Patrizia Tunici

Diamine and polyamine oxidase activities in phytohaemagglutinin-induced growth of rat small intestine

The activities of diamine and polyamine oxidases, two enzymes of polyamine catabolism, were studied in hyperplastic growth of rat small intestine induced by phytohaemagglutinin. This growth, evaluated by the elongation of Lieberkühns crypts, was more extensive in the proximal than in the distal parts of the gut. The activity of diamine oxidase was significantly reduced in the proximal (70%), medial (45%) and the distal (25%) parts. The activity of polyamine oxidase was doubled. The concentrations of putrescine, cadaverine and spermidine were significantly elevated in the three intestinal parts studied, whereas those of histamine and spermine were unchanged. It appears that changes in the activities of diamine and polyamine oxidases may contribute to the increased putrescine content, which is necessary to maintain active polyamine turnover for sustaining growth of the gut.

Transglutaminase activity in rat liver after acute ethanol administration.

The acute effect of ethanol on hepatic transglutaminase (EC 2.3.2.13) activity and polyamine levels were investigated in the rat. A high dose of ethanol (5 g/kg body weight, given by gastric intubation) caused in homogenate and cytosolic fraction an inhibition of 50-70% from 3 to 24 h which thereafter was reversible. Such a decrease may be in part responsible for the observed enhancement in putrescine and spermidine contents observed at the same times. Pyrazole, an inhibitor of alcohol dehydrogenase, prevented the ethanol-induced reduction in transglutaminase activity. Disulfiram, an inhibitor of aldehyde dehydrogenase, allowed detection of an inhibitory effect on enzyme activity even at a low dose of ethanol (2 g/kg), which per se did not modify transglutaminase activity. The hepatic cytosolic fraction, incubated in the presence of various concentrations of acetaldehyde, showed a dose-dependent inhibition of transglutaminase activity. All of these results suggest that acetaldehyde, the first and toxic metabolite of ethanol, inhibits hepatic transglutaminase activity, probably by its binding to the active thiol site of the enzyme. The reduction in transglutaminase may lead to an alteration of cytoskeleton, since the enzyme is known to be involved in tubuline polymerization and microfilament assembly.

POLYAMINE OXIDASE AND TISSUE TRANSGLUTAMINASE ACTIVATION IN RAT SMALL INTESTINE BY POLYAMINES

Polyamine degradation was studied in the small intestine from rats fed on a polyamine-supplemented diet. Lactalbumin diet was given to Hooded-Lister rats, with or without 5 mg rat(-1) day(-1) of putrescine or spermidine for 5 days. Polyamine oxidase activity increased with putrescine and spermidine in the diet, whereas spermidine/spermine N(1)-acetyltransferase and diamine oxidase activities were unchanged. We also studied the calcium-dependent and -independent tissue transglutaminase activities, since they can modulate intestinal polyamine levels. Both types of enzymes increased in the cytosolic fraction after putrescine (about 65%) or spermidine (80-100%). Our results indicate that exogenous polyamines stimulate intestinal polyamine oxidase and tissue transglutaminase activities, probably to prevent polyamine accumulation, when other pathways of polyamine catabolism (acetylation and terminal catabolism) are not activated.

Aging and polyamine acetylation in rat kidney

The acetylation of polyamines was investigated in rat kidney as a function of age. The activity of cytosolic spermidine/spermine N1-acetyltransferase, the rate-limiting enzyme in polyamine interconversion, increased from 3 to 36 months of age. The activity of cytosolic spermidine N8-acetyltransferase, an enzyme probably related to polyamine excretion, also increased. The activity of polyamine oxidase, which catalyzes the oxidative cleavage of polyamine N1-acetyl derivatives into putrescine, decreased until 24 months, when an accumulation of N1-acetylspermidine occurred. Subsequently, at 36 months, polyamine oxidase activity returned toward high values, in concomitance with the disappearance of N1-acetylspermidine, an increase in spermidine and putrescine, and a decline in spermine was observed. Our results show that in rat kidney during aging there is an activation of the acetylation and interconversion of higher polyamines into putrescine, which is considered an alternative pathway of spermidine and putrescine formation.

Distribution and activity of transglutaminase in rat brain carcinogenesis and in gliomas

Tissue transglutaminase is a calcium-dependent enzyme which may influence cell morphology, cytoskeletal processes and membrane functions. During rat brain carcinogenesis induced by transplacental administration of N-ethyl-N-nitrosourea to BD IX rats, cytosolic tissue transglutaminase activity was increased by about 140% at 30 days of extrauterine life and returned towards the control values at 3-5 months. In the particulate fraction, enzyme activity progressively increased, reaching values similar to those present in the developed gliomas. Tissue transglutaminase activity in gliomas had a behavior inverse to that observed in controls, with a decrease (about 50%) in the cytosol and a marked increase (380%) in the particulate fraction, indicating a redistribution of enzyme activity.

Response of intestinal transglutaminase activity to dietary phytohaemagglutinin

The behaviour of the activity of tissue transglutaminase, a calcium-dependent enzyme, and the levels of polyamines which are physiological substrates for the enzyme, were studied in rat small intestine induced to grow by lectin phytohaemagglutinin. Transglutaminase activity greatly increased in the homogenates and the cytosolic fractions of the intestinal mucosa of lectin-treated rats compared to that of untreated animals. The measurement of enzyme activity in the presence of monodansylcadaverine, a competitive inhibitor of transglutaminase, testified that the assayed enzyme activity was authentic transglutaminase. As regards polyamines, the level of spermine did not change, whereas putrescine and spermidine contents were enhanced. The activation of transglutaminase, which was probably due to Ca2+ accumulation in enterocytes, could have a role in maintaining enterocyte adhesion and intestinal cell homeostasis, and/or repairing lectin-induced damages of microvilli of the gut epithelium.

Oxidative degradation of polyamines in rat pancreatic hypertrophy.

In the hypertrophic pancreas, we studied the oxidative degradation of polyamines, which are endogenous polycations important for cell division, growth and differentiation. To induce pancreatic hypertrophy, rats were fed on a semi-synthetic diet containing a daily dose of 42 mg phytohaemagglutinin per rat for 5 or 10 days. In the model, the activities of polyamine oxidase (the enzyme that degrades spermidine, spermine and mainly their acetyl derivatives) and diamine oxidase (the key enzyme of terminal catabolism of polyamines in vivo) increased by 100-180% and 90-100%, respectively, parallel to an elevation in polyamine content (40-100%). The results suggest that in pancreas hypertrophy, which does not exhibit stimulation of spermidine/spermine N1-acetyltransferase activity, increases in the activity of polyamine and diamine oxidases are related events that lead to putrescine formation and removal of excess polyamines.

Polyamine acetylation in rat brain during N-ethyl-N-nitrosourea-induced cerebral carcinogenesis

The behavior of cerebral polyamine acetylation was examined in rat brain during N-ethyl-N-nitrosourea-induced carcinogenesis. Before tumor development, treated brains exhibited an enhancement in cytosolic spermidine/spermine N1-acetyl-transferase activity with concomitant accumulation of N1-acetylspermidine and increases in putrescine and spermidine. This indicates a stimulation of the interconversion pathway of polyamines into putrescine, an important molecule for cell growth. Our data also show the presence of a cytosolic spermidine N8-acetyltransferase activity in fetal rat brain, with values similar to those previously observed in gliomas. The detection of cytosolic spermidine N8-acetyltransferase activity in tumors may thus represent the expression of a fetal gene that does not seem to have a particular function during carcinogenesis.