Arginase induction by sodium phenylbutyrate in mouse tissues and human cell lines.

Hyperargininemia is a urea cycle disorder caused by mutations in the gene for arginase I (AI) resulting in elevated blood arginine and ammonia levels. Sodium phenylacetate and a precursor, sodium phenylbutyrate (NaPB) have been used to lower ammonia, conjugating glutamine to produce phenylacetylglutamine which is excreted in urine. The elevated arginine levels induce the second arginase (AII) in patient kidney and kidney tissue culture. It has been shown that NaPB increases expression of some target genes and we tested its effect on arginase induction. Eight 9-week old male mice fed on chow containing 7.5 g NaPB/kg rodent chow and drank water with 10 g NaPB/L, and four control mice had a normal diet. After one week all mice were sacrificed. The arginase specific activities for control and NaPB mice, respectively, were 38.2 and 59.4 U/mg in liver, 0.33 and 0.42 U/mg in kidney, and 0.29 and 1.19 U/mg in brain. Immunoprecipitation of arginase in each tissue with AI and AII antibodies showed the activity induced by NaPB is mostly AI. AII may also be induced in kidney. AI accounts for the fourfold increased activity in brain. In some cell lines, NaPB increased arginase activity up to fivefold depending on dose (1-5 mM) and exposure time (2-5 days); control and NaPB activities, respectively, are: erythroleukemia, HEL, 0.06 and 0.31 U/mg, and K562, 0.46 and 1.74 U/mg; embryonic kidney, HEK293, 1.98 and 3.58 U/mg; breast adenocarcinoma, MDA-MB-468, 1.11 and 4.06 U/mg; and prostate adenocarcinoma, PC-3, 0.55 and 3.20 U/mg. In MDA-MB-468 and HEK most, but not all, of the induced activity is AI. These studies suggest that NaPB may induce AI when used to treat urea cycle disorders. It is relatively less useful in AI deficiency, although it could have some effect in those patients with missense mutations.

Expression of arginase isozymes in mouse brain.

The two forms of arginase (AI and AII) in man, identical in enzymatic function, are encoded in separate genes and are expressed differentially in various tissues. AI is expressed predominantly in the liver cytosol and is thought to function primarily to detoxify ammonia as part of the urea cycle. AII, in contrast, is predominantly mitochondrial, is more widely expressed, and is thought to function primarily to produce ornithine. Ornithine is a precursor in the synthesis of proline, glutamate, and polyamines. This study was undertaken to explore the cellular and regional distribution of AI and AII expression in brain using in situ hybridization and immunohistochemistry. AI and AII were detected only in neurons and not in glial cells. AI presented stronger expression than AII, but AII was generally coexpressed with AI in most cells studied. Expression was particularly high in the cerebral cortex, cerebellum, pons, medulla, and spinal cord neurons. Glutamic acid decarboxylase 65 and glutamic acid decarboxylase 67, postulated to be related to the risk of glutamate excitotoxic and/or gamma-aminobutyric acid inhibitoxic injury, were similarly ubiquitous in their expression and generally paralleled arginase expression patterns, especially in cerebral cortex, hippocampus, cerebellum, pons, medulla, and spinal cord. This study showed that AI is expressed in the mouse brain, and more strongly than AII, and sheds light on the anatomic basis for the arginine-->ornithine-->glutamate-->GABA pathway.

The human arginases and arginase deficiency.

Arginase is the final enzyme in the urea cycle. Its deficiency is the least frequently described disorder of this cycle. It results primarily in elevated blood arginine, and less frequently in either persistent or acute elevations in blood ammonia. This appears to be due to a second arginase locus, expressed primarily in the kidney, which can be recruited to compensate, in part, for the deficiency of liver arginase. The liver arginase gene structure permitted study of the molecular pathology of patients with the disorder and the results of these studies and the inferences about the protein structure are presented. The conserved regions among all arginases allowed the cloning of AII, the second arginase isoform. It has been localized to the mitochondrion and is thought to be involved in ornithine biosynthesis. It shares the major conserved protein sequences, and structural features of liver arginase gene are also conserved. When AI and AII from various species are compared, it appears that the two diverged some time prior to the evolution of amphibians. The evidence for the role of AII in nitric oxide and polyamine metabolism is presented and this appears consonant with the data on the tissue distribution.

Cloning and characterization of the mouse and rat type II arginase genes.

Two forms of arginase, both catalyzing the hydrolysis of arginine to ornithine and urea, are found in animals ranging from amphibians to mammals. In humans, inherited deficiency of hepatic or type I arginase results in hyperargininemia, a syndrome characterized by periodic episodes of hyperammonemia, spasticity, and neurological deterioration. In these patients, a second extrahepatic or type II arginase activity is significantly increased, an induction that may partially compensate for the lack of AI activity and apparently mitigates some of the clinical effects of the condition. Cloning and characterization of the human AII cDNA was recently accomplished. The cloning, sequencing, and partial characterization of the mouse and rat AII cDNAs are reported herein. The DNA sequences predicted polypeptides of 354 amino acids, including a N-terminal mitochondrial import signal. Sequence homology to the human type II arginase, arginase activity data, and immunoprecipitation with an anti-AII antibody confirm the identity of these cloned genes as rodent extrahepatic type II arginases.

Cloning and characterization of the human type II arginase gene.

There are two forms of arginase in humans, both catalyzing the hydrolysis of arginine to ornithine and urea. Recent studies in animal models and in Type I arginase-deficient patients suggest that Type II arginase is inducible and may play an important role in the regulation of extra-urea cycle arginine metabolism by modulating cellular arginine concentrations. We PCR amplified and cloned the human Type II arginase gene, the first nonliver arginase gene reported in mammals. While sequence homology to Type I arginase, arginase activity data, and immunoprecipitation with an anti-AII antibody confirm the identity of this gene, Northern blot analysis demonstrates its differential expression in the brain, prostate, and kidney. Type II arginase may be an important part of the arginine regulatory system affecting nitric oxide synthase, arginine decarboxylase, kyotorphin synthase, and arginine-glycine transaminase activities and polyamine and proline biosynthesis.

Delivery of cytosolic liver arginase into the mitochondrial matrix space: a possible novel site for gene replacement therapy.

As a toxic metabolic byproduct in mammals, excess ammonia is converted into urea by a series of five enzymatic reactions in the liver that constitute the urea cycle. A portion of this cycle takes place in the mitochondria, while the remainder is cytosolic. Liver arginase (L-arginine ureahydrolase, A1) is the fifth enzyme of the cycle, catalyzing the hydrolysis of arginine to ornithine and urea within the cytosol. Patients deficient in this enzyme exhibit hyperargininemia with episodic hyperammonemia and long-term effects of mental retardation and spasticity. However, the hyperammonemic effects are not so catastrophic in arginase deficiency as compared to other urea cycle defects. Earlier studies have suggested that this is due to the mitigating effect of a second isozyme of arginase (AII) expressed predominantly in the kidney and localized within the mitochondria. In order to explore the curious dual evolution of these two isozymes, and the ways in which the intriguing, aspects of AII physiology might be exploited for gene replacement therapy of AI deficiency, the cloned cDNA for human AI was inserted into an expression vector downstream from the mitochondrial targeting leader sequence for the mitochondrial enzyme ornithine transcarbamylase and transfected into a variety of recipient cell types. AI expression in the target cells was confirmed by northern blot analysis, and competition and immunoprecipitation studies showed successful translocation of the exogenous AI enzyme into the transfected cell mitochondria. Stability studies demonstrated that the translocated enzyme had a longer half-life than either native cytosolic AI or mitochondrial AII. Incubation of the transfected cells with increasing amounts of arginine produced enhanced levels of mitochondrial AI activity, a substrate-induced effect that we have previously seen with native AII but never AI. Along with exploring the basic biological questions of regulation and subcellular localization in this unique dual-enzyme system, these results suggest that the mitochondrial matrix space may be a preferred site for delivery of enzymes in gene replacement therapy.

