Effects of phenylalanine and its metabolites on cytoplasmic free calcium in cortical neurons.
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).
Yu
Y G
YG
Department of Endocrinology and Genetic Metabolism, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Institute for Pediatric Research, Shanghai, China. guxuefan@gmail.com
Tang
F G
FG
Pan
J
J
Gu
X F
XF
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
D034741:RNA, Small Interfering; D010649:Phenylalanine; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium