Localization of endoplasmic reticulum and plasma membrane Ca2+-ATPases in subcellular fractions and sections of pig cerebellum.
Subcellular fractions and sections of the cerebellum were analysed to evaluate the relative activity and distribution of organellar and plasma membrane Ca2+-ATPases (SERCA and PMCA). Western blot analysis of the fractions with IID8 or Y/1F4 SERCA-specific antibodies or else with 5F10 or pbPMCA antibodies, specific to PMCA pump, revealed a major content of SERCA protein in microsomes and of PMCA protein in plasma membrane vesicles. The Ca2+-ATPase activity of microsomes was more sensitive to thapsigargin, a SERCA-specific inhibitor, whereas the activity of the plasma membrane vesicle fraction was inhibited more by vanadate, a blocker of PMCA activity. The SERCA and PMCA distribution analysed in cerebellar sections revealed IID8 antibody reactions in Purkinje cell cytoplasm, granule cells and cerebellar glomeruli. Y/1F4 gave immunostaining in Purkinje cells, molecular layer interneurons (basket and stellate cells) and glomeruli, but granule cells were not labelled. The 5F10 antibody reacted with Purkinje cells, including their dendritic spines, as well as cerebellar glomeruli, whereas the pbPMCA antibody labelled several processes in all three layers and some synaptic interaction sites. The differential content and localization of the two types of Ca2+ pumps in specific neuronal areas of pig cerebellum indicate precise Ca2+ requirements of specific cellular regions.
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain.
M Rosario
MR
Sepúlveda
Matías
M
Hidalgo-Sánchez
Ana M
AM
Mata
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000201:enzymology*; D002531:Cerebellum / Q000166:cytology* / Q000201:enzymology; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D053499:Plasma Membrane Calcium-Transporting ATPases; D011689:Purkinje Cells / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013347:Subcellular Fractions / Q000201:enzymology*; D013552:Swine; D016708:Synaptophysin / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D016708:Synaptophysin; D014638:Vanadates; D019284:Thapsigargin; D053499:Plasma Membrane Calcium-Transporting ATPases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases