Regulation of urea and citrulline synthesis under physiological conditions.

Information on the regulation of urea synthesis in vivo was obtained by examining the relationship between ureagenesis in vivo, citrulline synthesis in vitro, and two factors currently hypothesized to exert short-term regulation of this pathway: the liver mitochondrial content of N-acetylglutamate (NAG) and substrate availability. Rats meal-fed for 4 h every day (4-20 schedule) or for 8 h every other day (8-40 schedule) were used. (1) The citrulline-synthesizing capacity of mitochondria from livers of rats on the 8-40 schedule exceeded the corresponding velocity of urea synthesis in vivo at all time points studied. (2) Mitochondrial NAG in these livers increased from 127 +/- 32 pmol/mg of protein at 0 h to 486 +/- 205 pmol/mg at 3 h after the start of a meal, and decreased thereafter, but the correlation between NAG content and the velocity of citrulline synthesis was not simple, suggesting that NAG is not the only determinant of the state of activation of carbamoyl phosphate synthase I. (3) In rats on the 4-20 schedule killed 1 h after the start of the meal, the liver content of ornithine, citrulline, arginine, glutamate, alanine and urea increased 2.1-12-fold with respect to the values at 0 h; glutamine decreased by 39%. (4) The combined findings indicate that in vivo, moment-to-moment control of the velocity of urea synthesis is exerted by substrate availability. (5) Digestion limits the supply of substrate to the liver, and prevents its ureagenic capacity from being overwhelmed following a protein-containing meal.

Kinetic properties of carbamoyl-phosphate synthase (ammonia) and ornithine carbamoyltransferase in permeabilized mitochondria.

Previous studies using intact rat liver mitochondria have shown that the soluble matrix enzymes carbamoyl-phosphate synthase (ammonia) (CPS) and ornithine carbamoyltransferase (OCT) display some kinetic properties which would not be observed if they were homogeneously distributed in the matrix. In the present work we have extended these studies, using toluene-treated mitochondria which are fully permeable to substrates and inhibitors, yet retain 90% of their soluble enzymes. The results provide evidence of functional organization of CPS and OCT in situ. The major findings are as follows. (1) The apparent Km values of matrix OCT for carbamoyl phosphate and ornithine are respectively 8 and 2 times those measured for the soluble enzyme. delta-N-Phosphonacetyl-L-ornithine inhibits OCT in situ less than in solution, especially when carbamoyl phosphate is synthesized in the mitochondria rather than added to the medium. (2) During citrulline synthesis from endogenously generated carbamoyl phosphate, the concentration of the latter in permeabilized mitochondria is more than 10 times that in the medium, although the mitochondria are freely permeable to added molecules of this size. (3) Endogenously formed carbamoyl phosphate is used preferentially by OCT in situ; addition of a 200-fold excess of unlabelled carbamoyl phosphate has little effect on the conversion of labelled endogenously formed carbamoyl phosphate into citrulline by matrix OCT. (4) The synthesis de novo of carbamoyl phosphate from NH3, HCO3- and ATPMg is the same in the presence and absence of ornithine. (5) Studies with co-immobilized CPS and OCT gave results concordant with some of the above observations and with previous ones with intact mitochondria.

Channeling of urea cycle intermediates in situ in permeabilized hepatocytes.

Preferential use of endogenously generated intermediates by the enzymes of the urea cycle was observed using isolated rat hepatocytes made permeable to low molecular weight compounds with alpha-toxin. The permeabilized cells synthesized [14C]urea from added NH4Cl, [14C]HCO3-, ornithine, and aspartate, using succinate as a respiratory substrate; with all substrates saturating, about 4 nmol of urea were formed per min/mg dry weight of cells. Urea usually accounted for about 40-50% of the total (NH3 + ornithine)-dependent counts, arginine for less than 10%, and citrulline for about 30%. Very tight channeling of arginine between argininosuccinate lyase and arginase was shown by the fact that the addition of a 200-fold excess of unlabeled arginine to the incubations did not decrease the percentage of counts found in urea or increase that found in arginine, even though a substantial amount of the added arginine was hydrolyzed inside the cells. The channeling of argininosuccinate between its synthetase and lyase was demonstrated by similar observations; unlabeled argininosuccinate added in 200-fold excess decreased the percentage of counts in urea by only 25%. Channeling of citrulline from its site of synthesis by ornithine transcarbamylase in the mitochondrial matrix to argininosuccinate synthetase in the cytoplasmic space was also shown. These results strongly suggest that the three "soluble" cytoplasmic enzymes of the urea cycle are grouped around the mitochondria and are spatially organized within the cell in such a way that intermediates can be efficiently transferred between them.

Altered enzyme activities and citrulline synthesis in liver mitochondria from ornithine carbamoyltransferase-deficient sparse-furash mice.

