Abstract
Classic phenylketonuria (PKU) is characterized by brain lesions. However, its underlying neurotoxic mechanisms remain unknown. Based on our previous studies, we hypothesized that calcium might participate in PKU-associated neuropathy. In cultured cortical neurons, cytoplasmic free calcium concentration ([Ca2+]i) decreased dramatically when treatment with phenylalanine (Phe) and phenyllactic acid, while phenylacetic acid treatment immediately increased [Ca2+]i, which began to decrease after 3 min. Moreover, [Ca2+]i decreased dramatically after Phe treatment in the presence of EGTA suggesting that Phe might increase [Ca2+]i efflux. Phe-induced [Ca2+]i decrease was strongly inhibited by vanadate, a non-specific plasma membrane Ca2+-ATPase (PMCA) antagonist, suggesting that Phe might increase [Ca2+]i efflux throught modulating PMCA. These findings were further supported by the facts that Phe could increase membrance 45Ca-uptake capability and PMCA activity. In contrast, treatment of KBR7943 or thapsigargin, antagonists to Na/Ca Exchanger (NCX) and Sarco/Endoplasmic reticulum Ca2+-ATPase (SERCA), respectively, did not elicit any changes in [Ca2+]i. Specific siRNA against PMCA had an effect similar to vanadate. Since the brain injury induced by phenylalaninemia was thought to be a chronic process, we cultured cortical neurons in the presence of Phe for 2 weeks and measured [Ca2+]i, PMCA activity and 45Ca-uptake capability at days 3, 7, 9 and 14, respectively. PMCA activity and 45Ca-uptake capability decreased from day 9, at the same time [Ca2+]i increase was observed. In conclusion, PMCA participate in regulating Phe-induced initial rapid decrease in [Ca2+]i and subsequent long-term increase in [Ca2+]i.
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References
Glushakov AV, Dennis DM, Sumners C et al (2003) l-phenylalanine selectively depresses currents at glutamatergic excitatory synapses. J Neurosci Res 2:116–124
Glushakov AV, Glushakova O, Varshney M et al (2005) Long-term changes in glutamatergic synaptic transmission inphenylketonuria. Brain 128:300–307
Yang XW, Gu XF, Chen RG (2000) Toxic effects of phenylacetic acid to cultured rat cortical neurons. Chinese J Neurosci 16:330–332
Zhang HW, Lu JQ, Gu XF (2003) Expression of two neuron developmental associated genes induced by hyper-phenylalanine with real time quantitative RT-PCR. Chin Lab J 26:273–276
Zhang HW, Gu XF (2005) A study of gene expression profiles of cultured embryonic rat neurons induced by phenylalanine. Metab Brain Dis 20:61–72
Chu Z, Moenter SM (2006) Physiologic regulation of a tetrodotoxin-sensitive sodium influx that mediates a slow afterdepolarization potential in gonadotropin-releasing hormone neurons: possible implications for the central regulation of fertility. J Neurosci 26:11961–11973
Racay P, Kaplan P, Lehotsky J (1996) Control of Ca2+ homeostasis in neuronal cells. Gen Physiol Biophys 15:193–210
Mattson MP, LaFerla FM, Chan SL et al (2000) Calcium signaling in the RE: its role in neuronal plasticity and neurodegenerative disorders. Trends Neurosci 23:222–229
Doi T, Kuroda S, Michikawa T et al (2005) Inositol 1,4,5-trisphosphate- dependent Ca2+ threshold dynamics detect spike timing in cerebellar Purkinje cells. J Neurosci 25:950–961
Rossi ML, Prigioni I, Gioglio L et al. (2006) IP3 receptor in the hair cells of frog semicircular canal and its possible functional role. Eur J Neurosci 23:1775–1783
Penna A, Rassendren FA (2003) The TRP family of channels: a complex gallery of characters. J Soc Biol 197:249–258
Vale C, Alfonso A, Sunol C et al (2006) Modulation of calcium entry and glutamate release in cultured cerebellar granule cells by palytoxin. J Neurosci Res 83:1393–1406
Usachev YM, Marsh AJ, Johanns TM et al (2006) Kinase C in sensory neurons accelerates Ca2+ uptake into the endoplasmic reticulum. J Neurosci 26:311–318
Niggli V, Adunyah ES, Penniston JT et al (1981) Purified (Ca2+-Mg2+) ATPase of the erythrocyte membrane: reconstruction and effect of calmodulin and phospholipids. J Biol Chem 256:395–401
Niggli V, Adunyah ES, Carafoli E (1981) Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca2+-ATPase. J Biol Chem 256:8588–92
Sepulveda MR, Mata AM (2005) Localization of intracellular and plasma membrane Ca2+-ATPases in the cerebellum. The Cerebellum 4:82–89
Niggli V, Carafoli E (1981) Interaction of the purified Ca2+, Mg2+ - ATPase from human erythrocytes with phospholipids and calmodulin. Acta Biol Med Ger 40:437–442
Stains JP, Weber JA, Gay CV (2002) Expression of Na+/Ca2+ exchanger isoforms (NCX1 and NCX3) and plasma membrane Ca2+ ATPase during osteoblast differentiation. J Cell Biochem 84:625–635
Guerini D (1998) The Ca2+ pumps and the Na+/Ca2+ exchangers. Biometals 11:319–330
Gover TD, Moreira TH, Kao JP et al (2006) Calcium homeostasis in trigeminal ganglion cell bodies. Cell Calcium Oct 12
Dichter MA (1978) Rat cortical neurons in cell culture: culture methods, cell morphology, electrophysiology, and synapse formation. Brain Res 149:279–293
Janusz S, Iwona K, Jacek B et al (2004) The effect of antisense oligonucleotide treatment of plasma membrane Ca2+-ATPase in PC12 cells. Cell mole biol lett 9:451–464
Carmen Perez-Terzic, Marisa Jaconi , Lisa Stehno-Bittel (2004) Measurement of intracellular calcium concentration using confocal microscopy. In: David, Lambert (ed) Calcium signaling protocols. Humana press, pp 75–93
Salvador JM, Mata AM (1996) Purification of the synaptosomal plasma membrane (Ca2+-Mg2+)-ATPase from pig brain. Biochem J 315:183–187
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Stahl WL, Eakin TJ, Owens JW et al (1992) Plasma membrane Ca2+-ATPase isoforms: distribution of mRNAs in rat brain by in situ hybridization. Brain Res Mol Brain Res 16:223–231
Stahl WL, Keeton TP, Eakin TJ (1994) The plasma membrane Ca2+-ATPase mRNA isoform PMCA 4 is expressed at high levels in neurons of rat piriform cortex and neocortex. Neurosci Lett 178:267–270
Garcia ML, Usachev YM, Thayer SA et al (2001) Plasma membrane calcium ATPase plays a role in reducing Ca2+-mediated cytotoxicity in PC12 cells. J Neurosci Res 64:661–669
Heinonen JK, Lahti RJ (1981) A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal Biochem 113:313–317
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with Folin-phenol reagent. J Biol Chem 193:265–275
Bhatnagar K, Singh VP (2004) Ca2+ dependence and inhibitory effects of trifluoperazine on plasma membrane ATPase of thermoactinomyces vulgaris. Current Microbiol 49:28–31
Coletto L, Pinton P, Rizzuto R et al (2005) Inhibitory interaction of the 14-3-3{epsilon} protein with isoform 4 of the plasma membrane Ca2+-ATPase pump. J Biol Chem 280:37195–37203
Harald EM, Josef W, Ulrich B et al (2006) Brain imaging and proton magnetic resonance spectroscopy in patients with phenylketonuria. Pediatrics 2:1580–1583
Sener RN (2003) Diffusion MRI findings in phenylketonuria. Eur Radiol 13:226–229
Thinakaran G, Sisodia SS (2006) Presenilins and Alzheimer disease: the calcium conspiracy. Nat Neurosci 9:1354–1355
Marchetti C (2003) Molecular targets of lead in brain neurotoxicity. Neurotox Res 5:221–236
Thomas RC (2002) The effects of HCl and CaCl2 injections on intracellular calcium and pH in voltage-clamped snail (Helix aspersa) neurons. J Gen Physiol 120:567–579
Kip SN, Gray NW, Burette A et al (2006) Changes in the expression of plasma membrane calcium extrusion systems during the maturation of hippocampal neurons. Hippocampus 16:20–34
Yang LL, Wang C, Gros R et al (2003) Calcineurin-independent regulation of plasma membrane Ca2+ ATPase-4 in the vascular smooth muscle cell cycle. Am J Physiol Cell Physiol 285:88–95
Carter DS, Haider SN, Blair RE et al (2006) Altered calcium/ calmodulin kinase II activity changes calcium homeostasis that underlies epileptiform activity in hippocampal neurons in culture. J Pharmacol Exp Ther 319:1021–1031
Strehler EE, Zacharias DA (2001) Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 81:21–50
Lambeng N, Michel PP, Agid Y et al (2001) The relationship between differentiation and survival in PC12 cells treated with cyclic adenosine monophosphate in the presence of epidermal growth factor or nerve growth factor. Neurosci Lett 297:133–136
Strehler EE, Treiman M (2004) Calcium pumps of plasma membrane and cell interior. Curr Mol Med 4:323–335
Schuh K, Uldrijan S, Telkamp M et al (2001) The plasma membrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I. J Cell Biol 155:201–205
Gromadzinska E, Lachowicz L, Walkowiak B et al (2001) Calmodulin effect on purified rat cortical plasma membrane Ca2+-ATPase in different phosphorylation states. Biochim Biophys Acta 1549:19–31
Usachev YM, DeMarco SJ, Campbell C et al (2002) Bradykinin and ATP accelerate Ca2+ efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca2+ pump isoform 4. Neuron 33:113–122
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This work was supported by grant from the National Natural Science Foundation of China (No. 30471834).
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Yu, Y.G., Tang, F.G., Pan, J. et al. Effects of Phenylalanine and its Metabolites on Cytoplasmic Free Calcium in Cortical Neurons. Neurochem Res 32, 1292–1301 (2007). https://doi.org/10.1007/s11064-007-9303-3
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DOI: https://doi.org/10.1007/s11064-007-9303-3