Characterization of cationic amino acid transporter and its gene expression in rat hepatic stellate cells in relation to nitric oxide production.
OBJECTIVE
Nitric oxide is a potent mediator of hepatic sinusoidal hemodynamics and affects hepatic stellate cells (Ito cells, fat-storing cells). Although nitric oxide production may depend on the induction of inducible nitric oxide synthase and on transport of extracellular L-arginine, the precise mechanisms controlling nitric oxide production in stellate cells have not been well characterized.


METHODS
Using stellate cells prepared from the male Wistar rat, kinetic analysis of L-arginine transport and reverse transcription-polymerase chain reaction for cationic amino acid transporter were carried out. The effect of tumor necrosis factor-alpha and interferon-gamma on L-arginine transport, mRNA expression of cationic amino acid transporter and inducible nitric oxide synthase, and nitric oxide production of stellate cells was assessed.


RESULTS
The L-arginine transport system functioning in the transformed hepatic stellate cells was system y+, possibly mediated by cationic amino acid transporter-1 and cationic amino acid transporter-2B (Km approximately 50 microM). Tumor necrosis factor-alpha enhanced cationic amino acid transporter-2B mRNA expression and L-arginine transport, whereas cationic amino acid transporter-1 mRNA expression remained unchanged. Interferon-gamma induced the expression of inducible nitric oxide synthase mRNA without obvious changes in L-arginine transport. Interferon-gamma in combination with tumor necrosis factor-alpha induced nitric oxide production with an enhancement in cationic amino acid transporter-2B mRNA expression, inducible nitric oxide synthase mRNA expression, and L-arginine transport, while extracellular L-lysine competitively inhibited this nitric oxide production.


CONCLUSIONS
In transformed hepatic stellate cells, tumor necrosis factor-alpha and interferon-gamma have a crucial role in nitric oxide production, and extracellular L-arginine transport and inducible nitric oxide synthase expression are regulated in a differential cytokine-specific manner. As the estimated Km of L-arginine transporter in transformed hepatic stellate cells is very similar to the physiological L-arginine concentration in portal vein, we assume that increased portal L-arginine concentration may easily affect sinusoidal blood flow through enhancement of autocrine nitric oxide production in transformed hepatic stellate cells of diseased liver.
Ookawauchi
K
K
First Department of Medicine, Kochi Medical School, Nankoku, Japan.
Saibara
T
T
Yoshikawa
T
T
Chun-Lin
L
L
Hayashi
Y
Y
Hiroi
M
M
Enzan
H
H
Fukata
J
J
Onishi
S
S
D026923:Amino Acid Transport Systems, Basic; D000818:Animals; D001120:Arginine / Q000493:pharmacokinetics; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics; D002461:Cell Line, Transformed; D005786:Gene Expression Regulation / Q000502:physiology*; D007371:Interferon-gamma / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013237:Stereoisomerism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D026923:Amino Acid Transport Systems, Basic; D002352:Carrier Proteins; D008565:Membrane Proteins; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat