Dataset Information

Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine.

ABSTRACT: An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol x min(-1) x (mg of protein)(-1). Experiments with an increasing concentration (0- 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol x min(-1) x (mg of protein)(-1) for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol x min(-1) x g(-1)) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway.

SUBMITTER: Horyn O

PROVIDER: S-EPMC1138948 | biostudies-other | 2005 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:1. Isolated rat liver was perfused with 10 mM-15NH4Cl, 5 mM-lactate and 1 mM-ornithine, or with 3 mM-[15N]alanine and 1 mM-ornithine, in haemoglobin-free medium. The liver was physiologically stable for over 3 h and synthesized urea at the rate of 1.15 mumol.min-1.g of liver-1 (15NH4(+)-perfused) or 0.41 mumol.min-1.g-1 ([15N]alanine-perfused). 2. The perfused liver was continuously monitored by 15N n.m.r. spectroscopy at 20.27 MHz for 15N. Well-resolved 15N resonances of precursors and intermediates of the urea cycle, present at tissue concentrations of 0.2-3.0 mumol/g, were observed from the intact liver in 5-40 min of acquisition. Key metabolites in liver extract and the final perfusion medium were analysed by n.m.r. and by biochemical assays to determine fractional 15N enrichment and the total 15N recovery. 3. In 15NH4(+)-perfused liver (n = 6), 15N incorporation into glutamate and alanine (1.0-1.3 mumol/g), as well as progressive formation of [15N2]urea, was observed during the first 2 h of perfusion. In the second and third hour, hepatic concentrations of [omega-15N]citrulline and [omega,omega'-15N]argininosuccinate increased to n.m.r.-detectable levels (0.3-0.9 mumol/g). The [15N]aspartate pool was large in the absence of added ornithine, but on its addition was rapidly incorporated into argininosuccinate (n = 3). 4. In [15N]alanine-perfused liver, major metabolites were [15N]glutamate, [gamma-15N]glutamine and [15N]urea. Urea-cycle intermediates were undetectable. 5. The results suggest that, in intact liver provided with excess ammonia, low concentrations of cytosolic argininosuccinate synthetase and argininosuccinate lyase limited the rate of metabolite flux in the urea cycle. By contrast, in alanine-perfused liver at a physiological rate of urea synthesis, mitochondrial carbamoylphosphate synthetase was rate-limiting. 6. The potential utility of 15N n.m.r. for study of metabolite channelling through urea-cycle enzymes in intact liver is discussed.

Project description:A mathematical model was constructed to define the dynamics of incorporation of radioactivity into urea carbon and the guanidine carbon of arginine in plasma albumin after the rapid intraportal-venous administration of Na214CO3 in the isolated perfused rat liver. 2. The model was formulated in terms of compartmental analysis and additional experiments were designed to provide further information on subsystem dynamics and to discriminate between alternative model structures. 3. Evidence for the rapid-time-constant of labelling of intracellular arginine was provided by precursor-product analysis of precursor [14C]carboante and product [14C]urea in the perfusate. 4. Compartmental analysis of the dynamics of newly synthesized urea was based on the fate of exogenous [13C]urea, endogenous [14C]urea and the accumulation of [12C]urea in perfusate water, confirming the early completion of urea carbon labelling, the absence of continuing synthesis of labelled urea, and the presence of a small intrahepatic urea-delay pool. 5. Analysis of the perfusate dynamics of endogenously synthesized and exogenously administered [6-14C]arginine indicated that although the capacity for extrahepatic formation of [14C]-urea exists, little or no arginine formed within the intrahepatic urea cycle was transported out of the liver. However, the presence of a rapidly turning-over intrahepatic arginine pool was confirmed. 6. On the basis of these subsystem analyses it was possible to offer feasible estimations for the parameters of the mathematical model. However, it was not possible to stimulate the form and magnitude of the dynamics of newly synthesized labelled urea and albumin which were simultaneously observed after administration of [14C]carbonate on the basis of a preliminary model which postulated that both products were derived from a single hepatic pool of [16-14C]arginine. On the other hand these observed dynamics could be satisfied to a two-compartment arginine model, which also provided an explanation for discrepancies observed between albumin synthesis measured radioisotopically and immunologically. This was based on a relative overestimation of [14C]urea specific radioactivity resulting from the rapid dynamics of [14C]carbonate and the [14C]urea subsystem relative to the labelled albumin subsystem. The effects of arginine compartmentalization could be minimized in the model by minor slowing of the rate of [14C]carbonate turnover or by constant infusion of [14C]carbonate, both of which permitted valid determination of albumin-synthesis rates.

