Vasopressin and angiotensin II stimulate ureogenesis through increased mitochondrial citrulline production.

Vasopressin, angiotensin II, glucagon and epinephrine (through a cAMP-independent, alpha₁adrenergic mechanism), stimulate ureogenesis in isolated rat hepatocytes. Mitochondria, isolated from hepatocytes which were previously treated with these hormones, displayed an enhanced rate of citrulline synthesis in the presence of NH₄Cl as the nitrogen source. When mitochondria were incubated with glutamine as the nitrogen source, only those mitochondria isolated from hepatocytes previously treated with epinephrine or glucagon displayed an enhanced capacity to synthesize citrulline.When cells were incubated in the absence of extracellular calcium, the effects of vasopressin and angiotensin II on urea synthesis were abolished, whereas those of epinephrine and glucagon were only diminished. Mitochondria isolated from cells incubated under these conditions, showed that the effect of all these hormones on citrulline synthesis could still be observed. However, the effects of glucagon and epinephrine plus propranolol were larger than those of angiotensin II or vasopressin.Phosphatidylinositol labeling was significantly increased by epinephrine, vasopressin and angiotensin II both in the absence or presence of calcium. Cyclic AMP levels were significantly increased by glucagon or epinephrine but not by vasopressin or angiotensin II. The effect of epinephrine on cyclic AMP levels was blocked by propranolol both in the absence or presence of calcium.     

Effect of ornithine and lactate on urea synthesis in isolated hepatocytes.

1. In hepatocytes isolated from 24 h-starved rats, urea production from ammonia was stimulated by addition of lactate, in both the presence and the absence of ornithine. The relationship of lactate concentration to the rate of urea synthesis was hyperbolic. 2. Other glucose precursors also stimulated urea production to varying degrees, but none more than lactate. Added oleate and butyrate did not stimulate urea synthesis. 3. Citrulline accumulation was largely dependent on ornithine concentration. As ornithine was increased from 0 to 40 mM, the rate of citrulline accumulation increased hyperbolically, and was half-maximal when ornithine was 8-12 mM. 4. The rate of citrulline accumulation was independent of the presence of lactate, but with pyruvate the rate increased. 5. The rate of urea production continued to increase as ornithine was varied from 0 to 40 mM. 6. It was concluded that intermediates provided by both ornithine and lactate are limiting for urea production from ammonia in isolated liver cells. It was suggested that the stimulatory effect of lactate lies in increased availability of cytosolic aspartate for condensation with citrulline.     

The relationship between intramitochondrial N-acetylglutamate and activity of carbamoyl-phosphate synthetase (ammonia). The effect of glucagon

1. The relationship between urea synthesis, intracellular N-acetylglutamate and the capacity of rat-liver mitochondria to synthesize citrulline was investigated. 2. Treatment of rats with glucagon prior to killing results not only in an increased intramitochondrial ATP concentration and an increased capacity of the mitochondria to synthesize citrulline, but also in an increased concentration of intramitochondrial N-acetylglutamate. 3. Comparison of the rate of citrulline synthesis in mitochondria from glucagon-treated and from control rats, incubated under different conditions, shows that the increased N-acetylglutamate concentration after glucagon treatment is at least in part responsible for the observed increased capacity of the mitochondria to synthesize citrulline. 4. Ureogenic flux in isolated hepatocytes under different incubation conditions correlated with the intracellular concentration of N-acetylglutamate and with the capacity of the mitochondria to synthesize citrulline. 5. When isolated hepatocytes were incubated with NH3, ornithine, lactate and oleate, intracellular N-acetylglutamate increased about eightfold in the first 10 min; during this period the rate of urea synthesis increased considerably. 6. It is concluded that the concentration of intramitochondrial N-acetylglutamate plays an important role in the short-term control of flux through the urea cycle under different nutritional and hormonal conditions.     

Arginine uptake by isolated rat liver mitochondria

The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-¹⁴C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [¹⁴C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin-¹⁴C]carboxylic acid or [¹⁴C]sucrose There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH₄Cl, glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0°C. Mitochondria did not concentrate citrulline.     

A possible rate limiting factor in urea synthesis by isolated hepatocytes: the transport of ornithine into hepatocytes and mitochondria

The uptake of ornithine by isolated hepatocytes and by the particulate fraction of these cells was measured under various conditions of urea synthesis. Under conditions of maximum urea synthesis, i.e. in the presence of glucose, ornithine, ammonium chloride and oleate, the cytosolic concentration and the mitochondrial concentration of ornithine was extremely low, while citrulline accumulated in the cytosol. The data indicate that the rate of citrulline synthesis is limited by the availability of mitochondrial ornithine.     

Analysis of regulatory factors for urea synthesis by isolated perfused rat liver. I. Urea synthesis with ammonia and glutamine as nitrogen sources.

Urea synthesis was studied using the isolated liver perfusion with ammonium cholride and glutamine as nitrogen sources. The rate of urea formation increases with ammonium cholorde concentration up to 5mM, and the rate remained constant in the range between 5 and 20mM of ammonium chloride as the substrate. The concentration of ammonia in the medium to support the half-maximum velocity of urea formation was 0.7mM. The rate of urea formation was stimulated by the addition of 2.5mM ornithine, and the greater part of the ornithine which was taken up into the liver was accumulated as citrulline in the presence of ammonia. A considerable accelerating effect of N-acetylglutamate on the synthetic rate was observed, but a rather high concentration of N-acetylglutamate was required in order to obtain the maximum effect possibly, because its permeability into liver cells may be limited. A marked additive effect on the rate of urea formation was observed with the combined addition of ornithine and N-acetylglutamate. The metabolic conversion of glutamine nitrogen to urea in the perfused rat liver and the effect of several compounds which stimulated urea synthesis with ammonia were further examined. The process of conversion of glutamine nitrogen to urea might be composed of the following three steps. In the first lag phase, a small amount of glutamine was removed from the medium. In the second stage, the glutamine level decreased rapidly and ammonia was accumulated in the perfusate. The third stage was a period in which glutamine concentration remained at a constant low level, and the accumulated ammonia was rapidly conversed to urea. The rate of urea formation in this third stage was found to be much higher than that with ammonia as the substrate. The maximum rate of glutamine removal was obtained at pH 7.7 of the perfusate and at a concentration of 10mM glutamine. Urea formation with glutamine was also stimulated by the addition of ornithine, malate, or N-acetylglutamate, which had accelerating effects on the urea synthesis with ammonia. This stimulation was due to an effective conversion of ammonia to urea, but no change in the rate of removal glutamine was obtained.     

Inhibition of arginine synthesis by urea: A mechanism for arginine deficiency in renal failure which leads to increased hydroxyl radical generation

We have reported that (1) the synthesis of GSA, a uremic toxin, increases depending on the urea concentration and (2) GSA is formed from argininosuccinic acid (ASA) and the hydroxyl radical or SIN-1 which generates superoxide and NO simultaneously. However, an excess of NO, which also serves as a scavenger of the hydroxyl radical, inhibited GSA synthesis. We also reported that arginine, citrulline or ammonia plus ornithine, all of which increase arginine, inhibit GSA synthesis even in the presence of urea. To elucidate the mechanism for increased GSA synthesis by urea, we investigated the effect of urea on ASA and arginine, the immediate precursor of NO.Isolated rat hepatocytes were incubated in 6 ml of Krebs-Henseleit bicarbonate buffer containing 3% bovine serum albumin, 10 mM sodium lactate, 10 mM ammonium chloride and with or without 36 mM of urea and 0.5 or 5 mM ornithine at 37°C for 20 min. In vivo experiments, 4 ml/100 g body weight of 1.7 M urea or 1.7 M NaCl were injected intra-peritoneally into 5 male Wistar rats. Two hours after the intra-peritoneal injection of urea or 1.7 M NaCl, blood, liver and kidney were obtained by the freeze cramp method and amino acids were determined by an amino acid analyzer (JEOL:JCL-300).ASA in isolated hepatocytes was not detected with or without 36 mM (200 mgN/dl) urea, but the arginine level decreased from 36 to 33 nmol/g wet cells with urea. Ornithine which inhibits GSA synthesis, increased ASA markedly in a dose dependent manner and increased arginine. At 2 h after the urea injection the rat serum arginine level decreased by 42% (n = 5), and ornithine and citrulline levels increased significantly. Urea injection increased the ASA level in liver from 36–51 nmol/g liver but this was not statistically significant.We propose that urea inhibits arginine synthesis in hepatocytes, where the arginine level is extremely low to begin with, which decreases NO production which, in turn, increases hydroxyl radical generation from superoxide and NO. This may, also, be an explanation for the reported increase in oxygen stress in renal failure.     

Effect of propionate on the utilization of nitrogen from 15NH4Cl for urea synthesis in hepatocytes isolated from sheep liver

1. The effect of ornithine (2.0 mM) and propionate (5.0 mM) on the utilization of N from 15NH4Cl (5.0 mM) for urea synthesis in hepatocytes isolated from sheep liver was investigated. 2. The capacity of sheep hepatocytes to utilize [15N]ammonia in the absence of the other exogenous substrates was very low and amounted 132 +/- 37.3 mumol/hr per 1 g dry wt. 3. Ornithine failed to affect the total [15N]ammonia uptake and total urea synthesis, but at the same time it markedly increased the utilization of [15N]ammonia for ureagenesis and diminished the rate of urea synthesis from endogenous sources. 4. Propionate markedly increased total [15N]ammonia utilization and total urea formation; this increase resulted from the rise of ammonia utilization for urea synthesis and it was similar in the presence or absence of ornithine. 5. The capacity of sheep liver cells to utilize ammonia in the presence of propionate (in the presence or absence of ornithine) amounted to 256 mumol/hr per 1 g dry wt, thus being similar to the values in vivo. 6. It is concluded that in sheep hepatocytes both ornithine and propionate stimulate the utilization of ammonia for urea synthesis and these effects take place independently and occur by different mechanisms.     

Effects of inhibition of ornithine aminotransferase or of general aminotransferases on urea and citrulline synthesis and on the levels of acetylglutamate in isolated rat hepatocytes

Summary Canaline and gabaculine, inhibitors of γ-aminotransferases and thus of ornithine aminotransferase (E.C. 2.6.1.13), decreased the flow through ornithine carbamoyl transferase (E.C. 2.1.3.3) in isolated rat hepatocytes incubated with 10 mM NH₄Cl and ornithine. The levels of acetylglutamate, an essential activator of carbamoyl phosphate synthetase (ammonia) (E.C. 6.3.4.16), were also decreased, suggesting that the inhibitors had also caused a decrease in the rate of carbamoyl phosphate synthesis. Under these conditions, ornithine appears to be a precursor of acetylglutamate, via ornithine aminotransferase, possibly as a consequence of glutamate synthesis. The influence of aminooxyacetate, an aminotransferase inhibitor, has also been examined.     

The regulation of carbamoyl phosphate synthase activity in rat liver mitochondria.

The rate at which isolated rat liver mitochondria synthesized citrulline with NH4C1 as nitrogen source was markedly dependent on the protein content of the diet. 2. Citrulline synthesis was not rate-limited by substrate concentration, substrate transport or ornithine transcarbamoylase activity under the conditions used. 3. The intramitochondrial content of an activator of carbamoyl phosphate synthase, assumed to be N-acetyl-glutamate, varied markedly with dietary protein content. The variation in the concentration of this activator was sufficient to account for the observed variation in the rates of citrulline synthesis if this synthesis were rate-limited by the activity of carbamoyl phosphate synthase. 4. The rates of urea formation from NH4Cl as nitrogen source in isolated liver cells showed variations in response to diet that closely paralleled the variations in the rates of citrulline synthesis observed in isolated mitochondria. 5. These results are consistent with the postulate that when NH4Cl plus ornithine are present in an excess, the rate of urea synthesis is regulated at the level of carbamoyl phosphate synthase activity.     

Origin of the ammonia used for mitochondrial citrulline synthesis as revealed by 13C-15N spin coupling patterns observed by 13C NMR.

The sources of ammonia used by isolated, intact rat liver mitochondria in the production of citrulline have been investigated in situ using a novel methodology based on the analysis of 13C-15N heteronuclear couplings observed by 13C NMR. Isolated mitochondria from rat liver were incubated with ornithine, 13CO3H- and 15NH4Cl, using unlabeled glutamate or glutamine as alternative, intramitochondrial nitrogen donors. The production of (7-13C, 8-15N) or (7-13C, 8-14N) citrulline was determined in situ by 13C NMR and the relative proportions of 15N- and 14N-citrullines confirmed by high resolution 13C NMR analysis of the C-7 citrulline resonance observed in perchloric acid extracts prepared at the end of the incubations. The 15N fractional enrichment of the intramitochondrial NH3 pool was manipulated either by modifying the 15N enrichment of added 15NH4Cl, or by altering the concentration of the unlabeled nitrogen donors in the incubation medium. Fractional 15N enrichments measured in the N-8 nitrogen of the resulting (7-13C) citrulline closely paralleled those of the external 15NH4Cl with minor dilutions derived from the unlabeled nitrogen contribution from the alternative substrates. In the presence of 10 mM 15NH4Cl, 10 mM glutamate contributed 4% of the citrulline N-8 nitrogen. Under similar conditions, the contribution of nitrogen from 10 mM glutamine to N-8 citrulline was 6%. These results indicate that the primary source of ammonia used for citrulline synthesis by isolated, intact rat liver mitochondria is extramitochondrial, providing also an illustration of the use of 13C-15N spin coupling patterns observed by 13C NMR, as a new tool in the study of ammonia metabolism.     

