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Effect of phenylephrine on the compartmentation of inorganic phosphate in perfused rat liver during gluconeogenesis and urea synthesis: a 31P-n.m.r.-spectroscopic study.

ABSTRACT: The transport of Pi between the cytosol and the mitochondria was investigated in perfused rat liver stimulated with phenylephrine and metabolic precursors of glucose and urea: pyruvate, lactate, NH4+ and ornithine. The relative concentrations of phosphorus metabolites in the liver were measured by 31P-n.m.r. spectroscopy. When added simultaneously, phenylephrine and the precursors induced a decrease in the Pi level which in 4-5 min reached a new steady state at 73% of the control level. After 5 min or more of stimulation the ATP level had also decreased. When the stimulation ended, Pi and ATP returned to their initial levels within 15 min. In mitochondria isolated after 5 min of stimulation, Pi was increased more than 2-fold as compared with control mitochondria and, in addition, an accumulation of Pi from the perfusion buffer into the liver was observed. Phenylephrine by itself did not cause any significant changes in the ATP or Pi levels, whereas the glucose and urea precursors in the absence of phenylephrine induced a 9% decrease in Pi, while ATP remained constant. The Pi content of mitochondria isolated under these conditions was not significantly increased as compared with control mitochondria. These results showed that Pi accumulated into the mitochondria by a mechanism possibly involving exchange for malate, and that a major part of the intramitochondrial Pi was invisible by n.m.r.

SUBMITTER: Eriksson O

PROVIDER: S-EPMC1137977 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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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.

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Effect of phenylephrine on the compartmentation of inorganic phosphate in perfused rat liver during gluconeogenesis and urea synthesis: a 31P-n.m.r.-spectroscopic study.

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