Neurochemical Research

, Volume 32, Issue 8, pp 1292–1301 | Cite as

Effects of Phenylalanine and its Metabolites on Cytoplasmic Free Calcium in Cortical Neurons

Original Paper


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.


Phenylalanine(Phe) Plasma membrane Ca2+-ATPase (PMCA) Calcium Cortical neurons 



This work was supported by grant from the National Natural Science Foundation of China (No. 30471834).


  1. 1.
    Glushakov AV, Dennis DM, Sumners C et al (2003) l-phenylalanine selectively depresses currents at glutamatergic excitatory synapses. J Neurosci Res 2:116–124CrossRefGoogle Scholar
  2. 2.
    Glushakov AV, Glushakova O, Varshney M et al (2005) Long-term changes in glutamatergic synaptic transmission inphenylketonuria. Brain 128:300–307PubMedCrossRefGoogle Scholar
  3. 3.
    Yang XW, Gu XF, Chen RG (2000) Toxic effects of phenylacetic acid to cultured rat cortical neurons. Chinese J Neurosci 16:330–332Google Scholar
  4. 4.
    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 26:273–276Google Scholar
  5. 5.
    Zhang HW, Gu XF (2005) A study of gene expression profiles of cultured embryonic rat neurons induced by phenylalanine. Metab Brain Dis 20:61–72PubMedCrossRefGoogle Scholar
  6. 6.
    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 26:11961–11973PubMedCrossRefGoogle Scholar
  7. 7.
    Racay P, Kaplan P, Lehotsky J (1996) Control of Ca2+ homeostasis in neuronal cells. Gen Physiol Biophys 15:193–210PubMedGoogle Scholar
  8. 8.
    Mattson MP, LaFerla FM, Chan SL et al (2000) Calcium signaling in the RE: its role in neuronal plasticity and neurodegenerative disorders. Trends Neurosci 23:222–229PubMedCrossRefGoogle Scholar
  9. 9.
    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 25:950–961PubMedCrossRefGoogle Scholar
  10. 10.
    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 23:1775–1783PubMedCrossRefGoogle Scholar
  11. 11.
    Penna A, Rassendren FA (2003) The TRP family of channels: a complex gallery of characters. J Soc Biol 197:249–258PubMedGoogle Scholar
  12. 12.
    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 83:1393–1406PubMedCrossRefGoogle Scholar
  13. 13.
    Usachev YM, Marsh AJ, Johanns TM et al (2006) Kinase C in sensory neurons accelerates Ca2+ uptake into the endoplasmic reticulum. J Neurosci 26:311–318PubMedCrossRefGoogle Scholar
  14. 14.
    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 256:395–401PubMedGoogle Scholar
  15. 15.
    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 256:8588–92PubMedGoogle Scholar
  16. 16.
    Sepulveda MR, Mata AM (2005) Localization of intracellular and plasma membrane Ca2+-ATPases in the cerebellum. The Cerebellum 4:82–89PubMedCrossRefGoogle Scholar
  17. 17.
    Niggli V, Carafoli E (1981) Interaction of the purified Ca2+, Mg2+ - ATPase from human erythrocytes with phospholipids and calmodulin. Acta Biol Med Ger 40:437–442PubMedGoogle Scholar
  18. 18.
    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 84:625–635PubMedCrossRefGoogle Scholar
  19. 19.
    Guerini D (1998) The Ca2+ pumps and the Na+/Ca2+ exchangers. Biometals 11:319–330PubMedCrossRefGoogle Scholar
  20. 20.
    Gover TD, Moreira TH, Kao JP et al (2006) Calcium homeostasis in trigeminal ganglion cell bodies. Cell Calcium Oct 12Google Scholar
  21. 21.
    Dichter MA (1978) Rat cortical neurons in cell culture: culture methods, cell morphology, electrophysiology, and synapse formation. Brain Res 149:279–293PubMedCrossRefGoogle Scholar
  22. 22.
    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 9:451–464Google Scholar
  23. 23.
    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 75–93Google Scholar
  24. 24.
    Salvador JM, Mata AM (1996) Purification of the synaptosomal plasma membrane (Ca2+-Mg2+)-ATPase from pig brain. Biochem J 315:183–187Google Scholar
  25. 25.
    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 72:248–254PubMedCrossRefGoogle Scholar
  26. 26.
    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 16:223–231PubMedCrossRefGoogle Scholar
  27. 27.
    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 178:267–270PubMedCrossRefGoogle Scholar
  28. 28.
    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 64:661–669PubMedCrossRefGoogle Scholar
  29. 29.
    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 113:313–317PubMedCrossRefGoogle Scholar
  30. 30.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with Folin-phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  31. 31.
    Bhatnagar K, Singh VP (2004) Ca2+ dependence and inhibitory effects of trifluoperazine on plasma membrane ATPase of thermoactinomyces vulgaris. Current Microbiol 49:28–31Google Scholar
  32. 32.
    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 280:37195–37203PubMedCrossRefGoogle Scholar
  33. 33.
    Harald EM, Josef W, Ulrich B et al (2006) Brain imaging and proton magnetic resonance spectroscopy in patients with phenylketonuria. Pediatrics 2:1580–1583Google Scholar
  34. 34.
    Sener RN (2003) Diffusion MRI findings in phenylketonuria. Eur Radiol 13:226–229CrossRefGoogle Scholar
  35. 35.
    Thinakaran G, Sisodia SS (2006) Presenilins and Alzheimer disease: the calcium conspiracy. Nat Neurosci 9:1354–1355PubMedCrossRefGoogle Scholar
  36. 36.
    Marchetti C (2003) Molecular targets of lead in brain neurotoxicity. Neurotox Res 5:221–236PubMedCrossRefGoogle Scholar
  37. 37.
    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 120:567–579Google Scholar
  38. 38.
    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 16:20–34PubMedCrossRefGoogle Scholar
  39. 39.
    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 285:88–95Google Scholar
  40. 40.
    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 319:1021–1031PubMedCrossRefGoogle Scholar
  41. 41.
    Strehler EE, Zacharias DA (2001) Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 81:21–50PubMedGoogle Scholar
  42. 42.
    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 297:133–136PubMedCrossRefGoogle Scholar
  43. 43.
    Strehler EE, Treiman M (2004) Calcium pumps of plasma membrane and cell interior. Curr Mol Med 4:323–335PubMedCrossRefGoogle Scholar
  44. 44.
    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 155:201–205PubMedCrossRefGoogle Scholar
  45. 45.
    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 1549:19–31PubMedGoogle Scholar
  46. 46.
    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 33:113–122PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.Department of Endocrinology and Genetic Metabolism, Xinhua HospitalShanghai Jiaotong University School of Medicine, Shanghai Institute for Pediatric ResearchShanghaiChina

Personalised recommendations