Agmatine transport in brain mitochondria: a different mechanism from that in liver mitochondria.

The diamine agmatine (AGM), exhibiting two positive charges at physiological pH, is transported into rat brain mitochondria (RBM) by an electrophoretic mechanism, requiring high membrane potential values and exhibiting a marked non-ohmic force-flux relationship. The mechanism of this transport apparently resembles that observed in rat liver mitochondria (RLM), but there are several characteristics that strongly suggest the presence of a different transporter of agmatine in RBM. In this type of mitochondria, the extent of initial binding and total accumulation is higher and lower, respectively, than that in liver; saturation kinetics and the flux-voltage relationship also exhibit different trends, whereas idazoxan and putrescine, ineffective in RLM, act as inhibitors. The characteristics of agmatine uptake in RBM lead to the conclusion that its transporter is a channel with two asymmetric energy barriers, showing some characteristics similar to those of the imidazoline receptor I(2) and the sharing with the polyamine transporter.

Human mesenchymal stem cells as a two-edged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential.

BACKGROUND AND AIMS: Mesenchymal stem cells from bone marrow (MSCs) may have the potential to differentiate in vitro and in vivo into hepatocytes. We investigated whether transplanted human MSCs (hMSCs) may engraft the liver of non-obese diabetic severe combined immuno-deficient (NOD/SCID) mice and differentiate into cells of hepatic lineage. METHODS: Ex vivo expanded, highly purified and functionally active hMSCs from bone marrow were transplanted (caudal vein) in sublethally irradiated NOD/SCID mice that were either exposed or not to acute liver injury or submitted to a protocol of chronic injury (single or chronic intraperitoneal injection of CCl(4), respectively). Chimeric livers were analysed for expression of human transcripts and antigens. RESULTS: Liver engraftment of cells of human origin was very low in normal and acutely injured NOD/SCID mice with significantly higher numbers found in chronically injured livers. However, hepatocellular differentiation was relatively rare, limited to a low number of cells (ranging from less than 0.1% to 0.23%) as confirmed by very low or not detectable levels of human transcripts for alpha-fetoprotein, CK18, CK19 and albumin in either normal or injured livers. Finally, a significant number of cells of human origin exhibited a myofibroblast-like morphology. CONCLUSIONS: Transplanted hMSCs have the potential to migrate into normal and injured liver parenchyma, particularly under conditions of chronic injury, but differentiation into hepatocyte-like cells is a rare event and pro-fibrogenic potential of hMSC transplant should be not under-evaluated.

Aldehyde dehydrogenase from rat intestinal mucosa: purification and characterization of an isozyme with high affinity for gamma-aminobutyraldehyde.

In rat adrenal gland and gastric mucosa putrescine is efficiently oxidized to GABA via gamma-aminobutyraldehyde (ABAL) by action of diamine oxidase and aldehyde dehydrogenase. Having turned our attention on the rat intestinal mucosa, where putrescine uptake and diamine oxidase are active, we have purified and characterized an aldehyde dehydrogenase optimally active on gamma-aminobutyraldehyde. A dimer with a subunit molecular weight of 52,000, the native enzyme binds ABAL and NAD+ with high affinity: at pH 7.4, Km values are equal to 18 and 14 microM, respectively. Affinity for betaine aldehyde is much lower (Km = 285 microM), but the efficiency is equally good, thanks to a high value of V. Unaffected by disulfiram and Mg2+, the enzyme is activated by high NAD+ concentrations (Vnn = 1.6 x Vn) and is competitively inhibited by NADH. According to the best fitting model, the dimeric enzyme only binds one NADH and the mixed complex enzyme-NAD(+)-NADH is inactive. The increase of activity promoted by NAD+ can therefore be ascribed to an allosteric effect, rather than to the activation of a second reaction center. Highly stable at pH 6.8 in the presence of dithiothreitol and high phosphate concentrations, ABALDH is inactivated by ion-exchange resins and by cationic buffers. Our results show that the enzyme can be effectively involved in the metabolism of biogenic amines and, with a K(m) for ABAL lower than 20 microM, in the synthesis of GABA.

Kinetic and metabolic studies of 7-(1,3-dithiolan-2-ylmethyl)-1,3-dimethylxanthine in the rat.

