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

We investigated the ability of intracellular ornithine to alter both the biosynthesis of putrescine and the activity of ornithine decarboxylase in Reuber H35 hepatoma cells in culture incubated with 12-O- tetrade - canoylphorbol 13-acetate (TPA). In confluent cultures of H35 cells, the addition of TPA (1.6 microM) caused the activity of ornithine decarboxylase to increase by more than 100-fold within 4 h. When exogenous ornithine (0.1-1.0 mM) was added to the culture medium with TPA, a marked dose-dependent increase in the production of putrescine was observed. The activity of ornithine decarboxylase in the same cultures incubated with ornithine decreased in a similar dose-dependent manner. The addition of arginine (0.1-1.0 mM) (but not lysine or histidine) to the H35 cells in culture concomitant with TPA also led to a relative increase in putrescine biosynthesis and a decrease in ornithine decarboxylase activity compared to cultures not receiving the amino acids. A similar response to exogenous ornithine and TPA was observed in a series of less confluent rapidly growing cultures which were in culture for a shorter period of time. The confluent cultures possessed a basal level of arginase (55 units/mg protein) which increased approx. 2-fold upon treatment with TPA. The intracellular concentration of ornithine in the unstimulated cells was in the order of 0.02-0.03 mM. Upon incubation of the cells with exogenous ornithine or arginine, the intracellular pools of these amino acids increased 4- to 8-fold.

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

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)

Increased expression of transforming growth factor alpha precursors in acute experimental colitis in rats.

BACKGROUND AND AIM: Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), members of the EGF family of growth factors, protect rat gastric and colonic mucosa against injury. Having shown previously that exogenously applied EGF protects rat colonic mucosa against injury, the aim of the present study was to evaluate the endogenously expressed ligand mediating the protective effect of EGF/TGF-alpha in vivo. METHODS: In an experimental model of trinitrobenzene sulphonic acid (TNBS)/ethanol induced colitis in rats EGF and TGF-alpha expression was evaluated using a ribonuclease protection assay, northern blot analysis, western blot analysis, and immunohistochemistry. RESULTS: TGF-alpha mRNA increased 3-4 times at 4-8 hours after induction of colitis and returned to control levels within 24 hours. TGF-alpha immunoreactive protein with a molecular size of about 28 kDa representing TGF-alpha precursors increased markedly after induction of colitis with a peak at 8-12 hours. No fully processed 5.6 kDa TGF-alpha protein was detected in normal or inflamed colon tissue. Only a weak signal for EGF mRNA expression was detected in the rat colon and no EGF protein was observed by immunohistochemistry or western blot analysis. CONCLUSIONS: TGF-alpha precursors are the main ligands for the EGF receptor in acute colitis. It is hypothesised that TGF-alpha precursors convey the biological activity of endogenous TGF-alpha peptides during mucosal defence and repair.

Increased expression of epidermal growth factor-receptor in an experimental model of colitis in rats.

BACKGROUND: Epidermal growth factor and related proteins share some structural homology and bind to one common receptor. We have shown previously that exogenously applied EGF protects colonic mucosa against injury in an experimental model of colitis in rats and that the endogenously expressed ligands for the EGF-receptor are predominantly transforming growth factor alpha precursors. The aim of our present study was to evaluate the EGF-receptor expression in response to mucosal injury in the same model of colitis. METHODS: The trinitrobenzene sulphonic acid (TNBS)/ethanol-induced model of colitis in rats was used and EGF-receptor expression was evaluated using ribonuclease protection assay and Western blot analysis. The extent of mucosal injury and inflammation was characterized by using a microscopic and macroscopic damage score and by estimation of the myeloperoxidase activity in colonic specimens. RESULTS: Irritation of the colonic mucosa leads to severe colonic inflammation with tissue oedema, erosions and mucosal ulcers and to a significant increase in myeloperoxidase activity expressed by neutrophil granulocytes and macrophages. A significant increase in EGF-receptor mRNA expression was obtained at 8-24 h followed by an increased expression of the EGF-receptor protein at 1-5 days after the induction of colitis. On Western blot analysis only one immunoreactive band with a molecular weight of approximately 170 kDa was detected. CONCLUSIONS: Mucosal inflammation leads to a significant increase in the EGF-receptor expression in the early phases of colitis. These findings support our hypothesis that EGF and related proteins and their common receptor play a pivotal role in mucosal defence and repair.