A role for ornithine in the regulation of putrescine accumulation and ornithine decarboxylase activity in Reuber H35 hepatoma cells

Wu, Vincent; Byus, Craig V.

doi:10.1016/0167-4889(84)90102-2

Cited by 17 publications

(12 citation statements)

References 26 publications

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“…The addition of exogenous ornithine (0.5 mM) was accompanied by a 4-5-fold increase in intracellular steady-state ornithine levels and by a 6-8-fold increase in the presence of TPA and ornithine. In a manner identical to the serum-containing cultures (Wu and Byus (1984)) the addition of TPA and exogenous ornithine to the serum-free cells caused a dose-dependent increase in intracellular putrescine (up to 5-fold) and a concomitant decrease in ODC activity in comparison to stimulation with TPA alone. The addition of TPA led to a 3-5-fold increase in the incorporation of tritiated thymidine into DNA.…”

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confidence: 88%

“…In serum-containing cultures of Reuber H35 hepatoma cells, we have shown that the steady-state level of ornithine can markedly influence the level of ODC activity and polyamine accumulation following stimulation of these cells with the phorbolester tumor promoter TPA (Wu and Byus, 1984). Incubation of these cells with varying concentrations of extracellular ornithine led to a dose-dependent increase in the level of putrescine biosynthesis concomitant with a dose-dependent decrease in ODC activity.…”

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The level of substrate ornithine can alter polyamine‐dependent DNA synthesis following phorbolester stimulation of cultured hepatoma cells

Byus

1991

Journal Cellular Physiology

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Although the precise intracellular function(s) of the polyamines remain incompletely defined, a myraid of evidence now shows that the polyamines must accumulate or be maintained at a specific intracellular concentration in order for all mammalian cells to grow or divide. The initial step in polyamine biosynthesis normally involves the decarboxylation of ornithine by the enzyme ornithine decarboxylase (ODCase E.C. 4.1.1.17) to yield putrescine. Increases in the steady-state level of intracellular ornithine have been reported to markedly alter the accumulation of the polyamines following stimulation of Reuber H35 Hepatoma cells with 12-O-tetradecanoylphorbol-beta-acetate (TPA) in the presence of serum (Wu and Byus: (Biochem. Biophys. Acta 804:89-99, 1984); Wu et al.: (Cancer Res. 41:3384-3391, 1981). We wished to determine whether or not incubation of H35 hepatoma cells with exogenous ornithine would result in a stimulation of DNA synthesis following treatment with the mitogens TPA and insulin. For these studies, H35 cells were maintained under serum-free conditions for 2-3 days in order to obtain synchronous cultures suitable for analysis of the level of DNA synthesis. Cultures treated in this manner were highly viable, maintained similar growth rates, and possessed the equivalent levels of intracellular ornithine and polyamines as the serum-containing cultures. Arginine levels, however, were approximately twofold higher following culture under serum-restricted conditions for 3 days. The addition of exogenous ornithine (0.5 mM) was accompanied by a 4-5-fold increase in intracellular steady-state ornithine levels and by a 6-8-fold increase in the presence of TPA and ornithine. In a manner identical to the serum-containing cultures (Wu and Byus (1984] the addition of TPA and exogenous ornithine to the serum-free cells caused a dose-dependent increase in intracellular putrescine (up to 5-fold) and a concomitant decrease in ODC activity in comparison to stimulation with TPA alone. The addition of TPA led to a 3-5-fold increase in the incorporation of tritiated thymidine into DNA. In the presence of exogenous ornithine, TPA-induced DNA synthesis was further stimulated more than twofold in a dose-dependent manner. Insulin (10(-10)-10(-8) M) proved to be more efficacious as a mitogen in the H35 cells and led to greater stimulation of DNA synthesis than TPA. Insulin alone also resulted in a higher steady-state level of ornithine and putrescine in comparison with TPA alone.(ABSTRACT TRUNCATED AT 250 WORDS)

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The level of substrate ornithine can alter polyamine‐dependent DNA synthesis following phorbolester stimulation of cultured hepatoma cells

