Direct measurement of active transport systems for alanine in freshly isolated rat liver cells

Edmondson, James W.; Lumeng, Lawrence; Li, Ting‐Kai

doi:10.1016/0006-291x(77)91564-9

Cited by 46 publications

(14 citation statements)

References 13 publications

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“…the transport of the amino acid across the plasma membrane of the parenchymal cells, are as yet unknown. However, several groups have measured the maximal amount of alanine that can be accumulated intracellularly when alanine metabolism is inhibited by aminooxyacetate [3, 29,30]. In the absence of aminooxyacetate the maximal accumulation ratio is never reached either in cells from fasted rats [3] or in those from fed rats [3,4], indicating that under metabolising conditions the alanine transport system is out of equilibrium at all concentrations of alanine (0-6 mM) used in these studies.…”

Section: Discussionmentioning

confidence: 99%

Intracellular Compartment ation and Control of Alanine Metabolism in Rat Liver Parenchymal Cells

Groen

Sips

Vervoorn

et al. 1982

European Journal of Biochemistry

169

106

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The catabolism of alanine in isolated rat liver cells was studied using a perifusion system in order to titrate the cells with different concentrations of alanine.The Concentrations of the components of the alanine aminotransferase reaction in the cytosolic and mitochondrial compartments were measured after digitonin fractionation of samples of the cell suspension taken during each steady state. Comparison of the mass-action ratios with the equilibrium constant indicated that the cytosolic enzyme is poised towards pyruvate formation at all concentrations of alanine used, whereas the mitochondrial enzyme is always poised towards alanine formation. The calculated flux through cytosolic alanine aminotransferase, using literature data for the kinetic parameters of the enzyme and experimental data for the concentration of metabolites, was in close agreement with the measured carbon flux. It is concluded that the transamination step in alanine catabolism occurs in the cytosol.The concentration of the components of the glutamate dehydrogenase and 3-hydroxybutyrate dehydrogenase reactions in the mitochondrial compartment during each steady state were also measured. The dC of the combined reactions did not change appreciably on increasing the extracellular alanine concentration to 0.6 mM. At higher alanine concentrations AG became slightly more negative. As the extracellular Concentration of alanine was increased, the concentration of glutamate in both the cytosolic and the mitochondrial compartments increased. However, the cytosolic alanine concentration was always lower than that in the extracellular compartment even at the highest concentration of alanine (6 mM) infused. When increasing concentrations of pyruvate were infused together with a fixed concentration of alanine (0.6 mM), the cytosolic concentration of alanine increased to levels even higher than that in the perifusate. However, there was no significant effect in the rate of formation of nitrogenous products.It is concluded that the transport of alanine across the plasma membrane is the only rate-controlling step in alanine catabolism under the conditions used.Alanine plays an important role as a carrier of amino groups in the mammalian circulation. Thus amino nitrogen formed during muscle metabolism is transported to the liver mainly in the form of alanine (see [1,2] for reviews). It is, therefore, evident that the metabolism of alanine in the liver is a physiologically important process.The first step in alanine metabolism in liver is the translocation of the amino acid across the plasma membrane of the parenchymal cells. Our previous studies [3] have demonstrated that the transport step is rate-controlling in alanine degradation. Indeed, the agreement between the kinetics of alanine transport and that of alanine metabolism [3] suggested that at physiological concentrations of alanine in plasma (0.2-0.5 mM) the transport of the amino acid across the plasma membrane is the only rate-controlling step for its metabolism. These observations were confirmed by ...