Lack of effect of NH4Cl on protein synthesis in cultured fibroblasts

In experiments with isolated hepatocytes, Seglen [1] has shown that in the combined presence of NH₄Cl and high concentrations of valine, incorporation of this amino acid into cell protein is inhibited. He has proposed that NH₄Cl, in addition to inhibiting protein degradation in lysosomes, inhibits protein synthesis in these cells as part of a general toxic effect. To determine if NH₄Cl inhibits protein synthesis in cultured cells we incubated rat embryo fibroblasts, prelabeled with [¹⁴C]leucine, in the presence of 10 mM NH₄Cl and 15 mM leucine in both growth and serum-free media. We did not detect any effect of NH₄⁺ on protein synthesis or cell growth over a 3-day period. A partial inhibition of protein degradation was observed, particularly during the first 24 h of the experiment. In pulse-labeling experiments, NH₄Cl had no effect on the incorporation of [³H]leucine in the media. High concentrations of leucine, however, reduced re-utilization of endogenously derived leucine and inhibited the transport of valine into the cellular acid-soluble pool.These experiments show that at least in cultured fibroblasts 10 mM NH₄Cl shows no significant toxicity beyond an inhibition of lysosomal function. In addition these data suggest the possibility that high chase concentrations of one amino acid in the medium may be saturating a common transport mechanism, in effect reducing the transport of other amino acids utilizing this mechanism. A combined blockade by both NH₄Cl and a high concentration of a single amino acid may in certain sensitive cells result in a significant reduction in protein synthesis.     

Perifused monolayer cultures of rat hepatocytes as an improved in vitro system for studies on ureogenesis

A perifusion system has been developed for cultivation of adult rat hepatocytes, permitting continuous supply of medium to the cell monolayer instead of periodical changes as used in conventional culture technique (discontinuous culture). Additionally, a modification of Waymouth's MB 752/1 medium is described, which favoured the expression of several metabolic and regulatory events mentioned below and supported the maintenance of several enzymes involved in nitrogen metabolism. The improved nutritional conditions accelerated early monolayer formation and metabolic recovery, and favoured long-term cultivation of metabolically active and hormone responsive hepatocytes. Urea formation from substrates contained in the medium was found to be 2- to 3-fold higher and preserved for a considerably longer time than with discontinuously cultured cells, and was further enhanced by addition of tryptose phosphate broth or 4 mM NH₄Cl even after 10 days in culture. In the presence of glucagon (10⁻⁷ M) the urea production was more than doubled during a 24 h incubation period on the 4th day. Pretreatment with this hormone for 24 h also markedly stimulated the capacity of perifused cells for ureogenesis. Concomitantly, a rise in arginase activity up to 2-fold could be measured in response to glucagon, which was largely suppressed by simultaneous presence of leucine in concentrations between 5 and 10 mM.     

Role of anion translocation across the mitochondrial membrane in the regulation of urea synthesis from ammonia by isolated rat hepatocytes.

The regulation of urea synthesis from ammonia was investigated using isolated hepatocytes from fasted rats. Addition of ammonia alone produced only a small increase of urea formation, which was stimulated 2-fold by ornithine in conjunction with a fall of ATP levels and an accumulation of citrulline. Further addition of oleate or beta-hydroxybutyrate produced an additional 2-fold stimulation of urea formation to approximately 200 mumol/g dry weight/hour. The presence of oleate also protected against the inhibitory effect of 2,4-dinitrophenol on urea synthesis and the cellular ATP content. The data suggest that both the rate of of energy production and the rate of generation of reducing equivalents from endogensou substrates are insufficient to meet the requirements for optimal rates of urea synthesis. Urea formation from NH3 in the presence of ornithine and oleate, but iin the absence of gluconeogenic precursors, was inhibited by butylmalonate, a known inhibitor of malate-phosphate exchange across the mitochondrial membrane, and stimulated by theaddition of malate and other dicarboxylic acids and amino acids to the cell suspension...     

