Abstract
Arginase (EC 3.5.3.1), the final enzyme in the urea cycle, catalyses the hydrolysis of L-arginine to L-ornithine and urea. High activity of this enzyme in the liver indicates its primary role in ammonia detoxification. However, its wide tissue distribution suggests that this enzyme might perform other functions besides hepatic ureagenesis. Although the distribution and properties of arginase from many tissues of human, laboratory animals and some domestic animals have been studied, little is known about the pattern of distribution and physiological roles of this enzyme in the cat. The purpose of this study was to examine and compare the distribution of arginase in different tissues of the cat. A selection of tissue samples was assayed for arginase by the diacetyl monoxime method of determination of enzymatically formed urea. The protein content of tissues and enzymatic activities were calculated as units per gram tissue and units per milligram protein of the tissue. Results showed that the liver was the richest source of arginase followed by the oesophageal and tongue mucosal layers. Significant activity of this enzyme was found in the mucosa of the small intestine, kidney cortex, lung, testis and ovary. The results of this study will be discussed in terms of the involvement of arginase in several biochemical and physiological functions in this species.
Arginase (l-arginine amidinohydrolase, EC 3.5.3.1), the final enzyme in the urea cycle, catalyses the divalent cation-dependent hydrolysis of l-arginine to form the non-protein amino acid l-ornithine and urea (Aminlari and Vaseghi 1992, Cederbaum et al 2004). The highest activity of arginase is found in liver, the only organ containing all the enzymes of the urea cycle, which bespeaks its important role in ammonia detoxification occurring through this cycle (Cederbaum et al 2004). Significant arginase activity has been detected in a number of extrahepatic tissues which lack a complete urea cycle such as the kidney, prostate (Vockley et al 1996), lactating mammary gland (Yip and Knox 1972), pregnant uterus (Weiner et al 1996), nervous system (Spencer and Filbin 2004), intestine (Aminlari and Vaseghi 1992, Alican and Kubes 1996, Wu et al 1996), skeletal muscle (Aminlari and Vaseghi 1992) and in cancers of the stomach (Wu et al 1994), breast (Porembska et al 2003), colon (del Ara Rangel et al 2002) and prostate (Elgun et al 1999). This wide distribution in normal and pathological tissues suggests many other functions for arginase beside its role in ureagenesis in the liver. These include the biosynthesis of ornithine as a precursor of polyamines (spermine, spermidine and putrescine), glutamate (precursor of γ-amino butyric acid – GABA) and proline (Cederbaum et al 2004). Polyamines are vital for cell proliferation (Hakovirta et al 1993, Heby and Emanuelsfon 1981) and have been described as being modulators of ion channels and neurotransmitter receptors (Alican and Kubes 1996). GABA and l-proline have functions in cell signalling (Castillo et al 1993) and proline is needed for collagen synthesis. Other proposed roles for arginase include modulation of nitric oxide (NO) synthesis (Mori and Gotoh 2000), regulation of inflammatory and immunological responses (Mistry et al 2001), and wound healing and regulation of the airway smooth muscle relaxation (Que et al 1998).
This enzyme is widely distributed in nature from bacteria to man and studies of the distribution of this important enzyme in the various tissues of animal species are valuable in revealing its physiological roles. Although the distribution and properties of arginase from many tissues of human (Spector et al 1982, Zamecka and Porembska 1988), mouse (Spolaris and Bond 1988) and a few domestic animals (Aminlari and Vaseghi 1992) have been studied, little is known of the pattern of distribution and physiological roles of this enzyme in the tissues of cats. The purpose of this study was to examine and compare the tissue distribution of arginase in cats. The results of this study will help assess the role of this enzyme in different feline tissues and will be discussed in comparison with other animals and in its relation to the many physiological roles it might be involved in.
