The role of mitochondrially bound arginase in the regulation of urea synthesis: studies with [U-15N4]arginine, isolated mitochondria, and perfused rat liver.

The main goal of the current study was to elucidate the role of mitochondrial arginine metabolism in the regulation of N-acetylglutamate and urea synthesis. We hypothesized that arginine catabolism via mitochondrially bound arginase augments ureagenesis by supplying ornithine for net synthesis of citrulline, glutamate, N-acetylglutamate, and aspartate. [U-(15)N(4)]arginine was used as precursor and isolated mitochondria or liver perfusion as a model system to monitor arginine catabolism and the incorporation of (15)N into various intermediate metabolites of the urea cycle. The results indicate that approximately 8% of total mitochondrial arginase activity is located in the matrix, and 90% is located in the outer membrane. Experiments with isolated mitochondria showed that approximately 60-70% of external [U-(15)N(4)]arginine catabolism was recovered as (15)N-labeled ornithine, glutamate, N-acetylglutamate, citrulline, and aspartate. The production of (15)N-labeled metabolites was time- and dose-dependent. During liver perfusion, urea containing one (U(m+1)) or two (U(m+2)) (15)N was generated from perfusate [U-(15)N(4)]arginine. The output of U(m+2) was between 3 and 8% of total urea, consistent with the percentage of activity of matrix arginase. U(m+1) was formed following mitochondrial production of [(15)N]glutamate from [alpha,delta-(15)N(2)]ornithine and transamination of [(15)N]glutamate to [(15)N]aspartate. The latter is transported to cytosol and incorporated into argininosuccinate. Approximately 70, 75, 7, and 5% of hepatic ornithine, citrulline, N-acetylglutamate, and aspartate, respectively, were derived from perfusate [U-(15)N(4)]arginine. The results substantiate the hypothesis that intramitochondrial arginase, presumably the arginase-II isozyme, may play an important role in the regulation of hepatic ureagenesis by furnishing ornithine for net synthesis of N-acetylglutamate, citrulline, and aspartate.

nitric oxide-dependent and -independent pathways (5). Furthermore, arginine may regulate whole body nitrogen homeostasis following its up-regulation of hepatic N-acetylglutamate (NAG) 1 synthesis, and thereby ureagenesis (6 -11). However, as yet the mechanism(s) underlying the up-regulation of NAG is not completely understood. The liver is a major site for the uptake and metabolism of dietary arginine. Therefore, identification in the liver of the fate of arginine supplementation would enable understanding of the role of this amino acid in the regulation of NAG and urea synthesis as well as its action in normal and abnormal states.
In the urea cycle, arginine synthesis is balanced by arginine catabolism via the cytosolic arginase-I reaction. Thus, the hepatic urea cycle is not involved in the net synthesis or the net catabolism of arginine. Prior studies suggest that there is no equilibrium between dietary arginine and arginine formed in the urea cycle (12,13). Arginine entering the liver via the portal vein may be metabolized by four sets of enzymes: arginase, nitric-oxide synthase, arginine:glycine amidinotransferase, and arginine decarboxylase (14). Although arginine: glycine amidinotransferase can produce ornithine from arginine (14), arginase is the primary enzyme for generation of ornithine (5, 14 -18).
Ornithine is the key intermediary metabolite linking exogenous arginine with urea synthesis (13)(14)(15)(16)(17)(18). Thus, the catabolism of arginine via arginase may regulate urea synthesis by furnishing ornithine for synthesis of citrulline in the mitochondrial matrix. It has been shown that isolated mitochondria have a significant amount of arginase bound to the outer membrane (11,13). ϳ90% of the mitochondrially bound arginase was removed after washing of isolated mitochondria with 150 mM KCl (11). The remaining 10% of total arginase activity is present in the intramitochondrial matrix or membrane (11). More recent studies have identified this intramitochondrial arginase (IM-arginase) as the arginase-II isozyme (16, 19 -22). Arginase-II has a wide tissue distribution and may be involved in biosynthetic functions such as the formation of ornithine, glutamate, and polyamines (16, 19 -21). As yet, the extent of arginine hydrolysis to urea and ornithine via IM-arginase and its role in the regulation of NAG and ureagenesis are uncertain. In the current study we have examined the possibility that the IM-arginase is functionally advantageous for the synthesis of urea by providing ornithine for the synthesis of NAG and citrulline.
As illustrated in Fig. 1, we proposed that the mitochondrial catabolism of arginine via the IM-arginase reaction initiates a metabolic cascade that includes the formation of ornithine and thereby glutamate via the ornithine aminotransferase (OAT, EC 2.6.1.13) reaction. Concurrently, ornithine may be converted to citrulline, whereas glutamate would serve as precursor for both the synthesis of aspartate and NAG, an obligatory activator of carbamyl-phosphate synthetase-I (23). Aspartate would be transported into the cytosol to support the synthesis of argininosuccinate. Hence, the proposed metabolic cascade would result in increased: (i) availability of ornithine for mitochondrial synthesis of NAG and citrulline and (ii) availability of aspartate for cytosolic synthesis of argininosuccinate. This possibility is in accord with the notion that under physiological conditions ureagenesis depends upon mitochondrial ornithine availability and the transport of ornithine into hepatic mitochondria (24 -26). This is especially true when mitochondrial ornithine uptake is inhibited by other amino acids (26) or when the mitochondrial ornithine carrier is affected by H ϩ (27,28). Ureagenesis begins in mitochondria and finishes in the cytosol (18). Therefore, in the current studies we investigated hepatic arginine metabolism in isolated mitochondria and in a liver perfusion system. Experiments with isolated mitochondria and/or submitochondrial fractions provide valuable information concerning the location of arginase within the mitochondrion, its kinetic parameters, mitochondrial arginine metabolism, and the incorporation of its nitrogen into intermediates of the urea cycle (Fig. 1). Perfusion of the structurally intact liver with physiological concentrations of [U- 15 N 4 ]arginine and other metabolites would demonstrate the incorporation of the perfusate 15 N-labeled arginine into intermediates of the urea cycle and the production of 15 N-labeled urea isotopomers as illustrated in Fig. 1.
The possibility of hepatic zonation (29) is important in terms of the current hypothesis. It is not known whether arginase activity is located exclusively in the periportal hepatocytes, the site of urea synthesis, or may be present also in perivenous hepatocytes, the site of hepatic glutamine synthesis (29). If ornithine, the product of the arginase reaction, furnishes intermediates and/or activator (i.e. NAG) for urea synthesis, one would expect that the IM-arginase and the OAT reaction are located in close proximity to the zone where urea synthesis takes place. To address this question, separate perfusions were carried out with antegrade or retrograde flow, as previously indicated by Hä ussinger (29) and more recently by Brosnan and co-workers (30,31).
We used [U- 15 N 4 ]arginine as a precursor and gas chromatography-mass spectrometry (GC-MS) methodology to determine: 1) the time course of 15 N-labeled arginine catabolism in isolated mitochondria and, consequently, the incorporation of 15 N into glutamate, aspartate, citrulline, and NAG; 2) the dependence of 15 N-labeled metabolites production on arginine concentration; and 3) the relative uptake and catabolism of perfusate [U 15 N 4 ]arginine and thereby the production of 15 N-labeled metabolites and urea isotopomers by periportal hepatocytes (antegrade perfusion) or perivenous hepatocytes (retrograde perfusion).
The results demonstrate that in isolated mitochondria and liver perfusions 15 N-labeled ornithine, glutamate, citrulline, NAG, and urea were formed from external L-[U- 15 N 4 ]arginine. The data suggest that the catabolism of arginine via the IMarginase augments urea synthesis by furnishing the intermediate metabolites necessary for urea synthesis.

EXPERIMENTAL PROCEDURES
Materials and Animals-Male Sprague-Dawley rats (Charles River) were fed ad lib a standard rat chow diet. The chemicals were of analytical grade and were obtained from Sigma-Aldrich. Enzymes and cofactors for the analysis of urea, lactate, pyruvate, and ammonia were obtained from Sigma. L-[Guanidino-15 N 2 ]arginine, 99 mol % excess (MPE), was from Isotec, and L-[U- 15 N 4 ]arginine (99 MPE) was from ICON.
Preparation of Rat Liver Mitochondria and Mitochondrial Matrix-Mitochondria were isolated from the livers of overnight fasted rats by differential centrifugation as previously described (32). Briefly, the liver of an anesthetized rat was cannulated through the portal vein, rinsed with 0.9% NaCl solution (4°C), excised, and weighed. The minced liver was homogenized in a glass Potter-Elvehjem homogenizer with a Teflon pestle in 12.5 volumes of cooled isolation buffer consisting of 225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 5 mM Hepes, pH 7.4. The mitochondrial pellet was gently resuspended with isolation buffer to yield 50 -80 mg protein/ml. All of the manipulations were performed in a cold room, and the mitochondrial suspension was kept in an ice bath.
In a separate series of experiments, the mitochondrial pellet was washed twice: once with 150 mM KCl to remove arginase bound to the outer membrane of mitochondria as previously described (11) and then with isolation buffer to remove any residue of KCl. A separate preparation of KCl-washed mitochondria was used to obtain mitochondrial matrix. To this end, the KCl-washed mitochondrial pellet was resuspended in basic incubation medium (as indicated below) and subjected to three cycles of freezing in liquid nitrogen and thawing, as described (33). Mitochondrial membrane was separated from the matrix following centrifugation for 20 min at 40,000 ϫ g (33). Matrix was removed and added to the incubation flasks (as indicated below).
Metabolic Studies with Isolated Mitochondria-Mitochondrial suspension (3-4 mg protein/ml) was incubated in Erlenmeyer flasks (final volume, 2 ml) at 30°C, in a shaking water bath for the times with the addition of 15  To determine the time course of 15 N-labeled arginine catabolism and the incorporation of 15 N into other metabolites, the experiments were performed with KCl-washed and unwashed mitochondria. The incubations were carried out with basic medium and 2 mM L-[U- 15 N 4 ]arginine. This [Arg] is similar to that found in mitochondria isolated from livers obtained from overnight fasted rats (34). To establish the dependence of intermediary metabolites production on arginine concentration, mitochondrial incubations were carried out for 20 min with basic medium and with increasing concentrations (0 -5 mM) of [U-15 N 4 ]arginine.
At the indicated time, an aliquot (100 l) was taken for protein determination, and incubation was stopped with 100 -150 l of HClO 4 (60%). Metabolite measurements were done in neutralized extracts. Three to five independent experiments were carried out for each series.
Measurement of Mitochondrial Respiration-Respiratory control and oxygen consumption were determined in each mitochondrial preparation as previously described (34). Oxygen consumption usually was 2-3, 9 -12, and 2-3 nmol of O 2 /min/mg of protein for states 2, 3, and 4, respectively, and the V 3 /V 2 or V 3 /V 4 ratio was between 3 and 4. Metabolic studies were carried out with mitochondria having a V 3 /V 2 ratio greater than 3.
Experiments with Liver Perfusions-Livers from overnight fasted male rats were perfused in the nonrecirculating mode as previously described (12,35). We employed either antegrade or retrograde flow at the rate of 3-3.5 ml/g liver. The basic perfusion medium was Krebs saline (pH 7.4), continuously gassed with 95% O 2 , 5% CO 2 and containing lactate (2.1 mM) and pyruvate (0.3 mM) as metabolic fuels. pO 2 (in influent and effluent media) was monitored throughout, and oxygen consumption was calculated.
The first series of antegrade or retrograde single-pass perfusion was carried out with physiological concentrations of arginine and NH 4 Cl as nitrogen sources. After 20 min of preperfusion, the basic perfusion medium was replaced by one that contained, in addition to the lactate and pyruvate, 0. In each of the experiments outlined above, the perfusion was continued for 50 min. Samples were taken from the influent and effluent media for chemical and GC-MS analyses. At the end of the perfusion, the liver was freeze-clamped with aluminum tongs precooled in liquid N 2 . The frozen liver was ground into a fine powder, extracted into perchloric acid, and used for metabolite determination and 15 N enrichment. For measurement of the 15 N enrichment in urea or amino acids, the samples were prepared as previously described (35)(36)(37). Briefly, an aliquot of effluent, liver, or mitochondrial extract was purified by passage on an AG-50 (H ϩ ; 100 -200 mesh; 0.5 ϫ 2.5 cm) column and then converted into the t-butyldimethylsilyl derivatives. The m/z 231, 232, 233, and 234 ions of the urea t-butyldimethylsilyl derivative were monitored for singly (U mϩ1 ) and doubly (U mϩ2 ) 15 N-labeled urea isotopomers. Isotopic enrichment in glutamate, aspartate, and alanine was monitored using ratios of ions at m/z 433/432, 419/418, and 261/260, respectively. Enrichment in [2-15 N]glutamine was determined by monitoring m/z 259/258, and [5-15 N]glutamine was monitored by determining the difference between the m/z 432/431 and m/z 259/258 ratios. Additionally, doubly labeled glutamine was measured using the m/z 433/431 ratios.

GC-MS Methodology and Determination of 15 N-Labeled Metabolites-GC-MS
Because t-butyldimethylsilyl derivatives of arginine and citrulline have identical fragmentation and are not completely separated during GC-MS analysis, enrichment in [U-15 N 4 ]arginine, [␣,␦-15 N 2 ]ornithine, and [␣,␦-15 N 2 ]citrulline was determined using the N-dimethylaminomethylene methyl ester derivative (38). Briefly, an aliquot of a tissue extract was purified via an AG-50 (H ϩ ; 100 -200 mesh; 0.5 ϫ 2.5 cm) column, dried, and then converted into N-dimethylaminomethylene methyl ester derivatives (38). 15  The concentration and 15 N enrichment in N-acetylglutamate in liver or mitochondrial extracts were determined using GC-MS and an isotope dilution approach (12). In a few cases samples were spiked with 15 Nlabeled NAG, and the NAG concentration was calculated as indicated (40). The formation of [ 15 N]ammonia was determined as previously described (41). The concentration of amino acids was determined by high pressure liquid chromatography, utilizing precolumn derivatization with o-phthalaldehyde (42). The levels of ammonia and urea were measured (12,35). However, we have found that the colorimetric measurement of urea with phenol and hypochlorite is not sufficiently sensitive for determination of a low urea level in experiments with isolated mitochondria. Therefore, after the determination of the initial 15 N enrichment in urea (I 1 ), as indicated above, an aliquot (50 l) of mitochondrial extract was spiked with unlabeled urea, and a second (I 2 ) GC-MS measurement of 15 N enrichment was performed. The concentration of urea was calculated by isotope dilution as indicated for NAG.
Calculations and Statistical Analyses-Data obtained from mitochondrial incubations were analyzed with GraphPad Prism 4 software for linear and nonlinear curve fitting. Catabolism of [U- 15 N 4 ]arginine during the course of the incubation was fitted to a single exponential decay Y ϭ (I 0 Ϫ I 2 )e (Ϫkt) , where, I 0 represents the time 0 intercept and I 2 represents the concentration of [U- 15 N 4 ]arginine at the end of the incubation, respectively. The rate (nmol/min/mg protein) of [U-15 N 4 ]arginine catabolism (C r ) was calculated by the product of (I 0 Ϫ I 2 )*k. The production of [ 15 N 2 ]urea from [U- 15 N 4 ]arginine was used to calculate the flux through arginase (Q A ). In most cases, the rate of 15 N-labeled metabolites production (MP R ) was fitted to a one-phase exponential association (Y ϭ Y max *(1 Ϫ e (Ϫkt) )), and the production rate (nmol/min/mg protein) of each metabolite was calculated by the product of the corresponding Y max *k. In a few cases the production of 15 Nlabeled metabolites was best fitted to a linear regression analysis, and the production of 15 N-labeled metabolites was determined from the slope of the regression lines. The GraphPad Prism 4 software was also used to determine the best curve fit and to calculate the V max for the flux through arginase and the production of 15 N-labeled metabolites as well as the K m for arginine in experiments with an increasing concentration of arginine.
During liver perfusions, the rate of precursor-N uptake or the output of metabolites was determined by the measurement of metabolite concentration in the influent and effluent (nmol/ml), normalized to the flow rate (ml/min) and liver wet weight (35). The output of 15 N-labeled metabolites was calculated by the product of 15 N enrichment (MPE/100) times concentration (nmol/min/g wet wt) and is expressed as nmol 15 N metabolite/min/g wet wt.
The rate of perfusate arginine catabolism via the arginase reaction is represented by the output in the effluent of Each series of experiments was repeated three to five times with different mitochondrial preparations or three to four times with liver perfusion as outlined above. Statistical analysis was carried out using In-STAT 1.14 software for the Macintosh. A Student's t test or an analysis of variance test was employed to compare two groups or differences among groups as needed. A p value less than 0.05 was taken as indicating a statistically significant difference.

RESULTS AND DISCUSSION
An important feature of the current studies is the use of [U-15 N 4 ]arginine as a tracer to evaluate hepatic arginine metabolism and in particular the incorporation of 15 N into intermediary metabolites of the urea cycle by isolated mitochondria or a liver perfusion system. The sensitivity and precision of GC-MS make it a superb methodology to identify the primary metabolites of [U-15 N 4 ]arginine and their role in the regulation of urea synthesis, an advantage we have utilized in our previous studies dealing with the regulation of hepatic ureagenesis (12,(35)(36)(37)41).
Prior studies proposed that arginine acts as an allosteric activator of mitochondrial N-acetylglutamate synthetase (18). However, this concept has been the subject of disagreement (6 -11, 43, 44). Recently, we have proposed that agmatine, the product of the arginine decarboxylase reaction, may augment NAG synthesis and thereby urea synthesis (12). In the current investigation we have explored an alternative, but not mutually exclusive, possibility. As illustrated in Fig. 1, we propose that arginine entering the liver via the portal vein is metabolized by IM-arginase to form ornithine and urea. Subsequently, ornithine serves as substrate for glutamate and citrulline synthesis. Glutamate so formed is then used for the synthesis of NAG and/or is transaminated to form aspartate for the synthesis of argininosuccinate. Taken together, the current results support the hypothesis that mitochondrial arginine metabolism up-regulates ureagenesis by furnishing ornithine for net synthesis of NAG, citrulline, and aspartate. The following findings bear directly on this mechanism of arginine stimulation of hepatic urea synthesis.
Characterization of Mitochondrially Bound Arginase-The initial series of experiments was designed to determine the activity of arginase in submitochondrial fractions. In KClwashed mitochondria, unwashed mitochondria, mitochondrial matrix of KCl-washed mitochondria, and KCl-containing supernatant, arginase activity was 11.4, 94, 7.1, and 87 nmol/ min/mg protein, respectively. In unwashed mitochondria, the half-maximal activity of arginase (K m ) was achieved with [ar-ginine] at 10.4 Ϯ 1.1 mM with a V max of 657 Ϯ 24 nmol/min/mg protein. In KCl-washed mitochondria the K m value was 1.27 Ϯ 0.07 mM with a V max of 34 Ϯ 0.5 nmol/min/mg protein. These values are in good agreement with the prior study of Cheung and Raijman (11). The current study demonstrates that ϳ90% of mitochondrially bound arginase is located in the outer membrane, and 8% is in the mitochondrial matrix. Furthermore, the arginase activity in KCl-washed mitochondria is approximately that of the activity in isolated matrix. Thus, the arginase bound to the outer mitochondrial membrane presumably represents the activity of the arginase-I isozyme, whereas arginase in KCl-washed mitochondria, which is mainly located in the matrix, probably represents the activity of the arginase-II isozyme. This conclusion is in agreement with numerous studies indicating that the cytosolic arginase-I isozyme is located near the outer mitochondrial membrane (11,13,43), and the arginase-II isozyme is primarily intramitochondrial (19 -22).
Mitochondrial Catabolism of [U- 15 N 4 ]Arginine and Production of 15 Table I. In KCl-washed and unwashed mitochondria, the catabolism of [U-15 N 4 ]arginine was best fitted to a single exponential decay curve (r 2 ϭ 0.99; Fig. 2A). The ratio between the catab-olism rate (C r ) of [U- 15 N 4 ]arginine in KCl-washed and unwashed mitochondria was ϳ0.08 (Table I), in good agreement with matrix arginase activity as a fraction of total mitochondrially bound arginase. The production of 15 N-labeled urea and ornithine was inversely correlated to the disappearance of [U-15 N 4 ]arginine (Fig. 2). In KCl-washed mitochondria, 15 Nlabeled urea and ornithine increased almost linearly throughout the course of the incubation. In unwashed mitochondria, 15 N-labeled urea and ornithine increased linearly up to 7-10 min and then reached a plateau between 10 and 30 min (Fig. 2). The T1 ⁄2 values of arginine catabolism in experiments with KCl-washed and unwashed mitochondria were ϳ17 and 2 min, respectively. Hence, the plateau production of 15  the incubation (Fig. 2A). Because ornithine serves as an intermediary metabolite and is further metabolized in mitochondria (Fig. 1), the formation of [ 15 N 2 ]urea, an end product, was used to calculate the flux through the arginase reaction (Q A ). In KCl-washed and unwashed mitochondria, the values of Q A are 9.6 and 144 nmol/min/mg protein, respectively (Table I). The ratio between Q A values in experiments with KCl-washed and unwashed mitochondria is ϳ0.07, in good agreement with the percentage of matrix arginase of the total mitochondrially bound arginase velocity.  (Fig. 3B). The data indicate a rapid equilibrium between 15 N-labeled glutamate and aspartate. The production of aspartate in mitochondria and its translocation into cytosol may serve as a major source for argininosuccinate synthesis, consistent with a previous study using a liver perfusion system (35).
[ 15 N]Glutamate generated from [␣,␦-15 N 2 ]ornithine also was used to synthesize 15 N-labeled NAG by mitochondrial N-acetylglutamate synthetase (Fig. 3C). The addition to the medium of octanoic acid and CoA provided most acetyl-CoA required for NAG synthesis, a finding confirmed in experiments with 13 Clabeled octanoic acid (data not shown). In KCl-washed mitochondria, the production of 15 N-labeled NAG shows a lag period of ϳ5-7 min and then a linear increase during the course of the incubation (Fig. 3C). In unwashed mitochondria, the production of 15 N-labeled NAG linearly increased between 0 and 20 min and then reached a plateau. In both KCl-washed and unwashed mitochondria, the production of 15 N-labeled NAG (Fig. 3C) was linearly correlated to the production of [ 15 N]glutamate (Fig. 3A), indicating a dependence of NAG synthesis on mitochondrial [glutamate].
The mitochondrial level of NAG was ϳ0.3-0.4 nmol/mg protein. In experiments with unwashed mitochondria, this value was increased to ϳ1.2-1.5 nmol/mg (sum of 15 (Table I), fall within the physiological range and within the range of 4 -50 nmol/min/mg previously reported when isolated mitochondria were incubated with ornithine (46). Thus, the current observations, together with earlier data (6 -13, 25, 26, 43, 46), demonstrate that mitochondrial arginine catabolism provides ornithine and glutamate to promote NAG and citrulline synthesis. In addition, the current data demonstrate a linear relationship between the production of 15 N-labeled ornithine, NAG, and citrulline during the course of incubations (Figs. 2 and 3) or with an increasing concentration of [U-15 N 4 ]arginine (Fig. 4), consistent with the notion that the availability of mitochondrial ornithine supports the synthesis of citrulline and thus ureagenesis (24 -26, 28, 43, 46).  [U-15 N 4 ]arginine catabolism and production of 15 N-labeled metabolites during the course of mitochondrial incubation Production of 15 N-labeled metabolites (MP R ) by KCl-washed and unwashed mitochondria incubated with basic medium and 2 mM ͓U-15 N 4 ͔arginine as detailed under "Experimental Procedures." The rate constant for ͓U-15 N 4 ͔arginine catabolism (one-phase exponential decay) and production rates of 15 N-labeled metabolites were calculated with GraphPad Prism 4 software for linear or nonlinear (one-phase exponential) curve fitting using the data presented in Figs. 2 and 3. The data are the means Ϯ S.D. of three to five independent experiments. The k values are not applicable (NA), and in these cases MP R was calculated by the slope of the linear increase (7-30 min) of 15 N-labeled metabolites production after the lag period of approximately 5-7 min (Fig. 3).
The synthesis of citrulline was used as proxy for carbamyl phosphate synthesis (10,25,45). In both KCl-washed and unwashed mitochondria, the time course of [␣,␦-15 N 2 ]citrulline production was best fitted to a one phase exponential curve, reaching a similar Y max after ϳ20 min of incubation with [U-15 N 4 ]arginine (Fig. 3D). In experiments with unwashed mitochondria, which are similar to those previously described (9), the maximum production of [␣,␦-15 N 2 ]citrulline and thereby carbamyl phosphate occurred when [ 15 N-NAG] was ϳ150 pmol/mg mitochondrial protein (Fig. 3, C and D). In KCl-washed mitochondria the rate of [␣,␦-15 N 2 ]citrulline production was 2.24 Ϯ 0.2 nmol/min/mg protein, and in unwashed mitochondria it was 7.4 Ϯ 0.8 nmol/min/mg protein (Table I). These values are consistent with a previous study using isolated mitochondria of mouse or rat liver and 2 mM arginine (9). Furthermore, in unwashed mitochondria, the half-maximal synthesis of 15 N-labeled citrulline and NAG was achieved with ϳ0.11 and 0.47 mM arginine, respectively. The V max was 35 and 0.61 nmol/min/mg for citrulline and NAG, respectively (Table II). These values are in agreement with the K m and V max values obtained when isolated mitochondria were incubated with increasing concentrations of arginine (9). However, previous studies were unable to identify the mechanism(s) by which arginine stimulates NAG synthesis because the earlier investigations did not trace the metabolic fate of arginine, as was done in this study.
An observation of special importance is that the V max values for [␣,␦-15 N 2 ]citrulline synthesis are about the same in KClwashed and unwashed mitochondria (Table II). Thus, the catabolism of [U- 15 N 4 Table II) is sufficient to provide the required ornithine for citrulline synthesis. Therefore, although the IMarginase isozyme constitutes ϳ8% of the total mitochondrially bound arginase, it provides an adequate amount of ornithine for intramitochondrial synthesis of NAG and citrulline and thereby may have a key role in the up-regulation of ureagenesis. These observations, together with the time and dose dependence of [U-15 N 4 ]arginine incorporation into NAG, citrulline, and aspartate (Figs. 3 and 4 and Tables I and II), are in line with the hypothesis that mitochondrial arginine metabolism up-regulates ureagenesis by furnishing ornithine for net synthesis of NAG, citrulline, and aspartate.

]arginine via the IM-arginase reaction (Figs. 3 and 4 and
Characterization of [U- 15 N 4 ]Arginine Catabolism in the Perfused Liver-To demonstrate the incorporation of 15 N-labeled arginine into intermediates of the urea cycle and the production of U mϩ1 (Fig. 1) in an intact organ, the experiments were carried out with a liver perfusion system. We first employed a series of antegrade or retrograde single-pass perfusions with physiological concentrations of [U-15 N 4 ]arginine (0.25 mM) and unlabeled NH 4 Cl (0.3 mM) as a nitrogen source for urea synthesis. A second series of perfusions was performed with the addition of glutamine and ammonia to determine hepatic uptake and catabolism of arginine with an optimal supplementation of nitrogen sources for urea synthesis. Glutamine is a major precursor for urea nitrogen as well as a source of glutamate for NAG synthesis (36,37). Thus, the addition of unlabeled glutamine would provide information regarding the relationship between the activity of the urea cycle and the metabolism of perfusate [U-15 N 4 ]arginine. Results for this series of experiments are presented in Figs. 5-7. In the presence of 0.3 mM unlabeled ammonia, the uptake of [U-15 N 4 ]arginine was ϳ70 nmol/min/g (Fig. 5A). The direction of perfusion did not affect the uptake of arginine nor the flow rate (ϳ3-3.5 ml/min), oxygen consumption (ϳ2.5-3 mol/min/g), or total urea output (ϳ450 -500 nmol/min/g). Similarly, prior studies have shown that arginine metabolism, and thereby urea output and 14 CO 2 production from perfusate [U-14 C]arginine, were the same when livers were perfused in an antegrade or retrograde direction (30). Therefore, from this series of experiments we present only data  Similarly, in perfusions with glutamine, the uptake of [U- 15 N 4 ]arginine was ϳ80 nmol/min/g (Fig. 6A), indicating that glutamine did not affect hepatic uptake of arginine. In addition, the direction of perfusion did not affect the uptake of arginine (Fig. 6A) or oxygen consumption (ϳ2.5-3 mol/min/g). However, total urea output was ϳ2-fold higher with antegrade (ϳ800 nmol/min/g) versus retrograde (ϳ400 nmol/min/g) perfu-sions, demonstrating the high capacity of the periportal hepatocytes versus the perivenous hepatocytes in producing urea when glutamine was added to the perfusate.
Figs. 5 and 6 demonstrate an immediate production and output of the U mϩ2 isotopomer. The output of U mϩ2 must have occurred secondary to the catabolism of [U- 15 N 4 ]arginine to [ 15 N 2 ]urea and [␣,␦-15 N 2 ]ornithine. With antegrade flow, the enrichment in U mϩ2 was ϳ3 and 8 MPE in the presence and absence of glutamine in the perfusate, respectively. The rate of U mϩ2 was ϳ30 nmol/min/g with or without the addition of glutamine (Figs. 5B and 6B). Thus, the output of U mϩ2 was between 3 and 8% of total urea output. This fraction of U mϩ2 of total urea output is in good agreement with the velocity of matrix arginase in KCl-washed mitochondria, suggesting that the IM-arginase reaction may be responsible for generation of U mϩ2 . If perfusate [U- 15 N 4 ]arginine equilibrated with arginine formed in the urea cycle, then the enrichment in U mϩ2 would be above 50 MPE, similar to the enrichment in [U- 15 N 4 ]arginine in the freeze-clamped liver extract (Fig. 7). However, the data in Figs. 5-7 demonstrate that perfusate arginine did not equilibrate with arginine formed in the urea cycle.
An important question is whether during liver perfusion the output of U mϩ2 represents the activity of arginase-II or a mixture of arginase-I and arginase-II isozymes. The structure and function of both isozymes are nearly identical, and the available inhibitors (i.e. boronic acid, N -hydroxy-L-arginine) act similarly on both (22). Although antibodies against the arginase-I isozyme were previously used to differentiate between arginase-I and arginase-II (47), this approach cannot be used to determine the relative flux through each isozyme during liver perfusion. Thus, the current study cannot definitely determine whether during liver perfusion the production of U mϩ2 was solely mediated via the arginase-II reaction. On the other hand, the finding that the fraction of total urea output comprised by U mϩ2 (Figs. 5B and 6B) agrees closely with the percentage of activity of matrix arginase suggests that U mϩ2 was derived via the IM-arginase reaction. It was proposed that the IM-arginase might be associated with the ornithine transporter in the mitochondrial membrane (28). This arrangement may channel external arginine to arginase-II, thereby providing efficient mitochondrial production of ornithine, similar to the channeling between the argininosuccinate lyase and arginase-I reactions (13). The current study demonstrates that IM-arginase abets the synthesis of urea, as illustrated in Fig. 1. This conclusion is strongly supported by the data in Figs. 5-7, which demonstrates the production of [ 15 N]aspartate and U mϩ1 during liver perfusion with [U-15 N 4 ]arginine.
U mϩ1 was formed only during perfusion in the antegrade flow direction (Figs. 5B and 6B), apparently in periportal hepatocytes, the site of urea synthesis. As illustrated in Fig. 1 (Figs. 5B and 6B). In perfusions without glutamine, enrichment in U mϩ1 was ϳ2 MPE, indicating that ϳ5% of aspartate required for urea synthesis was derived from perfusate [U-15 N 4 ]arginine, and ϳ95% was from perfusate ammonia and internal sources, including proteolysis as previously suggested (35,41). In perfusions with unlabeled glutamine, the isotopic enrichment in U mϩ1 was ϳ1 MPE, indicating that in the presence of glutamine and ammonia, ϳ3% of aspartate utilized for urea synthesis was derived from perfusate [U-15 N 4 ]arginine.
Further evidence supporting the above conclusion is provided in Fig. 7, which represents the enrichment data for nitrogen-containing metabolites in freeze-clamped livers at the end of antegrade perfusions. It is evident that, when [U- 15 N 4 ]arginine was the labeled precursor, 15 N was incorporated into glutamate, aspartate, citrulline, and N-acetylglutamate. In perfusions without glutamine (Fig. 7A), the ratio between 15 N-labeled ornithine, citrulline, NAG, and aspartate and the precursor, [U- 15 N 4 ]arginine, indicates that ϳ70, 75, 8, 7, and 5% of hepatic ornithine, citrulline, glutamate, NAG, and aspartate, respectively, were derived from perfusate [U-15 N 4 ]arginine. Similar calculations in perfusions with glutamine (Fig. 7B), indicate that ϳ70, 65, 5, 3, and 7% of hepatic ornithine, citrulline, glutamate, aspartate, and NAG, respectively, were derived from perfusate [U-15 N 4 ]arginine. These values are not significantly different from the values obtained with perfusions without glutamine. Therefore, 0.25 mM perfusate arginine furnished between 7 and 10% of the hepatic NAG and 3-5% of the hepatic aspartate pool, even in the presence of glutamine, the major source of hepatic glutamate and thus NAG (36). Because the production of 15 N-labeled NAG and other metabolites was dose-dependent (Fig. 4), the production of NAG and/or aspartate would probably be many fold higher with therapeutic doses of arginine.
The current findings also provide a clue regarding the location of arginase-II and/or the OAT reaction within the liver acinus. Data in Figs. 5 and 6 demonstrate little differences in arginine uptake during liver perfusions with antegrade or retrograde flow. However, the output of U mϩ2 was ϳ2-fold higher with retrograde compared with antegrade flow, suggesting a 2-fold higher activity of arginase-II in perivenous than in periportal hepatocytes. Similarly, 15 N-labeled glutamate, NAG, citrulline, and aspartate from perfusate [U-15 N 4 ]arginine were generated both in antegrade or retrograde flow, indicating that OAT activity is present in both perivenous and periportal regions. This differs from the finding by Darnell and co-workers (48) suggesting that the OAT mRNA is mainly located in the perivenous hepatocytes where glutamine is formed. In the prior study experiments were carried out with mouse tissue, and the differences may reflect species differences in the expression and/or location of the OAT mRNA. Additionally, the in situ mRNA analysis used by Darnell and co-workers (48)  necessarily reflect the presence or absence of metabolic reactions as demonstrated here with 15 N-labeled arginine and GC-MS methodology. An additional point of interest is that we did not detect 15 N-labeled glutamine output during perfusions with [U- 15 N 4 ]arginine plus unlabeled ammonia, neither with retrograde nor antegrade flow. It is possible that the 15 N enrichment in glutamine was below the GC-MS detection limit (ϳ0.5 MPE).
In summary, the current study provides strong evidence to support the hypothesis that arginine stimulates ureagenesis secondary to its catabolism via intramitochondrial arginase, thereby supplying ornithine for synthesis of citrulline, glutamate, NAG, and aspartate. We previously demonstrated that agmatine, the product of arginine decarboxylation, stimulated the synthesis of NAG (12). The current findings, demonstrating that arginine furnishes substrates for NAG, citrulline, and aspartate, offer an additional and/or an alternative mechanism by which arginine may stimulate NAG synthesis and thus up-regulate ureagenesis.