogen ** ** 0.0 2.0 5.0 10.0 SCH 23390 (nM) control b) xenon nitrogen 025 10 sulpiride (nM) Figure 3 A. Effect of various concentrations of the D1-receptor antagonist SCH 23390 on cell survival after two-hour incubation A. Effect of various concentrations of the D1-receptor antagonist SCH 23390 on cell survival after two-hour incubation. No effect was observed on PC-12 cells maintained under normal conditions or in xenon, however, for cells kept in nitrogen, a con- centration-dependent decrease of cellular damage was found, indicating that the D1-receptor was involved to convey the cellu- lar damage. (n = 4; **P < 0.01 and *P < 0.05 with respect to untreated controls). B. Effect of various concentrations of the D2- receptor antagonist sulpiride on cell survival after two-hour incubation. No protective effect of sulpiride was found for cells incubated in nitrogen, even a slight increase of cellular damage was seen with increasing concentrations of sulpiride. Page 5 of 9 (page number not for citation purposes) LDH (mU/ml) LDH (mU/ml) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 *** *** *** *** *** 20 60 a b no dopamine 30 min dopamine 150 min dopamine control BAPTA Dopamine-indu Figure 4 ced cellular damage Dopamine-induced cellular damage. Cells were incubated for 2+ 150 min with and without 100 nM dopamine and LDH- Figure 5 Effects of intracellular Ca -buffering by BAPTA 2+ release was determined. (a) untreated control; (b) untreated Effects of intracellular Ca -buffering by BAPTA. If PC-12 cells kept for 30 min in normoxia followed by 120 min in cells were preloaded with 10 µM BAPTA, the neuroprotec- tive effect of xenon was strongly reduced. At the same time, xenon-atmosphere; (c) cells under normoxic conditions treated for 30 min with 100 nM dopamine followed by 120 however, a small reduction in cellular damage was observed min in normal medium; (d) cells under normoxic conditions in nitrogen-treated cells. (n = 5; P < 0.0001 analyzed by 2- treated for 30 min with 100 nM dopamine, followed by 120 way ANOVA and Bonferroni posttests between BAPTA- treated and untreated groups; no significant difference was min incubation in xenon-atmosphere in normal medium without dopamine; (e) cells under normoxic conditions found when the nitrogen-group was compared to the nitro- treated for 150 min with 100 nM dopamine; (f) cells under gen-BAPTA group). normoxic conditions treated for 30 min with 100 nM dopamine, followed by 120 min incubation in xenon-atmos- phere in medium containing 100 nM dopamine. No significant difference was found between (a) and (b) whereas the differ- ence between (c) and (d), and (e) and (f) was highly significant (P < 0.0001). calcineurin system that has been implicated in the regula- tion of monoamine release [24]. Alternatively, xenon might interact upstream of these regulatory systems with 2+ other Ca -dependent events required to occur in hypoxia-induced cell damage. Such a scenario is suggested damage and neurotransmitter release in a hypoxic situa- by our demonstration that the neuroprotective effect of tion thus qualifying it as an almost ideal early neuropro- xenon is strongly reduced if PC-12 cells are loaded with tectant. Concerning possible cellular targets for xenon, a BAPTA. Thus, at present all evidence obtained by us 2+ first indication for the participation of Ca -regulated [13,14,18,19] and others [15,16] establish a complex and events was obtained when it was shown that xenon composite picture of targets susceptible to xenon includ- 2+ blocked cells in metaphase and that the block could be ing NMDA receptors, Ca -regulating and -regulated sys- 2+ lifted by artificial small intracellular Ca - increases [18]. tems up to the activaton of transcription factors whereby Since the CaM KII complex is known to play a decisive such targets are probably not essentially and sequentially role in the metaphase/anaphase transition, it was tested if linked to each other. the CaMKII-inhibitor KN-93 had likewise metaphase- blocking properties. Such effects were obtained [19]. It is To summarize briefly our main findings: (1) The presence well known that in dopaminergic differentiated PC-12 of xenon blocks hypoxia-induced dopamine release in cells, the CaMKII complex is involved in the regulation of dopaminergic cells. (2) Hypoxia-induced dopamine neurotransmitter release [20-22] as well as its participa- increase is caused by an enhanced release of dopamine 2+ tion in a multitude of other Ca -dependent regulatory rather than a reduced uptake of dopamine. (3) When events [23]. Thus, it appears to be plausible that one of the measuring LDH release as a marker of cellular damage, targets for xenon might be the CaMKII complex, either xenon was found to block such release, which suggests 2+ directly or via interference with