Endotoxin stimulates lymphocyte glutaminase expression.
OBJECTIVE
Glutamine is the principal fuel used by lymphocytes. It is hydrolyzed by the glutaminase enzyme, which regulates the rate of intracellular glutamine metabolism. Since lymphocyte glutamine utilization is increased during infection to support cellular proliferation, we hypothesized that endotoxin regulates lymphocyte glutaminase expression at the molecular level.


METHODS
Adult rats received Escherichia coli endotoxin (one dose of 7.5 mg/kg) or saline. Total RNA from lymphocytes in the ileocolic lymph node chain was extracted for Northern hybridization and labeled with an alpha-phosphorus 32 rat glutaminase cDNA probe. The mRNA of the constitutively expressed gene beta-actin was the control for RNA loading. Quantitation of glutaminase transcripts was determined by densitometric scanning and values were normalized to actin. Glutaminase-specific activity (nanomoles per milligram of protein per hour) and glutaminase kinetic parameters were also determined.


RESULTS
Treatment with a single dose of endotoxin resulted in a 53% increase in glutaminase activity at 4 hours. Kinetic analysis showed that the increase in glutaminase activity was due to an 84% increase in Vmax (maximal enzyme velocity) with no change in Km (enzyme affinity). Endotoxin increased glutaminase mRNA twofold at 2 hours and more than fourfold at 4 hours. The increase in message preceded the increase in activity consistent with gene transcription prior to enzyme biosynthesis.


CONCLUSIONS
The increase in glutaminase activity provides lymphocytes in the mesenteric lymph nodes with more glutamine for energy and cellular proliferation during times of infection when the gut mucosal barrier may become compromised.
Sarantos
P
P
Department of Surgery, University of Florida College of Medicine, Gainesville.
Ockert
K
K
Souba
W W
WW
D000818:Animals; D004731:Endotoxins / Q000494:pharmacology*; D004926:Escherichia coli; D005972:Glutaminase / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D008198:Lymph Nodes / Q000187:drug effects / Q000201:enzymology; D008214:Lymphocytes / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D004731:Endotoxins; D012333:RNA, Messenger; D005972:Glutaminase