Byus

1991

Journal Cellular Physiology

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“…The steady-state intracellular level of L-ornithine markedly influences polyamine formation and DNA synthesis in neoplastic cells. 16,17 In addition, we have demonstrated that PDGF and lysophosphatidylcholineinduced polyamine synthesis and vascular SMC mitogenesis are dependent on the transcellular transport of L-ornithine. 12,18 The transport of cationic amino acids, such as L-ornithine, by vascular SMCs is mediated by the system y ϩ carrier.…”

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Thrombin Stimulates Vascular Smooth Muscle Cell Polyamine Synthesis by Inducing Cationic Amino Acid Transporter and Ornithine Decarboxylase Gene Expression

Durante

Liao

Peyton

et al. 1998

Circulation Research

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Abstract-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 ␣-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. (Circ Res. 1998;83:217-223.)

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“…Although this highly regulated inducible enzyme is often believed to be rate-limiting in the polyamine biosynthetic pathway, the availability of L-ornithine also plays a crucial role in regulating polyamine biosynthesis. Previous studies demonstrate that the steady state level of L-ornithine markedly influences polyamine formation in neoplastic cells (10,11). Moreover, exogenous L-ornithine administration further stimulates DNA synthesis in these cells, suggesting that the increase in DNA synthesis results from increased polyamine synthesis due to enhanced L-ornithine substrate availability (12).…”

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Platelet-derived Growth Factor Regulates Vascular Smooth Muscle Cell Proliferation by Inducing Cationic Amino Acid Transporter Gene Expression

Durante

Liao

Iftikhar

et al. 1996

Journal of Biological Chemistry

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Since recent studies demonstrated that platelet-derived growth factor (PDGF) induces vascular smooth muscle cell (SMC) proliferation by stimulating polyamine synthesis, we examined whether the transcellular transport of L-ornithine, the cationic amino acid precursor of polyamines, could regulate the mitogenic response of PDGF. Treatment of SMC with PDGF stimulated DNA and putrescine synthesis, and this was enhanced further by increasing the extracellular concentration of L-ornithine. The potentiating effect of Lornithine was reversed by the competitive inhibitor of cationic amino acid transport, methyl-L-arginine, or by preventing putrescine formation with ␣-difluoromethylornithine. Cationic amino acid uptake by SMC was Na ؉ -independent and was mediated by both a high and low affinity carrier system. Treatment of SMC with PDGF initially (0 -2 h) decreased basic amino acid transport, while longer exposures (6 -24 h) progressively increased uptake. Kinetic studies indicated that PDGF-induced inhibition was associated with a decrease in affinity for cationic amino acids, while the stimulation was mediated by an increase in transport capacity. Endogenous PDGF released by collagen-activated platelets likewise up-regulated cationic amino acid transport in SMC. Reverse transcriptase-polymerase chain reaction detected the presence of mRNA encoding two distinct cationic amino acid transporter (CAT) proteins, CAT-1 and CAT-2B. Treatment of SMC with PDGF strongly induced the expression CAT-2B mRNA and modestly elevated the level of CAT-1 mRNA. These results demonstrate that PDGF-induced polyamine synthesis and SMC mitogenesis are dependent on the transcellular transport of Lornithine. The capacity of PDGF to up-regulate the transport of L-ornithine by inducing the expression of the genes for CAT-1 and CAT-2B may modulate its mitogenic effect by providing SMC with the necessary intracellular precursor for polyamine biosynthesis.

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A role for ornithine in the regulation of putrescine accumulation and ornithine decarboxylase activity in Reuber H35 hepatoma cells

Cited by 17 publications

References 26 publications

The level of substrate ornithine can alter polyamine‐dependent DNA synthesis following phorbolester stimulation of cultured hepatoma cells

The level of substrate ornithine can alter polyamine‐dependent DNA synthesis following phorbolester stimulation of cultured hepatoma cells

Thrombin Stimulates Vascular Smooth Muscle Cell Polyamine Synthesis by Inducing Cationic Amino Acid Transporter and Ornithine Decarboxylase Gene Expression

Platelet-derived Growth Factor Regulates Vascular Smooth Muscle Cell Proliferation by Inducing Cationic Amino Acid Transporter Gene Expression

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