The kinetics (absorption, tissue distribution and excretion) of 7-(1,3-dithiolan-2-ylmethyl)-1, 3-dimethylxanthine(ABC 99) were studied in the rat. ABC 99 was administered orally at doses of 10, 30 and 100 mg/kg for the serum kinetic studies. Only the 30 mg/kg dose was used for the tissue distribution and excretion kinetic studies. ABC 99 is rapidly absorbed, metabolized in the liver and partially excreted in the urine. It is equally distributed in the tissues, including the brain, although in much lower amounts than those absorbed. Three metabolites were identified: theophylline, which forms in very small quantities, and two isomers (cis and trans) of the sulfoxide. The latter two compounds form in larger amounts with respect to theophylline, and the trans-isomer predominates. The metabolites are distributed to the tissues, but do not accumulate. Elimination is virtually complete at 24 h. The pharmacological activity of ABC 99 (antibronchospastic, mucoregulatory and anti-inflammatory) can be attributed the compound, which is absorbed in its original form. Similar activity of the two sulfoxides (met 1 and 2) cannot be excluded at present as they have a structure analogous to ABC 99.

Cytoprotective activity of deboxamet: a possible interference with prostaglandin and prostacyclin metabolism in rat gastric mucosa.

Deboxamet (5-methoxy-2-methyl-3-indolyl-acetohydroxamic acid) is a new synthetic drug with anti-ulcer and anti-secretory activity. The authors evaluated the ability of deboxamet to protect the rat gastric mucosa against the intensive necroses induced experimentally by absolute ethanol, NaCl (25%), HCl (0.6 N), acetylsalicylic acid plus HCl, and sodium taurocholate plus HCl. Deboxamet, as compared with pirenzepine, sulglycotide and PGE2, displayed a cytoprotective activity against these necrotizing agents. The involvement of deboxamet with prostacyclin metabolism was also investigated. In order to assess the presence of PGI2-like substances, extracts of mucosa from rats treated orally with deboxamet and sulglycotide were assayed i) on isolated rabbit mesenteric artery, ii) for hypotensive effect in anaesthetized rat, and iii) for anti-platelet activity. Deboxamet, like sulglycotide, was able to raise the availability of prostacyclins in the rat gastric mucosa, which is an important action in maintaining its cellular integrity. However, our results cannot determine whether this activity is due to an enhanced biosynthesis or a decreased degradation of prostacyclins.

Biological properties of pancreatic elastase in relation to atherosclerotic disease.

The inhibitory effect of elastase on experimental atherosclerosis has been reported in numerous studies. In our investigation, performed in the rat, a pancreatic extract provided with elastolytic activity has been shown to possess an anti-aggregative effect in vitro and ex vivo and anti-thrombotic properties. In addition, the elastase was capable of inhibiting endothelial exfoliation induced by the desquamatory agent sodium citrate. This agent was tested for its microhaemorrhoeological activity in acute and subacute experiments. In both these conditions, elastase was able to increase the flexibility of red blood cells and their resistance to lysis provoked by hypotonic solutions. In animals fed on an atherogenic diet, this substance limited the lipoprotein accumulation in the aorta wall. Moreover, it reduced the enhanced calcium content, induced by vitamin D administration, in the tissue of arteries. These data indicate that elastase can counteract some pathobiological aspects that characterize atherosclerotic events.

Synthesis, antilipidemic and platelet antiaggregatory activity of 2-aminobenzimidazole amide derivatives.

The synthesis and preliminary pharmacological evaluation of 2-aminobenzimidazole amide derivatives are reported. None of these compounds showed antilipidemic or platelet antiaggregatory activity comparable to that of drugs used in therapy.

In vivo effect of berenil on rat liver polyamine metabolism.

1. Berenil, administered to rats in vivo, promoted a decrease in liver SAMDC activity, but an increase in ODC and SAT activity. 2. Its effect on ODC was completely prevented by cycloheximide, that on SAT only partially. 3. Berenil had no effect on ODC activity in adrenalectomized rats. Adrenergic antagonists counteracted the effect of Berenil on ODC activity. 4. Polyamine content was increased. The maximum modification was observed for putrescine and N1-acetylspermidine.

JAR human placental choriocarcinoma cells actively synthesize, take up and release polyamines.