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Section: Discussionmentioning

confidence: 99%

Intracellular Compartment ation and Control of Alanine Metabolism in Rat Liver Parenchymal Cells

Groen

Sips

Vervoorn

et al. 1982

European Journal of Biochemistry

169

106

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“…Immediately following isolation, 95+1% (-SE, n = 8) of the cells exeluded trypan blue, andl 84+2% of the cells continued to exclude the dye following 2 h of incubation. Cells prepared in this manner exhibited high rates of gluconeogenesis (12), maintained intracellular K+ concentrations at 130-150 me(l/liter of cell water (13), and were active in protein synthesis (14) ancl amino acid transport (11).…”

Section: Methodsmentioning

confidence: 99%

“…The plasma content of pyridoxal-P aind( pyricloxic acidl increased 6-7-fold anid thatt of pyridoxal, 12-fold, but the pyridloxiine level did niot increase. Isolatedlhepatocytes, 1 g/15 ml, were incubated for 2 h with 3.33 ,uM [14C]pyridoxine (6 ,uCi/,umol).…”

mentioning

confidence: 89%

Plasma content of B6 vitamers and its relationship to hepatic vitamin B6 metabolism.

Lumeng¹,

Lui²,

Li³

1980

J. Clin. Invest.

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A B S T R A C T The plasnai content of B6 vitanmers is governed by, anmong other factors, dietary supply anid metabolic interconversion. This study examines the effect of pyridoxinie supplementation on the plasma content of B6 vitaimers anid pyridoxic acid in man, and the metabolic conversioni aind releatse of B6 compounlds in isolated rat hepatocytes. Six healthy humiian sulbjects were givein 100n)mg pyridoxine-HCI/d orally for 1-3 wk. Before pyridoxine stupplemenetationi, the mean total plasma level of B6 vitamners was 114+9 nM; acnd( pyridoxal-P, pyridoxamine-P, pyridoxal, pyridoxine, aind pyridoxaminie accounted for 54, 3, 11, 27, ani(d 5%, respectively. Plasmna level of pyridoxic acid was 40±7 nM. Thus, pyridloxal-P is the principal B6 vitaimer in plasma. Durinig pyridoxine stupplemiienitation, mealn plasnma levels of the B6 vitaimers anid pyridoxic acidl increasedl to 655±122 aindl 222±55 nM, respectively.The plasma content of pyridoxal-P aind( pyricloxic acidl increased 6-7-fold anid thatt of pyridoxal, 12-fold, but the pyridloxiine level did niot increase. Isolatedlhepatocytes, 1 g/15 ml, were incubated for 2 h with 3. 33 ,uM [14C]pyridoxine (6 ,uCi/,umol). At zero time, the cells containied about 35 nmol pyridoxal-P and 25 nmol pyridoxamiine-P. After 2 h incubation, the cellular content of' pyridoxal-P and pyridoxaminie-P dlid niot chIaInge significantly, but the meidiunm containetid 5.9 nmol pyridoxal-P, 0.3 nmol pyridoxamine-P, 7.2 nmol pyridloxal, 26.6 nmol pyridoxine, 0.3 nmol pyridoxaminie, and 7.5 iumol pyridoxic acid. Whereats the specific radlioaetivity of pyricloxal-P, pyridoxal, andl pyricloxic acid in the mediuimi approached that of [14C]pyridoxine, the specific radioactivity of celltular pyridoxal-P andl pyricloxamine-P was only 20% of that of pyricloxine. Thuis, newly syynthesized pyricloxal-P is not freely exchalngeable with endogenous pyridoxal-P, but is preferentiatlly releasedl or degraded to pyri(loxal aindl pyridoxic acidl. The latter B6 compounids are also released. These resuilts suggest that orally ingestedl pyri(loxine is rapidly Receivedffor publication 14 March 1980 anid in revised form 13 Junte 1980. 688 metabolized in liver and its products are released into the circulation in the form of pyridoxal-P, pyridoxal, acnd pyridoxic acid. INTRODUCTIONVitamin B6 is metabolized in vivo to multiple vitameric forms and isiultimnately excreted as pyridoxic acid in the urine. Pyridoxal-P anid pyridoxamnine-P, the coenzyme forms, are the maljor B6 comIIpoun(ls in tissues (1), but pyridoxal-P (2) acnd pyridoxal (3) appeatr to be the major form-iis in 10loo0( plasmla. The (quaintitative relationishlip between these B6 vitamers acnd( otlier B6 compounds in plasmia hals not been defined. Such knowle(dge is important to the understacnding of the process of tranlsport of vitaimin B6 aimionig the differenit orgains and(c tissues of the lbody.Reeiently, we have modified an existing method (4) for the separation and ancalysis of the B6 com11poun11dS.By using this method, we have miieasture(d ...

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“…However, when PLP was added in vitro to the dialyzed hemolysate supemate to provide binding to low affinity sites, PLP hydrolysis by the membrane ghosts occurred more rapidly. Acetaldehyde, 10 mM, further accelerated this degradation rate and about 30 Effect of acetaldehyde on hepatic tyrosine aminotransferase. Tyrosine aminotransferase has a relatively low affinity for PLP (15).…”

Section: Methodsmentioning

confidence: 94%

“…90-95% of the liver cells excluded trypan blue and exhibited intracellular K+ concentration of 134±+ 10.5 meq/liter (n = 10). The cells were morphologically intact by electron microscopy (9) and were active in amino acid transport (10), gluconeogenesis (10), and synthesis of enzymes inducible by hormones (11). These measures of viability were maintained with little change for at least 4 h after isolation.…”

Section: Methodsmentioning

confidence: 99%

The Role of Acetaldehyde in Mediating the Deleterious Effect of Ethanol on Pyridoxal 5′-Phosphate Metabolism

Lumeng¹

1978

J. Clin. Invest.

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A B S T R A C T Previous studies in vivo and with isolated perfused rat livers have suggested that the deleterious effect of ethanol on hepatic pyridoxal 5'-phosphate metabolism is mediated by acetaldehyde. Inasmuch as acetaldehyde has no effect on the synthesis of pyridoxal phosphate, it has also been postulated that acetaldehyde accelerates pyridoxal phosphate degradation by displacing this coenzyme from binding proteins, which protect it against hydrolysis. To test these hypotheses, studies have been performed with isolated rat hepatocytes, subcellular fractions of rat liver, and human erythrocytes. Ethanol oxidation lowered the pyridoxal phosphate content of isolated liver cells when acetaldehyde oxidation was inhibited by either disulfiram or prior treatment of rats with cyanamide. Additions of 7.5 mM acetaldehyde alone at 40-min intervals to cell suspensions decreased hepatic pyridoxal phosphate content only slightly because acetaldehyde was rapidly metabolized. However, when acetaldehyde oxidation and reduction were inhibited by cyanamide treatment and by 4-methylpyrazole and isobutyramide, respectively, a 40% decrease in hepatic pyridoxal phosphate content was observed in 80 min of incubation.In equilibrium dialysis experiments, acetaldehyde, 7.5 and 15 mM, displaced protein-bound pyridoxal phosphate in undialyzed hepatic cytosol and in hemolysate supernate containing added pyridoxal phosphate. In the presence of alkaline phosphatase, acetaldehyde accelerated the degradation of pyridoxal phosphate in dialyzed hemolysate supernate and hepatic cytosol with added pyridoxal phosphate. Acetaldehyde also inhibits tyrosine aminotransferase. The kinetics of inhibition were mixed competitive-noncompeti-

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Direct measurement of active transport systems for alanine in freshly isolated rat liver cells

Cited by 46 publications

References 13 publications

Intracellular Compartment ation and Control of Alanine Metabolism in Rat Liver Parenchymal Cells

Intracellular Compartment ation and Control of Alanine Metabolism in Rat Liver Parenchymal Cells

Plasma content of B6 vitamers and its relationship to hepatic vitamin B6 metabolism.

The Role of Acetaldehyde in Mediating the Deleterious Effect of Ethanol on Pyridoxal 5′-Phosphate Metabolism

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