Project description:Acute liver failure (ALF) is a rare but devastating disease associated with substantial morbidity and a mortality rate of almost 45%. Medical treatments, apart from supportive care, are limited and liver transplantation may be the only rescue option. Large animal models, which most closely represent human disease, can be logistically and technically cumbersome, expensive and pose ethical challenges. The development of isolated organ perfusion technologies, originally intended for preservation before transplantation, offers a new platform for experimental models of liver disease, such as ALF. In this study, female domestic swine underwent hepatectomy, followed by perfusion of the isolated liver on a normothermic machine perfusion device. Five control livers were perfused for 24 h at 37 °C, while receiving supplemental oxygen and nutrition. Six livers received toxic doses of acetaminophen given over 12 h, titrated to methemoglobin levels. Perfusate was sampled every 4 h for measurement of biochemical markers of injury (e.g., aspartate aminotransferase [AST], alanine aminotransferase [ALT]). Liver biopsies were taken at the beginning, middle, and end of perfusion for histological assessment. Acetaminophen-treated livers received a median dose of 8.93 g (8.21-9.75 g) of acetaminophen, achieving a peak acetaminophen level of 3780 µmol/L (3189-3913 µmol/L). Peak values of ALT (76 vs. 105 U/L; p = 0.429) and AST (3576 vs. 4712 U/L; p = 0.429) were not significantly different between groups. However, by the end of perfusion, histology scores were significantly worse in the acetaminophen treated group (p = 0.016). All acetaminophen treated livers developed significant methemoglobinemia, with a peak methemoglobin level of 19.3%, compared to 2.0% for control livers (p = 0.004). The development of a model of ALF in the ex vivo setting was confounded by the development of toxic methemoglobinemia. Further attempts using alternative agents or dosing strategies may be warranted to explore this setting as a model of liver disease.

Project description:Agmatine, a divalent diamine with two positive charges at physiological pH, is transported into the matrix of liver mitochondria by an energy-dependent mechanism the driving force of which is DeltaPsi (electrical membrane potential). Although this process showed strict electrophoretic behaviour, qualitatively similar to that of polyamines, agmatine is most probably transported by a specific uniporter. Shared transport with polyamines by means of their transporter is excluded, as divalent putrescine and cadaverine are ineffective in inhibiting agmatine uptake. Indeed, the use of the electroneutral transporter of basic amino acids can also be discarded as ornithine, arginine and lysine are completely ineffective at inducing the inhibition of agmatine uptake. The involvement of the monoamine transporter or the existence of a leak pathway are also unlikely. Flux-voltage analysis and the determination of activation enthalpy, which is dependent upon the valence of agmatine, are consistent with the hypothesis that the mitochondrial agmatine transporter is a channel or a single-binding centre-gated pore. The transport of agmatine was non-competitively inhibited by propargylamines, in particular clorgilyne, that are known to be inhibitors of MAO (monoamine oxidase). However, agmatine is normally transported in mitoplasts, thus excluding the involvement of MAO in this process. The I2 imidazoline receptor, which binds agmatine to the mitochondrial membrane, can also be excluded as a possible transporter since its inhibitor, idazoxan, was ineffective at inducing the inhibition of agmatine uptake. Scatchard analysis of membrane binding revealed two types of binding site, S1 and S2, both with mono-co-ordination, and exhibiting high-capacity and low-affinity binding for agmatine compared with polyamines. Agmatine transport in liver mitochondria may be of physiological importance as an indirect regulatory system of cytochrome c oxidase activity and as an inducer mechanism of mitochondrial-mediated apoptosis.

Dataset Information

Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets