Involvement of polyamines in epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and -beta 1 action on culture of L6 and fetal bovine myoblasts.

1. alpha-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase significantly abolished stimulation of protein synthesis evoked by EGF, TGF-alpha or -beta 1 in L6 and fetal bovine myoblasts. 2. The participation of polyamines in early events evoked by growth factors was shown by a significant stimulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase activity as well as increased concentration of spermidine and spermine in L6 cells exposed to TGF-alpha and EGF. 3. TGF-beta 1 at a high concentration (1 ng/ml) increased protein synthesis in L6 myoblasts but inhibited it in fetal bovine myoblasts. Metabolic effects of TGF-beta 1 in L6 cells was associated with an enhancement of decarboxylase activities, however there were no significant changes in cellular polyamine concentrations. Presented data suggest that polyamines are involved in the signal transduction pathway of EGF, TGF-alpha, and -beta 1 in L6 and fetal bovine myoblasts.

TGF-beta 1 inhibits polyamine biosynthesis in K 562 leukemic cells.

The present study proved that TGF-beta 1 significantly inhibited the growth of K 562 cells. The drop in cell numbers after 24 h incubation with increasing concentrations of TGF-beta 1 (0.01, 0.1, 1.0, 10.0 ng/ml) was accompanied by significant suppression of the activity of two key enzymes of polyamine biosynthesis: ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). In contrast to ODC and SAMDC activity, TGF-beta 1 did not significantly affect the absolute concentration of spermidine and spermine in K 562 cells. We suppose that the lack of an evident drop in concentration of spermidine and spermine in spite of a significant decrease in ODC and SAMDC activity in K 562 cells exposed to TGF-beta 1 resulted from the uptake of polyamines from the extracellular space.

Metabolic effect of orotic acid in calves.

Eight calves (males, Black and White crossbred with Holstein-Fresian) were fed milk and milk replacer without (control group) or with potassium orotate (3 mmol./l.) supplementation for 6 weeks after birth. Orotate depressed the biosynthesis of polyamines in mucosa of the gastrointestinal tract (rumen, omasum, abomasum, colon) by decreasing of ornithine decarboxylase activity with a simultaneous compensatory increase of S-adenosyl-methionine decarboxylase activity. A lower concentration of spermidine and spermine in the mucosa of the colon was also noted. The above changes were accompanied by increased urinary excretion of ornithine and arginine. Calf adaptation to a high OA intake was associated with an increased activity of the OA metabolizing enzyme complex (orotate phosphoribosyl transferase and orotidine monophosphate decarboxylase) in the liver, while urinary OA losses diminished with age. Increased concentrations of uracil and uridine in the liver and higher urinary excretion of pseudouridine in OA-fed calves was also observed. Stimulation of pyrimidine metabolism by OA depressed purine synthesis, which was reflected by a decrease of urate, hypoxanthine, and xanthine concentration in the liver. Interestingly OA enhanced urate excretion by the kidneys. OA strongly affected lipid metabolism in calves because total cholesterol, LDL-, and HDL-cholesterol, and triglycerides in blood plasma decreased while triglycerides accumulated in the liver of OA-fed calves. Milk OA in concentrations characteristic of cows with hereditary orotic aciduria exerts an unfavourable effect on the metabolism of polyamines, purines, and lipids in calf tissues.

Comparison of metabolic effects of EGF, TGF-alpha, and TGF-beta 1 in primary culture of fetal bovine myoblasts and rat L6 myoblasts.

1. Comparative studies of EGF, TGF-alpha, and TGF-beta 1 action on the synthesis of DNA and cellular proteins in rat L6 myogenic cells and fetal bovine myoblasts demonstrated considerable differences between particular growth factors, dependent on dose and target cells. 2. Among examined growth factors only EGF exerted mitostimulatory action, more pronounced at lower concentrations. EGF, progressively with dose, stimulated protein synthesis much more effectively in fetal bovine myoblasts than in L6 cells. 3. The dynamics of stimulation of protein synthesis by TGF-alpha was greater than by EGF in both examined types of cell cultures. 4. The maximal response of fetal bovine myoblasts to TGF-alpha in a concentration of 100 ng/ml reached 370%, whereas EGF in a 10 times higher concentration stimulated protein synthesis only to 123% of control. 5. In contrast to EGF, TGF-alpha significantly inhibits DNA synthesis. Inhibition of the mitogenic response with simultaneous stimulation of protein synthesis by TGF-alpha may indicate changes toward cell differentiation. 6. TGF-beta 1 in smallest concentration inhibits both DNA and protein synthesis. The suppressive action of TGF-beta 1 was more distinct in fetal bovine myoblasts than in the L6 cell line. 7. Increasing concentrations of TGF-beta 1 diminished its inhibitory effect, even leading to stimulation of protein synthesis at higher doses in L6 myoblasts.

Metabolic effect of orotic acid in rat L6 myoblasts.

Orotic acid (OA) is an intermediate in the pyrimidine pathway. The main source of OA in the human and animal diet is bovine milk and its products. OA significantly inhibited the stimulation of protein synthesis by FCS derived growth factors in L6 myoblasts. An increasing OA concentration (0.001 mM, 0.01 mM, and 0.1 mM) in a medium containing 2% FCS decreased the proliferation of L6 myoblasts (as measured by the number of cells) as well as the incorporation of traced thymidine into cellular DNA after 24 h incubation. This mitoinhibitory effect was accompanied by a significant reduction of ornithine decarboxylase (ODC) activity, an enzyme which is believed to be a valuable index of cell proliferation. A drop in the cytosol spermine level of L6 myoblasts treated with OA also occurred. A simultaneous, significant, compensative increase of S-adenosylmethionine decarboxylase (SAMDC) activity was noted. The addition of putrescine (2 microM), a product of ODC activity, abolished the depressional influence of OA on protein synthesis in L6 myoblasts, thus confirming its interference with the polyamine pathway. Dibutyryl-cAMP (0.05-0.2 mM) or adenine (25 microM) supplementation, regardless of OA concentration, significantly attenuated the mitoinhibitory effect and its inhibitory action on protein synthesis. This may suggest the existence of a purine deficiency in OA-treated myoblasts. These experiments indicate that increasing cellular OA concentration inhibits cell growth probably by disturbances in the chain of early events (synthesis of cyclic purine mononucleotides, and activity of the ODC/polyamine system), normally evoked by growth stimulating factors.

Mitogenic effect of lymph, amniotic and allantoic fluid in bovine fetal myoblasts.

Amniotic and allantoic fluids, as well as afferent lymph (tissue fluid), stimulated DNA synthesis in bovine fetal myoblasts (BFM) at low concentrations (5%). Higher doses abolished the mitostimulatory effect of these fluids and led toward inhibition of [3H]mTdR incorporation into DNA. These effects were much more evident (particularly in the case of amniotic fluid) when fluids were lyophylized prior to the experiment. BFM protein synthesis was accelerated by lymph in a dose dependent manner.