Loss of function mutations in conserved regions of the human arginase I gene.

We have utilized SSCP analysis to identify disease-causing mutations in a cohort with arginase deficiency. Each of the patient's mutations was reconstructed in vitro by site-directed mutagenesis to determine the effect of the mutations on enzyme activity. In addition we identified six areas of cross-species homology in the arginase protein, four containing conserved histidine residues thought to be important to Mn(2+)-dependent enzyme function. Mapping patient mutations in relationship to the conserved regions indicates that substitution mutations within the conserved regions and randomly occurring microdeletions and nonsense mutations have a significant effect on enzymatic function. In vitro mutagenesis was utilized to create nonpatient substitution mutations in the conserved histidine residues to verify their importance to arginase activity. As expected, replacement of histidine residues with other amino acids dramatically reduces arginase activity levels in our bacterial expression system.

Prediction of diabetic adherence using the BASIS-A inventory.

Medical and mental health professionals continue to strive for more effective ways of encouraging patients to adhere to suggested treatment plans. The purpose of this research was to explore the relationship between personality and adherence dynamics within a diabetic population. Specifically, we examined the relationship between Adlerian life style personality factors with 14 adherence behaviors in 38 patients with type I diabetes. Several statistically significant correlations showed that specific personality variables are associated with particular elements of the adherence regimen. The results indicate that strategies to increase adherence among patients should be individualized, and recommendations for how to increase adherence in patients with diabetes are given.

A method for quantification and correction of proteins after transfer to immobilization membranes.

A method is described for quantification of specific proteins after electrophoresis and transfer to immobilization membranes for Western blots. This method is analogous to methods used to correct the amounts of specific transcripts detected on Northern blots. Ponceau S staining of proteins bound to immobilization membranes is efficient and accurate compared to antibody binding in terms of time, effort and cost. Comparison of Ponceau S to other detection and staining methods for quantifying proteins and the basic chemistry of Ponceau S are summarized.

Co-induction of arginase and nitric oxide synthase in murine macrophages activated by lipopolysaccharide.

In view of studies showing that not only nitric oxide synthase (NOS) activity but arginase activity is induced in rodent macrophages by lipopolysaccharide (LPS), the objective of this study was to investigate the co-induction of these two enzymes and to ascertain whether common mechanisms are involved. RAW 264.7 cells were activated by 2 micrograms LPS/ml and incubated for up to 48 hr. Inducible NOS (iNOS) and inducible arginase II (AII) activities were monitored, respectively, by measuring NO2-/NO3- accumulation in cell culture media and formation of urea (as CO2) from L-arginine by cell lysates. AII activity increased linearly up to at least 48 hr, whereas NO2-/NO3- formation reached a plateau well before 48 hr. Immunoprecipitation experiments revealed that AII accounted for 90-100% of arginase activity in LPS-activated macrophages. The inhibitor of NF-kappa B activation, pyrrolidine dithiocarbamate, inhibited the induction of iNOS but not AII. Moreover, whereas IFN-gamma caused iNOS induction, AII induction was nearly abolished by IFN-gamma, perhaps by inhibiting transcription of the AII gene. These observations indicate that co-induction of iNOS and AII occurs by distinct transcriptional mechanisms, AII induction could diminish NO production by decreasing L-arginine availability, and IFN-gamma can prevent AII induction.

Subcellular location and differential antibody specificity of arginase in tissue culture and whole animals.

Studies in man and other mammals have demonstrated the existence of two forms of arginase, a cytoplasmic form located primarily in liver and a mitochondrial form expressed in lesser amounts in a larger number of organs, but especially kidney. They appear to be encoded in different gene loci. Using a colloidal silica gradient separation technique, we have now located arginase in H4 cells, a rat hepatoma-derived line, to the cytoplasm and the arginase in human embryonic kidney-derived line, to the mitochondrion. Antibody prepared against A1 precipitates all the arginase from liver, 50% from kidney and none of the activity from human embryonic kidney (HEK) cells. An antibody prepared against partially purified All, by contrast, precipitates > 90% of arginase activity from HEK cells, half from kidney and virtually none from H4 cells or rat liver.

