Thrombin stimulates vascular smooth muscle cell polyamine synthesis by inducing cationic amino acid transporter and ornithine decarboxylase gene expression.
Thrombin, a serine protease, is a potent mitogen for vascular smooth muscle cells (SMCs), but its mechanism of action is not known. Since L-ornithine is metabolized to growth-stimulatory polyamines, we examined whether thrombin regulates the transcellular transport and metabolism of L-ornithine by vascular SMCs. Treatment of SMCs with thrombin initially (0 to 2 hours) decreased L-ornithine uptake, whereas longer exposures (6 to 24 hours) progressively increased transport. Kinetic studies indicated that thrombin-induced inhibition was associated with a decrease in affinity for L-ornithine, whereas stimulation was mediated by an increase in transport capacity. Thrombin induced the expression of both cationic amino acid transporter (CAT)-1 and CAT-2 mRNA. Furthermore, thrombin stimulated L-ornithine metabolism by inducing ornithine decarboxylase (ODC) mRNA expression and activity. The stimulatory effect of thrombin on both L-ornithine transport and ODC activity was reversed by hirudin, a thrombin inhibitor, and was mimicked by a 14-amino acid thrombin receptor-activating peptide. Thrombin also markedly increased the capacity of SMCs to generate putrescine, a polyamine, from extracellular L-ornithine. The thrombin-mediated increase in putrescine production was reversed by N(G)-methyl-L-arginine, a competitive inhibitor of cationic amino acid transport, or by alpha-difluoromethylornithine (DFMO), an ODC inhibitor. DFMO also inhibited thrombin-induced SMC proliferation. These results demonstrate that thrombin stimulates polyamine synthesis by inducing CAT and ODC gene expression and that thrombin-stimulated SMC proliferation is dependent on polyamine formation. The ability of thrombin to upregulate L-ornithine transport and direct its metabolism to growth-stimulatory polyamines may contribute to postangioplasty restenosis and atherosclerotic lesion formation.
Houston VA Medical Center, Department of Medicine, Baylor College of Medicine, Tex 77030, USA.
W
W
Durante
L
L
Liao
K J
KJ
Peyton
A I
AI
Schafer
D026923:Amino Acid Transport Systems, Basic; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D000518:Eflornithine / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D009952:Ornithine / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000096:biosynthesis* / Q000235:genetics; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines / Q000378:metabolism*; D011700:Putrescine / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D013917:Thrombin / Q000494:pharmacology*; D019323:omega-N-Methylarginine / Q000494:pharmacology
D026923:Amino Acid Transport Systems, Basic; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009124:Muscle Proteins; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines; D012333:RNA, Messenger; D019323:omega-N-Methylarginine; D009952:Ornithine; D013917:Thrombin; D009955:Ornithine Decarboxylase; D011700:Putrescine; D000518:Eflornithine