title: Effects of phenylalanine and its metabolites on cytoplasmic free calcium in cortical neurons.
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 ([Ca(2+)](i)) decreased dramatically when treatment with phenylalanine (Phe) and phenyllactic acid, while phenylacetic acid treatment immediately increased [Ca(2+)](i), which began to decrease after 3 min. Moreover, [Ca(2+)](i) decreased dramatically after Phe treatment in the presence of EGTA suggesting that Phe might increase [Ca(2+)](i) efflux. Phe-induced [Ca(2+)](i) decrease was strongly inhibited by vanadate, a non-specific plasma membrane Ca(2+)-ATPase (PMCA) antagonist, suggesting that Phe might increase [Ca(2+)](i) efflux throught modulating PMCA. These findings were further supported by the facts that Phe could increase membrance (45)Ca-uptake capability and PMCA activity. In contrast, treatment of KBR7943 or thapsigargin, antagonists to Na/Ca Exchanger (NCX) and Sarco/Endoplasmic reticulum Ca(2+)-ATPase (SERCA), respectively, did not elicit any changes in [Ca(2+)](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 [Ca(2+)](i), PMCA activity and (45)Ca-uptake capability at days 3, 7, 9 and 14, respectively. PMCA activity and (45)Ca-uptake capability decreased from day 9, at the same time [Ca(2+)](i) increase was observed. In conclusion, PMCA participate in regulating Phe-induced initial rapid decrease in [Ca(2+)](i) and subsequent long-term increase in [Ca(2+)](i).
authors.affiliation: Department of Endocrinology and Genetic Metabolism, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Institute for Pediatric Research, Shanghai, China. guxuefan@gmail.com
authors.forename: Y G
authors.initials: YG
authors.lastname: Yu
authors.forename: F G
authors.initials: FG
authors.lastname: Tang
authors.forename: J
authors.initials: J
authors.lastname: Pan
authors.forename: X F
authors.initials: XF
authors.lastname: Gu
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D003593:Cytoplasm / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D010661:Phenylketonurias / Q000378:metabolism; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D034741:RNA, Small Interfering; D010649:Phenylalanine; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium