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

127

Control of urea synthesis was studied in rat hepatocytes incubated with physiological mixtures of amino acids in which arginine was replaced by equimolar amounts of ornithine. The following observations were made.1. Intramitochondrial carbamoyl phosphate was always below 0.1 mM. Only when ornithine was absent and when, in addition, the concentration of amino acids was higher than four times their plasma concentration, intramitochondrial carbamoyl phosphate rose up to about 3 mM ; under these conditions ammonia accumulated in the medium.2. The relationship between ornithine-cycle flux and the concentration of the cycle intermediates at varying amino acid concentration indicated that under near-physiological conditions the ornithine-cycle enzymes are far from being saturated with their substrates.3. Moderate concentrations of norvaline had no effect on the rate of urea synthesis unless the cells were severely depleted of ornithine.4. Activation of carbamoyl-phosphate synthetase (ammonia) by addition of N-carbamoylglutamate only slightly stimulated urea production at all amino acid concentrations. However, in the presence of the activator the curve relating ornithine-cycle flux to the steady-state ammonia concentration was shifted to lower concentrations of ammonia.5. The intramitochondrial concentration of carbamoyl phosphate in rat liver in vivo was below 0.1 mM. This value is far below the concentration required for substantial inhibition of carbamoyl-phosphate synthetase.6. It is concluded that in vivo the function of activity changes in carbamoyl-phosphate synthetase, via the welldocumented alterations in the intramitochondrial concentration of N-acetylglutamate, is to buffer the intrahepatic ammonia concentration rather than to affect urea production per se. At constant concentration of ammonia the rate of urea production is entirely controlled by the activity of carbamoyl-phosphate synthetase.Short-term control of urea-cycle flux has in the past been the subject of many studies. In reviewing the literature on this topic [I] we have concluded that: (a) the capacity of the urea cycle, i.e. with saturating intracellular concentrations of ammonia, ornithine and aspartate, is determined by the V of argininosuccinate synthetase; and (b) that in vivo, with nonsaturating substrate supply, flux through the urea cycle must be primarily controlled by the activity of carbamoyl-phosphate synthetase, a conclusion based on qualitative rather than on quantitative considerations. It is also clear from the literature that in vivo modulation of carbamoyl-phosphate synthetase activity occurs by variations in the intramitochondrial concentration of N-acetylglutamate, the essential activator for the enzyme [l -31. However, this latter concept has been questioned [4].

Section: Discussionmentioning

confidence: 99%

“…Hepatocytes were isolated from the livers of male Wistar rats (200-250 g; fasted for 24 h) using the method of Berry and Friend [I 31 with the modifications described by Groen et al [14].…”

Section: Methodsmentioning

confidence: 99%

Control of ureogenesis

Meijer

Lof

Ramos

et al. 1985

127

“…Hepatocytes were isolated from 20 -24-h-starved male Wistar rats (200-250 g) according to Berry and Friend [21] with the modifications described by Groen et al [22].…”

Section: Methodsmentioning

confidence: 99%

Control by pH of urea synthesis in isolated rat hepatocytes

Boon¹,

Meijer²

1988

Control by pH of urea synthesis has been studied in isolated rat hepatocytes incubated with a physiological mixture of amino acids.Inhibition of urea synthesis by decreasing the pH of the medium was caused by diminished production of ammonia and not, as suggested in the literature, by inhibition of entry of ammonia into the ornithine cycle. The decrease by low pH of the rate of degradation of the added amino acids, that of alanine being quantitatively the most important, was accompanied by a decrease in their intracellular concentration.It is concluded that inhibited transport of amino acids across the plasma membrane of the hepatocyte is responsible, at least in part, for the fall in urea synthesis with decreasing pH.It is proposed that inhibition by low pH of other steps in the ureogenic pathway, distal to the production of ammonia, does not affect flux through the ornithine cycle per se, but rather contributes to the buffering of the intrahepatic concentration of ammonia.Synthesis of urea has been shown to decrease at low pH, both in vivo [l -31 and in vitro, with isolated hepatocytes incubated with NH4CI or with livers perfused with NH4C1 [7]. Several mechanisms have been proposed that might contribute to the inhibition of urea synthesis at low pH. These include a decreased rate of production of N-acetylglutamate [8], the essential activator of carbamoyl-phosphate synthetase, a decrease in the concentration of unprotonated NH3 [6, 91, which is the true substrate for carbamoyl-phosphate synthetase [lo], and a decrease in the activity of mitochondrial carbonic anhydrase [7] ; the latter enzyme must produce HCO; from metabolically generated C 0 2 for synthesis of carbamoyl phosphate Ell], because carbamoyl-phosphate synthetase uses HCO;, and not C 0 2 , as the substrate [12] and because the mitochondrial inner membrane is impermeable to HCO; [13].In vivo, 70-75% of total urea synthesis is derived from the intrahepatic degradation of amino acids; the remainder is derived from portal ammonia [14, 151. Therefore, breakdown of amino acids can be an additional site for pH regulation. For example, the rate of transport of amino acids across the plasma membrane of the hepatocyte is known to decrease at acidic pH [16, 171. Indeed, degradation of at least one amino acid, glutamine, is strongly depressed in acidosis, not only because of inhibition of glutaminase activity [I81 but also because of inhibition of glutamine transport across the plasma membrane of the hepatocyte [19]. Hepatic alanine consumption is also pH-sensitive, both in vivo [3] and in the isolated perfused liver [20].The experiments described in this paper were designed to answer the question of which of the above-mentioned mechanisms is primarily responsible for the inhibition by low pH of urea synthesis from amino acids. The data indicate that, under our experimental conditions, transport of amino acids across the plasma membrane of the hepatocyte is the main target for pH control of ureogenesis. MATERIALS AND METHODSHepatocytes were isolated from 20 ...

“…Hepatocytes from male Wistar rats (200-250 g), starved for 20 -24 h, were isolated by the method of Berry and Friend [14] as modified by Groen et al [15]. Liver cells were perifused by the method of Van der Meer and Tager [ll] with the modifications described by Groen et al [9, 151.…”

Section: Methodsmentioning

confidence: 99%

“…Metabolites were measured in the protein-free neutralised extracts. For the measurements of the distribution of aspartate and glutamate across the mitochondria1 membrane, cells were fractionated using the digitonin technique described by Zuurendonk and Tager [I61 with the modifications described by Groen et al [15].…”

Section: Methodsmentioning

confidence: 99%

The malate/aspartate shuttle and pyruvate kinase as targets involved in the stimulation of gluconeogenesis by phenylephrine

Leverve

Verhoeven

Groen

et al. 1986

Self Cite

The mechanisms responsible for the stimulation by phenylephrine of gluconeogenesis from dihydroxyacetone and glycerol were studied in perifused rat hepatocytes.1 . The stimulation by phenylephrine of glucose formation from dihydroxyacetone was biphasic. Transient stimulation of about 25% after 3 min was followed by a stable stimulation of about 15% 7 min later.Concomitantly there was a transient inhibition by phenylephrine of pyruvate kinase flux during the first few minutes followed by a more stable inhibition after about 10 min. The stable inhibition could quantitatively account for the stable stimulation of gluconeogenesis by the hormone when dihydroxyacetone was the gluconeogenic substrate. The effects of phenylephrine were independent of CAMP. The flow in the perifusion chamber (12 ml) was 5 ml/min. The perifusion fluid was Krebs-Ringer bicarbonate buffer containing 1.3 mM Ca2+. After 45 min, when the cells had reached a steady state, phenylephrine was added. In order to have an initial concentration of exactly 10 pM, 120 nmol phenylephrine was injected directly into the perifusion chamber at the start of the infusion. Glucose, lactate, pyruvate and ethanol were measured in the perifusate. For the determination of intracellular metabolites, samples of the cell suspension were taken from the chamber without interrupting the flow. For this purpose, a syringe was introduced into the chamber