Arginase deficiency manifesting delayed clinical sequelae and induction of a kidney arginase isozyme.

Deficiency of liver arginase (AI) is characterized clinically by hyperargininemia, progressive mental impairment, growth retardation, spasticity, and periodic episodes of hyperammonemia. The rarest of the inborn errors of urea cycle enzymes, it has been considered the least life-threatening, by virtue of the typical absence of catastrophic neonatal hyperammonemia and its compatibility with a longer life span. This has been attributed to the persistence of some ureagenesis in these patients through the activity of a second isozyme of arginase (AII) located predominantly in the kidney. We have treated a number of arginase-deficient patients into young adulthood. While they are severely retarded and wheelchair-bound, their general medical care has been quite tractable. Recently, however, two of the oldest (M.U., age 20, and M.O., age 22) underwent rapid deterioration, ending in hyperammonemic coma and death, precipitated by relatively minor viral respiratory illnesses inducing a catabolic state with increased endogenous nitrogen load. In both cases, postmortem examination revealed severe global cerebral edema and aspiration pneumonia. Enzyme assays confirmed the absence of AI activity in the livers of both patients. In contrast, AII activity (identified by its different cation cofactor requirements and lack of precipitation with anti-AI antibody) was markedly elevated in kidney tissues, 20-fold in M.O. and 34-fold in M.U. Terminal plasma arginine (1500 mumols/l) and ammonia (1693 mmol/l) levels of M.U. were substantially higher than those of M.O. (348 mumols/l and 259 mumols/l, respectively). By Northern blot analysis, AI mRNA was detected in M.O.'s liver but not in M.U.'s; similarly, anti-AI crossreacting material was observed by Western blot in M.O. only. These findings indicate that, despite their more long-lived course, patients with arginase deficiency remain vulnerable to the same catastrophic events of hyperammonemia that patients with other urea cycle disorders typically suffer in infancy. Further, unlike those other disorders, an attempt is made to compensate for the primary enzyme deficiency by induction of another isozyme in a different tissue. Such substrate-stimulated induction of an enzyme may be unique in a medical genetics setting and raises novel options for eventual gene therapy of this disorder.

Molecular genetic study of human arginase deficiency.

We have explored the molecular pathology in 28 individuals homozygous or heterozygous for liver arginase deficiency (hyperargininemia) by a combination of Southern analysis, western blotting, DNA sequencing, and PCR. This cohort represents the majority of arginase-deficient individuals worldwide. Only 2 of 15 homozygous patients on whom red blood cells were available had antigenically cross-reacting material as ascertained by western blot analysis using anti-liver arginase antibody. Southern blots of patient genomic DNAs, cut with a variety of restriction enzymes and probed with a near-full-length (1,450-bp) human liver arginase cDNA clone, detected no gross gene deletions. Loss of a TaqI cleavage site was identified in three individuals: in a homozygous state in a Saudi Arabian patient at one site, at a different site in homozygosity in a German patient, and in heterozygosity in a patient from Australia. The changes in the latter two were localized to exon 8, through amplification of this region by PCR and electrophoretic analysis of the amplified fragment after treatment with TaqI; the precise base changes (Arg291X and Thr290Ser) were confirmed by sequencing. It is interesting that the latter nucleotide variant (Thr290Ser) was found to lie adjacent to the TaqI site rather than within it, though whether such a conservative amino acid substitution represents a true pathologic mutation remains to be determined. We conclude that arginase deficiency, though rare, is a heterogeneous disorder at the genotypic level, generally encompassing a variety of point mutations rather than substantial structural gene deletions.

Differential expression of the two human arginase genes in hyperargininemia. Enzymatic, pathologic, and molecular analysis.