Materials and methods
Organs and tissue samples were obtained post-mortem from 12 apparently healthy adult male and non-pregnant female domestic cats involved in road traffic accidents that were referred to the veterinary school hospital by biopsy or autopsy 0–1 h after death. All samples were kept on ice and transferred within 30 min to the laboratory. Tissues were separated, stripped of fat and extraneous materials, washed a few times with physiological saline and then blotted. Tissues were weighed and extracts were prepared by freezing in liquid nitrogen, homogenising with a hand homogeniser and diluting the homogenate in nine parts of 0.025 M sodium phosphate buffer (pH=7.2). The suspensions were centrifuged for 15 min at 4000 g in a high-speed refrigerated centrifuge (MSE, Scientific Instruments, Sussex, UK). The supernatants were used as the source of enzyme. Arginase was assayed as described by Natelson (1971). Briefly, to 220 μl solution containing 0.02 M arginine monohydrochloride and 1% MnCl2, pH=9.5, was added 20 μl of tissue extract and the mixture was incubated at 37°C. After 20 min 1 ml of stop solution (0.15 M sulphuric acid and 2.2% sodium tungstate) was added to the mixture. After centrifugation at 4000 g for 15 min, 500 μl of solution (containing 2% diacetyl monoxime in 2% acetic acid) and 2.5 ml of acid mixed solution (containing 1.85 ml 85% phosphoric acid, 0.65 ml sulphuric acid and 0.65 mg ferric ammonium sulphate) were added to 1 ml of supernatant. The mixture was incubated in boiling water for 30 min and after cooling to room temperature, the concentration of urea was determined by reading the absorbance at 450 nm against the blank containing all components except tissue extract. One unit of arginase activity is defined as the amount of enzyme that produces 1 μmol of urea from arginine in 1 min at 37°C at pH 9.5. Total protein was assayed according to Lowry et al (1951) using crystalline bovine serum albumin as a standard. All chemicals were of analytical grade and were supplied by Sigma (St Lewis, Mo, USA). Statistical analysis was performed by one-way analysis of variance and Duncan test using SPSS software for multiple comparisons of the means of arginase specific activity of different tissues.
Results
Arginase activity in 30 different tissues of cats is shown in Table 1. All of the tissues studied contain arginase activity. The liver had the highest activity of this enzyme. Oesophageal and tongue mucosal layers had significantly higher arginase activity than all other tissues except liver (P<0.05). Other tissues with relatively high arginase activity were diaphragm, oesophagus (whole tissue), duodenal mucosa, tongue (whole tissue) and kidney cortex. In tissue from the oesophagus, duodenum and jejunum, the specific activity of arginase was significantly higher in the mucosal layer than submucosal layers (P<0.05) but the differences were not significant between mucosal and submucosal layers of stomach, ileum and rectum. Very low arginase activity was observed in stomach, heart, submucosal layers of jejunum, duodenum, ileum, trachea, rectum, pancreas, uterus and spleen. In tissue from the small intestine, the specific activity of arginase showed gradual significant decreases from duodenum to ileum.
Mean (SD) arginase activity*
Sample size=12 and was the same for all tissues.
Tissues were weighted after washing with normal saline and blotting with blotter paper.
Values with different superscripts are significantly different (P<0.05).
Discussion
Arginase activity showed widespread distribution in all tissues studied. Liver tissue had the highest level of this enzyme activity. A similar pattern was reported earlier in other domestic animals including dog, cattle, sheep, camel, horse and donkey (Aminlari and Vaseghi 1992). In mammals, the liver is the organ in which a full urea cycle is functional (Greenberg 1960) and the only organ containing all of the enzymes of urea cycle (Cederbaum et al 2004). Arginase is the final enzyme of urea cycle that is responsible for the hydrolysis of arginine to urea and ornithine and liver arginase deficiency has been reported to cause hyperammonia indicating that its primary role in the liver is ammonia detoxification via urea cycle (Cederbaum et al 2004).
Specific activity of arginase in the liver of cats was significantly higher than those reported for cattle, sheep, camel, horse, donkey and dog (Aminlari and Vaseghi 1992). This difference might be related to the differences in feeding habits of the cat. The cat has specific nutritional requirements that are different from other animals. For example, among all mammals, cats have the highest protein requirement (Morris and Rogers 1991, Hendriks et al 1997, Zoran 2002). The liver urea cycle in cats has a very high and constant activity rate which is minimally affected by the dietary protein level (Hendriks et al 1997, Russel et al 2000, Zoran 2002). The difference between the arginase activities in the livers of the cat and the dog may be related to an important difference in the pattern of regulation of urea cycle between these two species. The activity of the urea cycle in cats is constant and continuous (Morris and Rogers 1991, Zoran 2002). The difference can also be attributed to different metabolic aspects between cat and dog. Cats are metabolically adapted to use protein and fat for energy production and for maintenance of blood glucose concentration even when sources of protein in the diet are limiting (Hendriks et al 1997). As a result, the protein requirement of cats is more than twice that of dogs (Russel et al 2000). This fact might explain a more apparently active urea cycle in cat than dog.
A higher level of hepatic arginase activity than other tissues, suggests that this enzyme may be used as a good marker for the diagnosis of liver diseases in cats. Aminlari et al (1994) reported that serum arginase activity rose rapidly in dogs, cattle and sheep after experimental hepatic necrosis. In this respect, due to higher hepatic arginase activity in cat, the serum arginase activity might even be more useful as a marker for hepatocellular damages in this species.
The existence of arginase in various extrahepatic tissues that do not have complete urea cycle indicates that this enzyme may be functional in various physiological processes in cats. A source of variation in arginase activity in different feline tissues might be related to the existence of different forms of this enzyme in different tissues as demonstrated in other species (Spolaris and Bond 1988, Zamecka and Porembska 1988, Que et al 1998, Mistry et al 2001, Cederbaum et al 2004). It will be interesting to find the correlation between arginase activity with the level of mRNA of different forms of arginase to elucidate the pattern of arginases gene expression at the level of transcription. Next to the liver, oesophageal and tongue mucosal layers are the richest sources of arginase among the tissues studied. This study is the first report that shows oesophageal mucosa and tongue mucosa as important sources of arginase. Specific activity of arginase in oesophageal mucosa was significantly higher than oesophageal whole tissue and oesophageal submucosa. Also this activity in tongue mucosa was significantly higher than tongue whole tissue. This result indicates that arginase may have important role(s) in these mucosal layers. As described before, arginase hydrolyses l-arginine to urea and ornithine. Ornithine can be decarboxylated by the enzyme ornithine decarboxylase to produce putrescine which serves as a precursor for the synthesis of polyamines. Polyamines have been described as being modulators of ion channels (Williams 1997). It has been demonstrated that cats respond to salty, sour, and bitter stimuli as well as to amino acids and nucleotides, but do not show neural responses to sucrose and several other sugars (Li et al 2005). In this respect arginase might be involved in the modulation of taste due to the indirect effect on ion channels of apical microvilli of taste receptor cells. The taste transduction pathway depends on ion channels in the apical microvilli of taste receptor cells (Gilbertson et al 2000). On the other hand polyamines are vital for cell proliferation and tissue repair (Heby and Emanuelsfon 1981, Tabor and Tabor 1984, Hakovirta et al 1993, Kepka-Lenhart et al 2000). In man and in many other species, the taste buds on the surface of the tongue are constantly assaulted by abrasion against the teeth and hard palate (Beidler and Smallman 1965). Therefore, arginase may be essential for cell proliferation, differentiation and repair of these mucosal layers.
The specific activity of arginase is significantly higher in mucosal layers of the duodenum and jejunum in comparison with the submucosal layers of these regions. Significant differences were not observed between the arginase activity of mucosal and submucosal layers of the ileum and rectum. Specific activity of arginase in the mucosal layers of the small intestine showed gradual and significant decreases from the duodenum to the ileum. Gradual decreases of arginase activity in the small intestine have been reported previously in the rat (Konarska and Tomaszewski 1975). Arginase is functional in some physiological phenomena of the small intestine. Intestinal arginine is degraded primarily by arginase and to a much lesser extents, by nitric oxide synthase (NOS) (Wu et al 1996). In the enterocytes, arginine-derived ornithine is converted mainly into proline by the enzyme ornithine aminotransferase and pyrroline-5-carboxylate reductase (Davis and Wu 1998). In these cells, proline, ornithine, citrulline and CO2 account for 56, 37, 4, and 1% of the metabolised arginine carbons, respectively, and polyamines and NO are quantitatively minor products of arginine (Wu et al 1996). Results from studies (Windmuller and Spaeth 1976) in rats and in adult humans (Castillo et al 1993) revealed that substantial amounts of dietary arginine are not available to extraintestinal tissues. According to this information, roles of arginase in the small intestine of cat can be related to ornithine production for synthesis of proline as an important amino acid and synthesis of polyamines which are essential for proliferation, differentiation and repair of intestinal epithelial cells (Luk et al 1980). In addition, arginase might be involved in regulating the synthesis of NO which plays an important role in regulation of intestinal blood flow, integrity, secretion and epithelial cell migration (Alican and Kubes 1996). Finally arginase might participate in modulation of the entry of absorbed dietary arginine into portal circulation (the role of intestinal amino acid catabolism was extensively reviewed by Wu (1998).
Table 1 shows that arginase activity in the kidney of cat is similar to other domestic animals previously reported (Aminlari and Vaseghi 1992). Arginine is essential for growth in the kitten and because of the resulting hyperammonaemia, in the adult cat and other carnivores, an arginine-free diet is life threatening (Morris and Rogers 1978). The relative inability of cats to synthesise arginine is explained by the very low activities of four enzymes in their enterocytes: ornithine aminotransferase, carbamoyl phosphate synthase, pyrroline-5-carboxylate synthase and ornithine carbamoyl transferase (Rogers and Phang 1985). It has been shown that the mammalian kidney contains several enzymes involved in arginine metabolism (argininosuccinate synthase, argininosuccinate lyase and arginase (Ratner and Petrack 1953, Porembska et al 1971, Ratner 1973). Dhanakoti et al (1990) demonstrated that the arginine produced by the kidney is released into the venous blood and is used in the synthesis of NO guanidino compounds, proteins and other biochemical compounds. Some of arginine produced in the kidney might, however, be hydrolysed by renal arginase and used for thesynthesis of these compounds in kidney. NO has been known to be involved in many biochemical and physiological functions of kidney (for example, see Ishii et al 2004). As the level of NO is controlled by arginase (Mori and Gotoh 2000, Ishii et al 2004), arginase might be considered to be indirectly involved in many physiological functions in kidney mediated by NO. Specific activity of arginase is significantly higher in the renal cortex in comparison with the renal medulla. Levillain et al (1996) reported that in cat kidney, arginase activity was restricted to the proximal tubules. Our study, however, showed arginase activity in both the renal cortex and medulla. This may be related to the anatomical structure of the cat kidney. All nephrons in the feline kidney are juxtamedullary and not cortical. In juxtamedullary nephrons, proximal tubules extend into the medulla (Reece 1996) hence, explaining the presence of arginase in both the cortex and medulla.
Feline lung contains substantial arginase activity in comparison to many other tissues and is considerably higher than that found in other animals (Aminlari and Vaseghi 1992). Lung arginase is involved in attenuation of the inhibitory non-adrenergic non-cholinergic (iNANC) nerve-mediated airway relaxation. The iNANC nervous system is the most effective bronchodilating neural pathway of the airways. Regulation of this novel regulatory mechanism of airway responsiveness might be involved in the pathophysiology of some allergic diseases (Maarsingh et al 2005). Another role that has been proposed for arginase in the lung is its involvement in the recovery phase of lung injury and lung responsiveness to inflammatory stimuli (Que et al 1998).
No significant difference between arginase specific activity of the testis and ovary was observed. The value of cat testicular specific activity of arginase is similar to that reported for normal men (0.016±0.005 U/mg protein) (Elgun et al 2000). It seems that the main role of arginase in testis and ovary is regulation of NO. Excessive NO production, which may be related to low arginase activity, has been reported to account for oxyradical-mediated tissue damage (Nathan 1997), cell death either through apoptosis or necrosis (Bonfoco et al 1995) and impairment of spermatogenesis in gonads (Taneli et al 2005). Lowered NO concentration as a result of increased arginase activity, leads to increased sperm motility but negative correlation was established between sperm count and arginase activity (Elgun et al 2000). Therefore, like other animals, testicular arginase may play important roles in regulating spermatogenesis and fertility in the male cat and in many functions of ovary and female reproduction processes via regulation of NO and polyamines production.
In summary, this study showed widespread distribution of arginase activity in hepatic and extrahepatic tissues of the cat. Distribution of arginase in various extrahepatic tissues that do not have a complete urea cycle suggests that this enzyme may be functional in various physiological phenomena in the cat. Future studies are needed to clarify involvement of arginase in various physiological processes and pathophysiological conditions in cat.
Footnotes
Acknowledgement
This research was financially supported by grant number 84-GR-VT-11 of Shiraz University Research Council.