Uptake of polyamines has been investigated extensively in many cells, but not in placenta, where the polyamine-polyamine oxidase system is supposed to have an immunoregulatory function in pregnancy. Due to the importance of the transfer in this tissue, we have started this study. JAR human placental choriocarcinoma cells in monolayer at confluency were used as a model for measuring the key enzymes of polyamine synthesis and interconversion, rate of uptake and efflux, and the polyamine content. Polyamines were taken up by JAR cells and released by an independent mechanism. Ornithine decarboxylase and spermidine acetyltransferase activities and the rate of transport in and out of the cell were much higher than in other cells, such as L1210 cells. However the systems used for uptake and release appear in many respects to be similar to those observed in L1210 cells, but different from others. The uptake appears to be regulated by an inhibitory protein. Moreover, protein kinase C appears to be involved in the process. The efflux also is regulated as in L1210 cells, through control of H+ and Ca2+ concentration. In conclusion, this study shows that, in JAR cells, ornithine decarboxylase and spermidine acetyltransferase activities were much higher than in other cells, and so was the rate of transport in and out of the cells. As a result, a much higher polyamine content was observed.

Preliminary studies on the absorption, distribution and excretion of 2-(7'theophyllinemethyl)-1,3-dioxolane in the rat.

Preliminary studies in the rat were carried out on the absorption, distribution and excretion of 2-(7'-theophyllinemethyl)-1,3-dioxolane (ABC 12/3), a new compound with anti-bronchospastic activity. Our results show that ABC 12/3 is rapidly absorbed and remains in the blood for about 6 hours. The distribution of this compound in the various tissues appears quite rapid, reaching good concentrations in the lung. Urinary excretion is complete within 12 hours after the administration of the compound, which is metabolized like a theophylline derivative.

Influence of non-steroidal anti-inflammatory compounds on the hepatic tryptophan pyrrolase activity in hypo- and hyperthyroid rats.

Hypothyroid state induced in the rat by thyroparathyroidectomy does not modify the activity of hepatic tryptophan pyrrolase (TPO) while hyperthyroidism, obtained after daily injection of 3, 3', 5-triiodo-L-thyronine, inhibits significantly the liver TPO activity. Treatment with oxametacine, indomethacin, phenylbutazone, flufenamic acid and acetylsalicylic acid, increases the activity of hepatic TPO in hypothyroid state, whereas, in hyperthyroid rats, the same drugs are able to restore the normal enzymatic activity, except for acetylsalicylic acid. During the development of an acute inflammatory process, provoked by carrageenan injection, the treatment with non-steroidal anti-inflammatory agents induces an enhancement of hepatic TPO activity. On the contrary, such enzymatic activity appears unaffected in chronic inflammation induced by cotton-pellet implantation.

Doxofylline in rat brain in relation to locomotor activity.

Doxofylline is a new xanthine derivative with significant bronchodilatatory activity. We have studied in HPLC the distribution of doxofylline in various areas of rat brain (cortex, cerebellum, limbic system) and its activity on the central nervous system by using a spontaneous motility test comparing it with aminophylline administered orally in equimolar doses (4.7 - 9.4 - 19 x 10(-5) mol/kg). Doxofylline is absorbed 3 or 4 times less than aminophylline at the same doses. Nevertheless, the quantity of doxofylline that goes to the brain is equivalent to that absorbed, whereas the quantity of aminophylline is about one-third. This is due to the greater liposolubility of doxofylline in comparison to aminophylline. In spite of the fact that doxofylline is easily distributed in the brain, spontaneous motility in animals is not modified, whereas aminophylline increases this activity significantly. The low affinity of doxofylline with adenosine receptors (A1 and A2) in comparison with aminophylline explains the lack of side effects on the central nervous system which has been amply documented for theophylline and for other methylxanthine derivatives.

Oxygen regulation of rat hepatocyte iNOS gene expression.

BACKGROUND/AIMS: The human iNOS promoter contains a consensus sequence for binding the hypoxia inducible factor. The aim of this study was to see whether iNOS gene expression is triggered by oxygen tension in rat hepatocytes exposed in vivo to high (periportal) and low (perivenous) oxygen tension. METHODS: Hepatocytes transfected or not with a plasmid containing rat iNOS promoter linked to chloramphenicol acetyltransferase were cultured at 21% and 5% oxygen tension. In normal hepatocytes, iNOS protein, mRNA and activity were detected. In transfected cells, chloramphenicol acetyltransferase activity was measured. RESULTS: In cells cultured in a hypoxic environment, both iNOS protein and mRNA increased, whereas the nitrite level in the medium decreased. However, electron paramagnetic resonance analysis and in vitro iNOS activity indicated that iNOS was active. Transfection experiments showed that the expression of chloramphenicol acetyltransferase driven by iNOS promoter was increased in cells maintained at low oxygen tension. CONCLUSIONS: Our experiments show that in rat hepatocytes: 1) iNOS is induced by low oxygen tension; 2) the modification occurs at the transcriptional level; 3) the enzyme at 5% oxygen is able to catalyze the synthesis of NO, although no nitrites are accumulated in the medium. These findings could have physiopathological relevance, e.g. in determining the resistance of perivenous hepatocytes to ischemia injury.

Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase.

Agmatine has been proposed as the physiological ligand for the imidazoline receptors. It is not known whether it is also involved in the homoeostasis of intracellular polyamine content. To show whether this is the case, we have studied the effect of agmatine on rat liver cells, under both periportal and perivenous conditions. It is shown that agmatine modulates intracellular polyamine content through its effect on the synthesis of the limiting enzyme of the interconversion pathway, spermidine/spermine acetyltransferase (SSAT). Increased SSAT activity is accompanied by depletion of spermidine and spermine, and accumulation of putrescine and N1-acetylspermidine. Immunoblotting with a specific polyclonal antiserum confirms the induction. At the same time S-adenosylmethionine decarboxylase activity is significantly increased, while ornithine decarboxylase (ODC) activity and the rate of spermidine uptake are reduced. This is not due to an effect on ODC antizyme, which is not significantly changed. All these modifications are observed in HTC cells also, where they are accompanied by a decrease in proliferation rate. SSAT is also induced by low oxygen tension which mimics perivenous conditions. The effect is synergic with that promoted by agmatine.

Metabolism of 7-(1,3-dithiolan-2-ylmethyl)-1,3-dimethylxanthine by rat liver microsomes. Diastereoselective metabolism of the 1,3-dithiolane ring.

The metabolic transformation of the antibronchospastic compound ABC-99 [7-(1,3-dithiolan-2-ylmethyl)-1,3-dimethylxanthine] was studied in vitro with a rat liver microsomal preparation containing an NADPH-generating system. Thirty percent of the ABC-99 was metabolized and the only metabolic pathway observed as the oxidation of the 1,3-dithiolane ring. Two distinct sulfoxides were formed diastereoselectively, the trans isomer being the major product in the ratio 7:3. In contrast to the 1,3-dioxolane ring of doxophylline, the 1,3-dithiolane ring of ABC-99 did not undergo oxidative opening through acetal carbon oxidation. Furthermore no N-dealkylation to theophylline was observed. This high regioselectivity in in vitro metabolism was most likely due to the nucleophilicity of the sulfur atom. The diastereoselective sulfoxidation was apparently catalyzed by flavin-dependent monooxygenases, as no effect was observed with CO treatment, whereas selective thermal inactivation significantly reduced the rate of sulfoxidation.

Transport and metabolism of agmatine in rat hepatocyte cultures.

Rat hepatocytes in culture take up [14C]-agmatine by both a high-affinity transport system [KM = 0.03 mM; Vmax = 30 pmol x min x (mg protein)-1] and a low-affinity system. The high-affinity system also transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4-guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C-4-aminobutyrate is also accumulated in the presence of an inhibitor of 4-aminobutyrate transaminase.

Agmatine induces apoptosis in rat hepatocyte cultures.

BACKGROUND/AIMS: Agmatine, the compound formed by decarboxylation of arginine, is believed to be an endogenous neurotransmitter through interaction with the imidazoline receptors. However, it also appears to regulate rat hepatocyte polyamines by modifying both their synthesis and their catabolism. As the decrease in polyamine content has been correlated with apoptosis, we examined the possibility that agmatine has an effect on this phenomenon. METHODS: Apoptotic cells were detected by visualizing nuclear shrinkage/fragmentation in hepatocytes cultured at 21 and 5% oxygen tension. Caspase-3 activity, cleavage of PARP, release of cytochrome c and mitochondrial swelling were therefore measured in the two conditions and in the presence or not of agmatine. RESULTS: In rat hepatocytes agmatine promoted apoptosis, procaspase 3 processing and increase of caspase-3 like activity. This occurred through mitochondria swelling and release of cytochrome c. Cyclosporin A and catalase blocked the swelling. CONCLUSIONS: Our experiments show that agmatine, besides all the known biological effects, has also part, at least in hepatocytes, in the modulation of programmed cell death.