Previous studies in our laboratory and others have demonstrated in humans and other mammals two isozymes of arginase (AI and AII) that differ both electrophoretically and antigenically. AI, a cytosolic protein found predominantly in liver and red blood cells, is believed to be chiefly responsible for ureagenesis and is the one missing in hyperargininemic patients. Much less is known about AII because it is present in far smaller amounts and localized in less accessible deep tissues, primarily kidney. We now report the application of enzymatic and immunologic methods to assess the independent expression and regulation of these two gene products in normal tissue extracts, two cultured cell lines, and multiple organ samples from a hyperargininemic patient who came to autopsy after an unusually severe clinical course characterized by rapidly progressive hepatic cirrhosis. AI was totally absent (less than 0.1%) in the patient's tissues, whereas marked enhancement of AII activity (four times normal) was seen in the kidney by immunoprecipitation and biochemical inhibition studies. Immunoprecipitation-competition and Western blot analysis failed to reveal presence of even an enzymatically inactive cross-reacting AI protein, whereas Southern blot analysis showed no evidence of a substantial deletion in the AI gene. Induction studies in cell lines that similarly express only the AII isozyme indicated that its activity could be enhanced severalfold by exposure to elevated arginine levels. Our findings suggest that the same induction mechanism may well be operative in hyperargininemic patients, and that the heightened AII activity may be responsible for the persistent ureagenesis seen in this disorder. These data lend further support to the existence of two separate arginase gene loci in humans, and raise possibilities for novel therapeutic approaches based on their independent manipulation.

Isolation of human liver arginase cDNA and demonstration of nonhomology between the two human arginase genes.

A human liver cDNA library was screened by colony hybridization with a rat liver arginase cDNA. The number of positive clones detected was in agreement with the estimated abundance of arginase message in liver, and the identities of several of these clones were verified by hybrid-select translation, immunoprecipitation, and competition by purified arginase. The largest of these human liver arginase cDNAs was then used to detect arginase message on northern blots at levels consistent with the activities of liver arginase in the tissues and cells studied. The absence of a hybridization signal with mRNA from a cell line expressing only human kidney arginase demonstrated the lack of homology between the two human arginase genes and indicated considerable evolutionary divergence between these two loci.

Comparison of arginase activity in red blood cells of lower mammals, primates, and man: evolution to high activity in primates.

Arginase activity in red blood cells (RBC) of various mammalian species including man was determined. In nonprimate species, the activity generally fell below the level of detectability of the assay: less than 1.0 mumol urea/g hemoglobin per hr. Activities in higher nonhuman primates were equal to or of the same order of magnitude as those in man (approximately 950 mumol/g hemoglobin per hr). RBC arginase deficiency with normal liver arginase activity has been shown to segregate as an autosomal codominant trait in Macaca fascicularis established and bred in captivity. This study confirms the presence of this polymorphism in wild populations trapped in several geographic areas and demonstrates the absence of immunologically cross-reactive material in the RBC of RBC arginase-deficient animals. These data when taken together suggest that the expression of arginase in RBC is the result of a regulatory alteration, has evolved under positive selective pressure, and is not an example of the vestigial persistence of an arcane function. The expression of arginase in the RBC results in a marked drop in the arginine content of these cells.

Differential expression of multiple forms of arginase in cultured cells.

Arginase (EC 3.5.3.1), the final enzyme in the urea cycle, catalyzes the cleavage of arginine to orthinine and urea. At least two forms of this enzyme, AI and AII, have been described and are probably encoded by discrete genetic loci. The expression of these separate genes has been studied in mammalian cells grown in culture. The permanent rat-hepatoma line H4-II-E-C3 contained exclusively the AI enzyme; the form in mammals comprising about 98% of the arginase activity in liver and erythrocytes but catalyzing only about one half of that reaction in kidney, gastrointestinal tract, and brain. By contrast, human-embryonic-kidney and -brain cells, after transformation with the human papovavirus BK, contained only the AII species of arginase, which form contributes the remaining half of that catalysis in those mammalian tissues in vivo. We report here the results of an extensive study on the properties of these two forms of arginase in the three cell lines, including Km values for arginine, behavior on polyacrylamide gels under non-denaturing conditions, and cross-reactivity with lapine antibodies against the arginases from either rat or human liver.