other Ca -dependent sys- that xenon reduces hypoxia-induced cellular damage. (4) 2+ tems. One of those may be the Ca /calmodulin-activated Increased extracellular dopamine can damage Page 6 of 9 (page number not for citation purposes) LDH (mU/ml) LDH (mU/ml) control xenon control+10µM BAPTA xenon+10µM BAPTA N +10µM BAPTA 2 BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 ** control a) ** nitrogen 0 30 60 90 120 time (min) ** control nitrogen xenon 30 b) A Figure 6 . Dopamine release from mesencephalic dopaminergic cells under normoxic conditions, in N , or in xenon A. Dopamine release from mesencephalic dopaminergic cells under normoxic conditions, in N , or in xenon. Mesencephalic cells containing dopaminergic neurons were exposed on day 14 after preparation for two hours either to normal atmosphere, or xenon or nitrogen. Dopamine was not released from cells maintained under normal conditions or in xenon whereas a signif- icant amount of dopamine was liberated from cells maintained in nitrogen. B. Cellular damage in mesencephalic dopaminergic cells after two-hour incubation. If cells were kept in normal air, or in xenon, only a small amount of LDH was released. Much higher cellular damage was found when cells were incubated in nitrogen. (n = 4; **P < 0.01). Page 7 of 9 (page number not for citation purposes) LDH (mU/ml) dopamine (nM) BMC Neuroscience 2004, 5:55 http://www.biomedcentral.com/1471-2202/5/55 dopaminergic cells directly. This is mainly mediated by chased from Molecular Probes, (Leiden, The Nether- D1 receptor agonism rather than D2. (5) Such direct extra- lands), and all standard chemical products were obtained cellular dopamine-induced damage can be reduced by the from Merck (Berlin, Germany). presence of xenon, even when the increase in extracellular dopamine has not been caused by an episode of cellular Statistical analysis hypoxia. (6) The above described protective effects of All experiments were repeated at least five times, i.e. in xenon depend on the presence of calcium ions. five different plates on five different days. The data were presented as means ± SEM. The results of multiple groups Further studies will show if indeed in the hypoxic cell were analyzed using one-way ANOVA with Dunnett's multiple intracellular targets exist for xenon and how they multiple comparison post test or two-way ANOVA with are orchestrated together to result in cellular protection. Bonferroni posttests using GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego California USA. Differences with p values less than 0.05 were consid- Conclusions Based on the present results obtained with NGF-differen- ered statistically significant. tiated PC-12 cells and on the literature cited in this paper, xenon appears to be a neuroprotectant for a broad spec- Authors' contributions trum of neuronal cells; given its proven non-toxicity based CP conceived the study, participated in its design and on its long clinical use, it may come close to fulfilling the coordination, carried out the cellular studies involving the requirements for an ideal or "gold standard" various gas treatments, participated in the preparation of neuroprotectant. the cells, and drafted the manuscript. PB and WS partici- pated in the design of the experiments and the gas appli- Methods cations, JM performed neurotransmitter analysis and LDH Cells determinations, and WK participated in the design of the Rat pheochromocytoma cells (PC-12) were maintained in study and performed the statistical analysis. All authors RPMI 1640 medium containing 5% fetal calf serum, 10% read and approved the final manuscript. horse serum, at 37°C, 5% CO . For experiments, cells were seeded in 24-well plates at a density of 1 × 10 cells/ Acknowledgements The partial support of this work by Linde Gas Therapeutics is gratefully well and nerve-growth-factor (Promega, Heidelberg, Ger- acknowledged. many) was added (0.4 µg/ml) whereupon cells entered differentiation. They were used on day five after the addi- References tion of growth factor [25]. Primary dopaminergic cells 1. Lipton P: Ischemic cell death in brain neurons. Physiol Rev 1999, from rat embryonic brain were prepared as described (26) 79:431-1568. and used on day 14. 2. Dirnagl U, Iadekola C, Moskowitz MA: Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 1999, 22:391-397. 3. 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Andreeva N, Ungethüm U, Heldt J, Marschhausen G, Altmann Th, Andersson K, Gross J: Elevated potassium enhances glutamate vulnerability of dopaminergic neurons developing in mesen- cephalic cell cultures. Exp Neurol 1996, 137:255-262. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes)

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Published: Dec 8, 2004

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