The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity.
In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.
Franchi-Gazzola
Renata
R
Dipartimento di Medicina Sperimentale, Sezione di Patologia Generale e Clinica, Università degli Studi di Parma, Via Volturno 39, 43100 Parma, Italy. renata.franchi@unipr.it
Gaccioli
Francesca
F
Bevilacqua
Elena
E
Visigalli
Rossana
R
Dall'Asta
Valeria
V
Sala
Roberto
R
Varoqui
Hélène
H
Erickson
Jeffrey D
JD
Gazzola
Gian C
GC
Bussolati
Ovidio
O
D026924:Amino Acid Transport System A / Q000138:chemical synthesis* / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D019921:Biotinylation; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology*; D007150:Immunohistochemistry; D007700:Kinetics; D008970:Molecular Weight; D010758:Phosphorus / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D011868:Radioisotopes; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000187:drug effects
D026924:Amino Acid Transport System A; D006982:Hypertonic Solutions; D012333:RNA, Messenger; D011868:Radioisotopes; D010758:Phosphorus; D004004:Dichlororibofuranosylbenzimidazole; D011392:Proline