H. Pösö

Sensitive Enzymic determination of methylglyoxal bis(guanylhydrazone) in cultured cells and in animal tissues

Methylglyoxal bis(guanylhydrazone) (MGBG) is an antiproliferative compound used clinically in cancer chemotherapy [l-3]. Earlier the mechanism of action of MGBG was often related to the metabolism of polyamines, especially because its antiproliferative effects were counteracted by spermidine 141: it was only in [5] that MGBG was shown to be a remarkably potent and specific inhibitor of eukaryotic S-adenosylL-methionine decarboxylase (EC 4.1 .1.50), an enzyme directly involved in the synthesis of spermidine and spermine. Since then the compound has been widely used to produce intracellular polyamine deprivation in a number of experimental systems including cultured cells and animal tissues (for references see [6]). In all systems employed, the growth-inhibitory effect of MGBG can be prevented by PM levels of spermidine and spermine [6]. This finding has been taken as evidence indicating that the antiproliferative action exerted by MGBG is mediated through an inhibition of intracellular accumulation of spermidine and spermine. However, a straightforward interpretation of these studies is complicated by the fact that MGBG, spermidine and spermine appear to compete for a common cellular uptake system [7,8]. It is thus possible that the prevention of MGBG-induced toxicity by polyamines could partly result from a reduction of the intracellular concentration of the drug, and is not necessarily due to a normalization of cellular polyamine pattern. With the possible exception of a few studies [9 ,101, the effect of polyamines on the intracellular concentrations of MGBG has not been checked properly. This apparently is related to the lack of sensitive methods for determination of

Stabilization of ornithine decarboxylase in rat liver.

The response of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity to cyloheximide has been measured in rat liver after various stimuli known to enhance the enzyme activity. While a single injection of cycloheximide almost totally (> 95%) inhibited the activity of ornithine decarboxylase in livers of growth-hormone treated rats at 45 min after the administration of the drug, this inhibition was much less striking in livers of partially hepatectomized (80%), of thioacetamide-treated (60%) and especially of carbon-tetrachloride treated (15%) animals thus suggesting a stabilization of the enzyme after the latter treatments. The measurement of the apparent half-life of ornithine decarboxylase after cycloheximide revealed a substantial decrease in the decay rate of the enzyme in livers of thioacetamide-treated rats. The apparent half-life of S-adenylsyl-l-methionine decarboxylase (EC 4.1.1.50) likewise appeared to be longer in rats previously treated with thioacetamide or carbon tetrachloride than in partially hepatctomized animals. Injection of thioacetamide also resulted in an initial stabilization of liver ornithine decarboxylase against 1,3-diaminopropane, a potent indirect inhibitor of the enzyme. However, 30 min after diaminopropane injection the enzyme activity started to fall extremely rapidly. This sudden drop in ornithine decarboxylase activity following diaminopropane could be largely prevented with cycloheximide. This finding clearly indicates that a protein-synthesis dependent formation of macromolecular inhibitors of ornithine decarboxylase may also be an early event after amine treatments. The striking stabilization of ornithine decarboxylase activity following these treatments, which are known to produce an initial liver necrosis (especially carbon tetrachloride and thioacetamide), may indicate that the system(s) responsible for the rapid inactivation of liver ornithine decarboxylase is more sensitive to tissue necrosis than the enzyme itself.

Inhibition of ornithine decarboxylase by diamines in regenerating rat liver. Evidence for direct action on the accumulation of the enzyme protein.

The regulation of mammalian ornithine decarboxylase (EC 4.1 .l .17) in vivo by natural polyamines (putrescine, spermidine and spermine) as well as by structurally related but unphysiologic amines (1,3-diaminopropane) appears to involve several distinctly different mechanisms [l-5]. The prevention of ornithine decarboxylase induction by putrescine and diaminopropane [2,6] in rat liver immediately after partial hepatectomy resembles the action achieved with inhibitors of RNA synthesis [6] whereas, during later periods of liver regeneration, the administration of the diamines results in an extremely swift decay of ornithine decarboxylase activity, suggesting a direct translational inhibition [2,5,6]. Another mechanism, fundamentally different from the concept of ‘classic repression’, has been recently advanced by E. S. Canellakis et al. [7,8]. According to their observations, it appears that the administration of amines in vivo [8] or in vitro in cell cultures [7] results in an appearance of macromolecular inhibitors of ornithine decarboxylase. The formation of such nondialyzable inhibitors after application of putrescine in H-35 hepatoma cells has been confirmed by Clark and Fuller [9]. However, they were unable to detect similar inhibitors in normal or transformed Swiss 3T3 cells or cultured human fibroblasts after exposure of the cells to putrescine [9]. We have likewise been unable to obtain’ any convincing evidence that the swift decay of ornithine decarboxylase in regenerating rat liver after injection of diamines would be based

Inhibition of prereplicative polyamine accumulation in regenerating rat liver

Abstract Injections of 1,3-diaminopropane, an inhibitor of mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, during the first 10 h after partial hepatectomy markedly delayed the stimulation of liver DNA synthesis from [3H]thymidine normally occurring at the beginning of second day of liver regeneration. Inhibition of ornithine decarboxylase by repeated injections (every 2 h) of diaminopropane for 10 h after partial hepatectomy abolished the enhancement in DNA synthesis at 20 h postoperatively, where shorter periods of postoperative diaminopropane treatment resulted in less complete prevention of the synthesis of DNA. Under these experimental conditions the rate of liver DNA synthesis in vivo from [3H]thymidine showed a highly significant positive correlation with the concentration of tissue spermidine but not with that of putrescine or spermine. It was also possible to prevent the accumulation of spermidine and to depress liver DNA synthesis at 24 h postoperatively by starting the treatment with diaminopropane after varying lag periods following partial hepatectomy. Treatment with diaminopropane started as late as at 12 h after partial hepatectomy still prevented any accumulation of liver spermidine and resulted in a profound inhibition (about 85%) of DNA synthesis at 24 h postoperatively. The inhibition of DNA synthesis was gradually subsiding when the commencement of the amine treatment was moved more far from the time of the operation.

Regulation of ornithine decarboxylase by diamines in regenerating rat liver.

1. Introduction Unlike the inducible enzymes in bacteria, many of the mammalian enzymes, which regulate the flow of substrates through metabolic pathways rapidly chang ing their activity in response to a variety of stimuli, are labile proteins with short molecular half-lives [

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo.

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activity in rat kidney. Administration of diaminopropane 60 min before partial hepatectomy only marginally inhibited ornithine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant. An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed S-adenosyl-L-methionine decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy. Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals. Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life. Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [14C]methionine 4 h after partial hepatectomy or after administration of porcine growth hormone. Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornithine decarboxylase activity and spermidine synthesis.

Inhibition of ornithine decarboxylase in regenerating rat liver by acute ethanol treatment

Abstract The activity of ornithine decarboxylase (EC 4.1.1.17) in the liver was significantly inhibited by gastric intubation of ethanol 4 h after partial hepatectomy. This inhibition was apparent only when ethanol was administered 1 h before, at the same time, or 1 h after the operation Similarly, ethanol inhibited ornithine decarboxylase 24 h after partial hepatectomy if the treatment was continued for 5 h. When ethanol was given 1 to 2 h before the death of the animal it only marginally inhibited the ornithine decarboxylase. In the kidneys of operated rats the ornithine decarboxylase is greatly stimulated and this effect was also inhibited by ethanol. The time dependence of ethanol inhibition in the kidneys was similar to that in the liver. In contrast to ornithine decarboxylase the treatment with ethanol did not inhibit the activity of alanine aminotransferase (EC 2.6.1.2), used as a control enzyme of long half-life, either in liver or in kidney. By using inhibitors of alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) it was shown that the accumulation of acetaldehyde or the change in the free NADH free NAD + ratio was not necessary for the inhibition of ornithine decarboxylase to occur. Thus it appears likely that ethanol itself can inhibit the synthesis of new protein in regenerating rat liver. Although the exact mechanism of the effects of ethanol on protein synthesis remains to be determined our results suggest that regenerating rat liver is a good model to study the inhibitory effect(s) of alcohol on protein synthesis.