Inhibition of ureagenesis by valproate in rat hepatocytes. Role of N-acetylglutamate and acetyl-CoA.

Valproate (0.5-5 mM) strongly inhibited urea synthesis in isolated rat hepatocytes incubated with 10 mM-alanine and 3 mM-ornithine. Valproate at the same concentrations markedly decreased concentrations of N-acetylglutamate, an essential activator of carbamoyl-phosphate synthetase I (EC 6.3.4.16), in parallel with the inhibition of urea synthesis by valproate. This compound also lowered the cellular concentration of acetyl-CoA, a substrate of N-acetylglutamate synthase (EC 2.3.1.1); glutamate, aspartate and citrulline were similarly decreased. Valproate in a dose up to 2 mM did not significantly affect the cellular concentration of ATP and had no direct effect on N-acetylglutamate synthesis, carbamoyl-phosphate synthetase I and ornithine transcarbamoylase (EC 2.1.3.3) activities.     

Rate-limiting factors in urate synthesis and gluconeogenesis in avian liver

1. Urate synthesis and other metabolic characteristics of isolated chicken hepatocytes were studied. 2. The distinction is made between immediate precursors of the purine ring (glycine, glutamine, aspartate, formyltetrahydrofolate, bicarbonate) and ultimate precursors from which the immediate precursors are formed in the liver. 3. In hepatocytes from well-fed chickens the rate of urate synthesis was not greatly increased by the addition of amino acids or NH₄Cl, but in hepatocytes from 72h-starved chickens the rate was much increased when alanine or asparagine was added as the only substrate. Other amino acids, when added alone, did not affect the rate. The exceptional effect of alanine and asparagine is due to the ready formation of the immediate precursors. 4. Conditions are described under which glutamine, serine, glycine plus formate, ribose and glucose increased the rate of urate synthesis. 5. At 1mm-NH₄Cl (a concentration not much higher than that of blood plasma) the rate of urate synthesis in the presence of lactate was increased, but higher concentrations inhibited urate synthesis in the presence of lactate or alanine; with alanine even 1mm-NH₄Cl was inhibitory. 6. Glucose synthesis from lactate, alanine or dihydroxyacetone was also inhibited by 1mm-NH₄Cl. 7. NH₄Cl inhibition of urate and glucose synthesis was paralleled by an increased rate of glutamine synthesis. Thus in the presence of NH₄Cl the gluconeogenic precursors are diverted from the pathway of gluconeogenesis to that of glutamate and glutamine synthesis. This implies that the synthesis of these amino acids is the primary process in the detoxication of ammonia in the avian liver. 8. Urate synthesis, like urea synthesis, can be looked on as a cyclic process with either phosphoribosyl pyrophosphate or ribose acting as the carrier on which the purine ring is assembled. 9. The energy requirements of urate synthesis depend on whether phosphoribosyl pyrophosphate is regenerated from IMP by pyrophosphorylase or by phosphorylation and pyrophosphorylation of ribose. It is 6 or 9 pyrophosphate bonds of ATP respectively.     

Channeling of extramitochondrial ornithine to matrix ornithine transcarbamylase

In the presence of citrulline synthesis, we made the following observations. External ornithine is channeled between its transporter and ornithine transcarbamylase; mitochondria preloaded with cold ornithine, then incubated with [3H]ornithine, produced citrulline of the same specific radioactivity as that of external ornithine, while matrix ornithine remained essentially unlabeled. The channeling of ornithine suggests that some soluble enzymes are organized within the mitochondrial matrix. The rate of ornithine transport can be greater than 80 nmol/min/mg. At rates of carbamyl phosphate synthesis of 10-50 nmol/min/mg, the rate of citrulline synthesis is controlled by external ornithine in the range 0.03-0.2 mM; at greater than or equal to 0.2 mM ornithine, transport is not limiting for citrulline synthesis. At external ornithine concentrations less than or equal to 1 mM, i.e. within the physiological range, this amino acid is undetectable in the matrix. Given the rates of citrulline and urea synthesis which occur in vivo and the concentrations of ornithine present in the liver, our findings indicate that ornithine may contribute to the physiological regulation of urea synthesis. Preliminary reports of parts of this work have been published (Raijman, L., Cheung, C-W., and Cohen, N. S. (1984) Fed. Proc. 43, 1831; Cohen, N. S., Cheung, C-W., and Raijman, L. (1986) Fed. Proc. 45, 2677).     

Interrelations between ureogenesis and gluconeogenesis in isolated hepatocytes. The role of antion transport and the competition for energy.

1. Glucose synthesis from lactate plus pyruvate and from lactate plus alanine was measured in the presence or absence of 1mM-oleate or 2mM-octanoate at low (2mM) or high (8mM) concentrations of NH4Cl. 2. Both fatty acids alone or with 2mM-NH4Cl doubled glucose production from lactate plus pyruvate. Glucose synthesis from lactate plus alanine, in the presence of oleate, was decreased 16% by 2mM-NH4Cl. 3. In the presence of fatty acids, 8mM-NH4Cl decreased gluconeogenesis by 60-65% from both lactate plus pyruvate and lactate plus alanine. This inhibition was correlated with a high accumulation of aspartate and a drastic decrease in 2-oxoglutarate and malate in the cells. 4. In the presence of 2mM- or 8 mM-NH4Cl, oleate and glucogenic precursors, the addition of 2.5mM-ornithine stimulated urea synthesis. 5. This was paralleled by a decrease of 16% in glucose synthesis from lactate plus pyruvate in the presence of 2mM-NH4Cl and had no effect at 8mM-NH4Cl. In the system producing glucose from lactate plus alanine, ornithine completely reversed the inhibition caused by 2mM-NH4Cl and only partly that by 8mM-NH4Cl. 6. Gluconeogenesis from pyruvate was also inhibited by 2mM-NH4Cl in the presence of oleate or ethanol. This way due to the decrease of malate, which is the C4 precursor of glucose in this system. 7. The limitation of gluconeogenesis by 2-oxoglutarate and malate concentrations in the liver cell and the competition for energy between glucose and urea synthesis is discussed.     

Metabolic and enzymatic characteristics of adult rat liver parenchymal cells in non-proliferating primary monolayer cultures

Parenchymal cells from normal adult rat liver, prepared with high yield (30 × 10⁶ cells/g liver) and viability index (>96%) by a non-perfusion method, were maintained in non-proliferating monolayer culture. Several metabolic functions were investigated for 7 days to evaluate functional integrity of the cultured hepatocytes. Leucine was linearly incorporated into protein for 4.5 h at each day of cultivation and the incorporation rate increased up to 2-fold after 3 days. Urea production was maintained at a rate of 0.5 μmoles/mg protein × h for at least 7 days, and its amount was enhanced 2-fold within 24 h by the addition of 3 mM NH₄Cl. Glucose was formed during the first days by the hepatocytes and was then taken up with increasing amount from the surrounding medium. Lactate consumption, on the other hand, was replaced by lactate production after one day of cultivation.Variations in enzyme levels of lactate dehydrogenase, arginase, glutamine synthetase and glucose-6-phosphatase were also studied during the whole culture period. Cell leakage, which was detected only in the case of lactate dehydrogenase (LDH), occurred through the 4th day along with a concomitant loss of intracellular LDH activity. After 4 days, however, the enzyme activity returned to the initial level. Arginase was maintained throughout the cultivation period and was stimulated 2- to 3-fold within 24 h by NH₄Cl. Glutamine synthetase declined within the first 4 h of cultivation and then remained in the hepatocytes with a transitory rise after 2 days. Its activity was also found to be inversely related to the concentration of glutamine in the culture medium up to 4 mM. Glucose-6-phosphatase gradually decreased during the cultivation period, the enzyme activity, however, was stimulated by glucagon within 24 h.     

Inhibition of CA V decreases glucose synthesis from pyruvate

The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 5 mM pyruvate, 5 mM lactate, 3 mM ornithine, 10 mM NH4Cl, 1 mM oleate; with these inclusions both urea and glucose synthesis start with HCO3- -requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated Ki for each approximately 0.1 mM. In other experiments hepatocytes were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 10 mM glutamine, 1 mM oleate; with these inclusions glucose synthesis no longer starts with a HCO3- -requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated Ki of 0.1 mM, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 degrees C, with 7.5 mM pyruvate, 3 mM ATP, and 10 mM NaHCO3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.