Male mice carrying the spfash mutation have 5-10% of the normal activity of ornithine carbamoyltransferase, yet are only slightly hyperammonaemic and develop quite well. A study of liver mitochondria from normal and spfash males showed that they differ in important ways. (1) The spfash liver contains about 33% more mitochondrial protein per g than does normal liver. (2) The specific activities of carbamoyl-phosphate synthetase (ammonia) and glutamate dehydrogenase are about 15% lower than normal in mitochondria from spfash mice, whereas those of beta-hydroxybutyrate dehydrogenase and cytochrome oxidase are 22% higher and 30% lower respectively. (3) In the presence of 10 mM-ornithine and the substrates for carbamoyl phosphate synthesis, coupled and uncoupled mitochondria from spfash mice synthesize citrulline at unexpectedly high rates, about 25 and 44 nmol/min per mg respectively. Though these are somewhat lower than the corresponding rates obtained with normal mitochondria, the difference does not arise from the deficiency in ornithine carbamoyltransferase, but from the lower carbamoyl-phosphate synthetase activity of the mutant mitochondria. (4) At lower external [ornithine] (less than 2 mM), a smaller fraction of the carbamoyl phosphate synthesized is converted into citrulline in spfash than in normal mitochondria. These studies show that what appears to be a single mutation brings about major adaptations in the mitochondrial component of liver. In addition, they clarify the role of ornithine transport and of protein-protein interactions in citrulline synthesis in normal mitochondria.

Measurements of mitochondrial volumes are affected by the amount of mitochondria used in the determinations.

Mitochondrial water spaces were determined by centrifugal filtration, by using 3H2O and [14C]-sucrose, -mannitol, -inulin and -dextran. The volume (in microliter/mg of mitochondrial protein) of each of the spaces was inversely proportional to the amount of mitochondria (mg of protein) centrifuged. The dextran space (representing extramitochondrial water carried down with the mitochondria) decreased the most, and accounted for most of the changes observed in the other spaces. However, the calculated matrix and intermembrane spaces also decreased when increasing amounts of mitochondria were centrifuged. For each space, the same value was obtained when centrifugal filtration was done at 8000 and at 15,600 g, and when the mitochondria were incubated with the markers for 15 s to 5 min, indicating that sucrose, mannitol and inulin do not penetrate the matrix, nor does dextran penetrate the intermembrane space, under the incubation and centrifugation conditions generally used to measure mitochondrial spaces.

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).

The apparent Km of ammonia for carbamoyl phosphate synthetase (ammonia) in situ.

Experiments with carbamoyl phosphate synthetase (ammonia) in solution and in isolated mitochondria are reported which show the following. NH3 rather than NH4+ is the substrate of the enzyme. The apparent Km of NH3 for the purified enzyme is about 38 microM. The apparent Km for NH3 measured in intact isolated mitochondria is about 13 microM. This value was obtained for both coupled and uncoupled mitochondria and was unchanged when the rate of carbamoyl phosphate synthesis was increased 2-fold by incubating uncoupled mitochondria in the presence of 5 mM-N-acetylglutamate. According to the literature, the concentration of NH3 in liver is well below the measured apparent Km. On the basis of this and previous work we conclude that, quantitatively, changes in liver [NH3] and [ornithine] are likely to be the most important factors in the fast regulation of synthesis of carbamoyl phosphate and urea. This conclusion is consistent with all available evidence obtained with isolated mitochondria, isolated hepatocytes, perfused liver and whole animals.

Mitochondrial-reticular cytostructure in liver cells.

This study examines the structural relationship of mitochondria and the endoplasmic reticulum in liver. Livers of rat and Japanese quail were homogenized and fractionated in media of 0.25 M-sucrose, either 5mM or 50 mM in sodium Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid], pH 7.4 (2.2 mM or 22 mM in Na respectively), designated here as low- and high-salt media. Three particulate fractions were prepared by sequential centrifugation. A nuclear pellet sedimenting at 300 g was obtained as described by Shore & Tata [(1977) J. Cell Biol. 72, 714-725], and from the resulting supernatant thereof a low-speed pellet (1100-1500 g) and a high-speed pellet (8000-10 000 g) were prepared. In the low-salt medium the yields of mitochondrial matrix enzymes (citrate synthase, glutamate dehydrogenase, ornithine carbamoyltransferase) and their specific activities in the low-speed pellet were over twice those in the high-speed pellet. In the high-salt medium the yield of matrix enzymes was 4-5 times, and the specific activities were up to 3 times, higher in the low-speed pellet than in the high-speed pellet. Oxygen uptake and respiratory control ratio were also much higher in the low-speed pellets in both media. Some 50-65% of the microsomal marker enzyme glucose 6-phosphatase was in the supernatant from the high-speed pellet, and the rest sedimented with the mitochondria. Repeated washing with the high-salt medium removes only a limited amount of reticulum. Washing with salt-free sucrose removes most of the reticulum, but a fraction remains strongly bound to mitochondria. Homogenates from quail and rat liver were fractioned isopycnically on Percoll gradients in either 0.25 M-sucrose or 0.25 M-sucrose/50 mM-sodium Hepes. Up to five particulate bands were separated and assayed. Mitochondria were present in two to three bands and were associated with endoplasmic reticulum. As seen in the phase-contrast microscope the mitochondria prepared in the low-salt medium consist of separate organelles. In the high-salt medium the mitochondria appear as chains of from three to ten organelles not touching each other. On addition of univalent ions at concentrations above 20 mM, the mitochondria aggregate into chains, and at higher ionic strength larger multidimensional aggregates are formed. The dispersion and aggregation of mitochondria are reversible. Negatively stained electron micrographs reveal a branched mitochondrial structure, with mitochondria held together by strands of reticulum.(ABSTRACT TRUNCATED AT 400 WORDS)

Mitochondrial carbamyl phosphate and citrulline synthesis at high matrix acetylglutamate.

Matrix acetylglutamate of uncoupled rat liver mitochondria increased about 10-fold, to 4.3 nmol/microliters, upon incubation with 5 mM concentrations of that compound. Uncoupled mitochondria incubated with the reagents needed for carbamyl phosphate and citrulline synthesis and 5 mM acetylglutamate synthesized citrulline at velocities which reached 99 nmol/min/mg of protein; simultaneously, as much as 47 nmol/min/mg of carbamyl phosphate accumulated and was distributed between matrix and medium. Maximal total carbamyl phosphate synthesis was, therefore, 146 nmol/min/mg, similar to the activity measured in liver homogenates. Without added acetylglutamate, some carbamyl phosphate accumulated when citrulline synthesis was about 40 nmol/min/mg. The finding that ornithine transcarbamylase can be limiting for citrulline synthesis shows that the activity of this enzyme is greatly restricted in mitochondria. The stimulation by ornithine of mitochondrial carbamyl phosphate synthesis was prevented when ornithine transcarbamylase was inhibited more than 96% by 5 mM delta-N-phosphonacetyl-L-ornithine, suggesting that the normal stimulatory effect of ornithine on carbamyl phosphate synthetase occurs via ornithine transcarbamylase. Lower concentrations of delta-N-phosphonacetyl-L-ornithine were required to achieve a given inhibition of citrulline synthesis from added carbamyl phosphate from endogenously synthesized carbamyl phosphate. The results reported suggest the existence of interactions between carbamyl phosphate synthetase and ornithine transcarbamylase in the matrix.

The effects of ornithine on mitochondrial carbamyl phosphate synthesis.

When carbamyl phosphate is synthesized by isolated liver mitochondria in the absence of ornithine, the following is observed. 1. Carbamyl phosphate synthesis is progressively inhibited during the first 2 min of incubation, after which it reaches a steady rate which is 8% of that observed with ornithine. 2. Within 2 min, carbamyl phosphate accumulates in the matrix to very high levels (16 nmol/microliter) which are inhibitory to carbamyl phosphate synthetase; afterwards the levels decline, and by 15 min they are essentially the same as those found in the presence of ornithine (3 to 4 nmol/microliter). 3. The decrease in matrix carbamyl phosphate is the result of decreased synthesis and of leakage from mitochondria; 90% of the total carbamyl phosphate is in the medium after 10 min. 4. Addition of ornithine after 5 to 15 min results in rates of carbamyl phosphate synthesis essentially identical with those found when ornithine is added at the start of the incubation. 5. ATP synthesis appears to be somewhat inhibited. 6. When the accumulation of carbamyl phosphate in the medium is prevented by converting that compound to carbamyl aspartate, the total synthesis of carbamyl phosphate is not increased. Three factors appears to be involved in the low capacity of mitochondria to synthesize carbamyl phosphate in the absence of ornithine: a. inhibition of carbamyl phosphate synthetase by matrix carbamyl phosphate early in incubations; b. slight inhibition of ATP synthesis throughout the incubations; c. quantitatively most important, a very low activity of carbamyl phosphate synthetase even when matrix carbamyl phosphate is low and ATP is not limiting.

Citrulline synthesis in rat tissues and liver content of carbamoyl phosphate and ornithine.

Rat liver ornithine carbamoyltransferase appears to be located exclusively in the mitochondria; the activity that is found in the soluble fraction is indistinguishable from mitochondrial ornithine carbamoyltransferase by simple kinetic criteria, and seems to result from breakage of mitochondria during homogenization. Of several rat tissues studied, only the liver and the mucosa of small intestine contain significant amounts of ornithine carbamoyltransferase; the activity in intestinal mucosa is less than one thousandth of that in liver. Qualitatively, this distribution coincides with that of carbamoyl phosphate synthetase I and its cofactor, acetylglutamate. The rat liver contents of carbamoyl phosphate and ornithine were 0.1 and 0.15mumol/g wet wt. of tissue respectively. On the basis of these values, it is proposed that in vivo the ornithine carbamoyltransferase activity of liver may be much lower than its maximal activity in vitro might suggest.