Relationship between the phosphorylation state and the rate of ethanol elimination in regenerating rat liver.

It has been suggested that ATP consunlption by affecting the rate of NADH reoxidation in the respiratory chain may influence the rate of ethanol elimination [ 1,2] . On the other hand, the phosphorylation state (ATP/ADP X Pi) of the liver cytoplasm rather than concentration of ATP or ADP, has been claimed to control the activity of the respiratory chain (3,4]. We made an experimental study of this supposed relationship between the phosphorylation state and the rate of ethanol elimination using partially hepatectomized rats. In regenerating rat liver the activity of the respiratory chain is enhanced [S] and ethanol elimination rate is higher than in u~loperated control rats [6]. It was found that the changes in ethanol elimination rate corresponded to those in phosphorylation state. This suggests that the part played by phosphorylation state of the liver cytoplasm in regulation of the rate of ethanol elinli~~ation is important.

Inhibition of rat ovarian ornithine decarboxylase by ethanol in vivo and in vitro

Intragastric administration of ethanol greatly inhibited ovarian ornithine decarboxylase (L-ornithine carboxy-lyase EC 4.1.1.17) stimulated by human chorionic gonadotropin in vivo. The inhibition occurred only if the treatment with ethanol was started before the injection of hormone. The use of inhibitors for alcohol dehydrogenase and aldehyde dehydrogenase clearly showed that the observed inhibition was a direct effect of ethanol itself. When rat ovarian cells were incubated in vitro with human chorionic gonadotropin the activity of ornithine decarboxylase was also markedly stimulated. This stimulation could also be inhibited by ethanol. Moreover, actinomycin D and alpha-amanitin inhibited the stimulation of ornithine decarboxylase, showing that the enhanced activity in vitro resulted from the synthesis of new mRNA for ornithine decarboxylase. The time dependence of the inhibition caused by ethanol addition resembled that after addition of actinomycin D. This supports the view that one site where ethanol inhibits protein synthesis is at the transcriptional level.

Inhibition by aliphatic alcohols of the stimulated activity of ornithine decarboxylase and tyrosine aminotransferase occurring in regenerating rat liver

Abstract Inhibition of the synthesis of ornithine decarboxylase and tyrosine aminotransferase by ethanol, 1-propanol, 2-propanol and 2-methyl-2-propanol was studied in partially hepatectomized rats. The inhibition by ethanol appeared to have a direct dependence upon the dose. Other alcohols tested were even more efficient inhibitors than ethanol of the stimulation of ornithine decarboxylase and tyrosine aminotransferase, in regenerating liver remnant after partial hepatectomy. In addition, the stress of the operation was found to stimulate the activity of ornithine decarboxylase in the kidneys and to a lesser extent in the brain. In kidneys, but not in the brain, ethanol inhibited the increase in the activity of ornithine decarboxylase in a dose-dependent manner. 2-Methyl-2-propanol, similarly to the higher dose of ethanol, inhibited ornithine decarboxylase in the kidneys, whereas other alcohols appeared to have no effect. Since 2-propanol and 2-methyl-2-propanol do not produce aldehydes in the liver or change the NADH/NAD + redox state, the results suggest that the synthesis of ornithine decarboxylase and tyrosine aminotransferase was inhibited by ethanol molecules. The dose dependence of the ethanol inhibition, both in the liver and kidneys, also supports this suggestion.