Growth conditions differentially regulate the expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured neurons.
We have studied the expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K+ (10, 25, or 40 mM) with or without 100 microM N-methyl-D-aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mM K+ (K10) and increased dramatically in cultures grown in 25 mM K+ (K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 + NMDA). In cultures grown in 40 mM K+ (K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and -3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA-stimulated 45Ca2+ influx and 4 beta-[3H]phorbol 12,13-dibutyrate binding in intact cells. Both functional parameters increased along with the K+ concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits.(ABSTRACT TRUNCATED AT 250 WORDS)
Condorelli
D F
DF
Institute of Biochemistry, University of Catania, Italy.
Dell'Albani
P
P
Aronica
E
E
Genazzani
A A
AA
Casabona
G
G
Corsaro
M
M
Balázs
R
R
Nicoletti
F
F
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016202:N-Methylaspartate / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D016376:Oligonucleotides, Antisense; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*; D051381:Rats; D018091:Receptors, AMPA / Q000096:biosynthesis*; D017470:Receptors, Glutamate / Q000032:analysis / Q000096:biosynthesis; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D046911:Macromolecular Substances; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D015240:Phorbol 12,13-Dibutyrate; D016202:N-Methylaspartate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D011188:Potassium; D002118:Calcium