Nirmalendu Saha

Ureogenesis in Indian air-breathing teleosts: adaptation to environmental constraints

Abstract Most of the Indian air-breathing teleosts are primarily ammoniotelic, but appear to have retained the genes for the urea cycle enzymes, since a full complement of urea cycle enzymes have been reported for many of them. The ability to synthesize urea by these fish is probably due to their amphibious nature, and their normal habitat of swamps, where the water ammonia level may to be quite high, is uninhabitable to any typical freshwater teleosts. One of these air-breathing species, the singhi catfish ( Heteropneustes fossilis ), can tolerate very high ambient total ammonia concentrations (up to 75 mM ammonium chloride) for weeks without any deleterious effects. Transition from ammoniotelism to ureotelism occurs in some of these species of air-breathing fish when exposed to apparently stressful conditions such as higher ambient ammonia, to air, and also when they live in semidry condition inside mud during habitat drying. Although the real mechanism(s) of regulation of ureogenesis is not clear in these fish, given available data, it is hypothesized that the accumulation of ammonia within the body per se under the above stressful conditions is likely the internal modulator for enhanced ureogenesis mainly to avoid any build up of ammonia to a level that can be toxic to these fish. An active urea cycle is believed to predominate over uricolysis as a source of urea, even though both pathways are present in these air-breathing fish. The presence of significant levels of both carbamyl phosphate synthetase (CPS), CPS I-like and CPS III activities, reported in some air-breathing catfishes, may represent intermediate scenarios for a proposed evolutionary transition from CPS III to CPS I, or may play an important physiological adaptive role in the tolerance of these fish to high concentrations of ambient ammonia

Induction of ornithine-urea cycle in a freshwater teleost, Heteropneustes fossilis, exposed to high concentrations of ammonium chloride

Abstract An ammoniotelic freshwater teleost, Heteropneustes fossilis , tolerated ambient ammonium chloride concentration up to 75 mM. Ammonia accumulated significantly in all the tissues within 7 days of treatment and the concentration remained high throughout the 4-week period of treatment. The activity of enzymes of the ornithine-urea (o-u) cycle were induced within 7 days, and thereafter remained high in both the liver and kidney of the fish. Urea accumulated significantly in various tissues simultaneous with the induction of o-u cycle enzymes. Accumulated ammonia induced the activity of the enzymes of the o-u cycle for its metabolic conversion to urea. This helped the freshwater fish to avoid toxaemia and to tolerate high concentrations of ammonia in the ambient medium.

Role of amino acid metabolism in an air-breathing catfish, Clarias batrachus in response to exposure to a high concentration of exogenous ammonia.

The air-breathing ureogenic walking catfish (Clarias batrachus) faces various environmental constraints throughout the year leading to the problem of accumulation of toxic ammonia. In the present study, the possible role of conversion of accumulated ammonia to various non-essential free amino acids (FAAs) was tested in this fish under hyper-ammonia stress caused by exposing the fish at 25 mM NH(4)Cl for 7 days. Significant accumulation of ammonia of approximately two- to threefold was observed in different tissues (except in the brain), which was accompanied with the significant accumulation of non-essential FAAs in the NH(4)Cl-exposed fish. There was approximately two- to threefold increase of non-essential FAAs in different tissues and in the plasma of the NH(4)Cl-exposed fish compared to the control fish after 7 days of exposure, which was mainly attributable to the increase of Asp, Ala, Gly, Glu, Gln and taurine (Tau) concentrations in general, with certain tissue-specific variations. This was also accompanied with significant increase of activity of certain amino acid metabolism-related enzymes such as the glutamine synthetase (approx. two- to threefold), glutamate dehydrogenase (ammonia utilizing direction) (approx. twofold), aspartate and alanine aminotransaminases (approx. twofold) mainly in the liver, kidney and muscle of the NH(4)Cl-exposed fish. Thus, it appears that the walking catfish has the capacity of active conversion of accumulated ammonia to non-essential FAAs under condition of high concentrations of external ammonia. However, the increase of urea excretion rate due to active conversion of ammonia to urea via the induced urea cycle appears to be quantitatively much more important pathway than the increase of tissue levels of FAAs in dealing with a severe ammonia load.

Enhanced expression of basolateral multidrug resistance protein isoforms Mrp3 and Mrp5 in rat liver by LPS

Abstract Lipopolysaccharide (LPS) induces hepatocellular downregulation and endocytic retrieval of multidrug resistance protein 2 (Mrp2, Abcc2). Basolateral Mrp isoforms may compensate for the intracellular metabolic changes in cholestasis. Therefore, the effect of LPS on the zonal localization of Mrp2 and Mrp3 and the expression of Mrp3, Mrp4, Mrp5, and Mrp6 mRNA were investigated in rat liver. In normal rat liver Mrp3 was found in pericentral hepatocytes also expressing glutamine synthetase. In LPS-treated rat liver the decrease in Mrp2 protein was most pronounced in pericentral hepatocytes, with only minor down-regulation in periportal hepatocytes. Conversely, induction of Mrp3 was found in pericentral hepatocytes with a low expression of Mrp2. Furthermore, we found a strong induction of Mrp5 mRNA. Likewise, Mrp6 mRNA was upregulated, however Mrp6 protein expression was not significantly altered. It is concluded that Mrp3 is inversely regulated to Mrp2 in a zonal pattern and may compensate for the LPSinduced loss of Mrp2 in the perivenous area. Induction of pericentral Mrp3 and upregulation of Mrp5 mRNA may play an important role in the hepatocellular clearance of cholephilic substances and cyclic nucleotides accumulating after LPS treatment.

Changes in free amino acid synthesis in the perfused liver of an air‐breathing walking catfish, Clarias batrachus infused with ammonium chloride: A strategy to adapt under hyperammonia stress

The changes in the free amino acid (FAA) levels, the rate of efflux of FAAs from the perfused liver, and the activity of some enzymes related to amino acid metabolism such as glutamate dehydrogenase (GDH, both reductive amination and oxidative deamination), glutamine synthetase (GS), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were studied in the liver of a freshwater air-breathing teleost, the walking catfish, Clarias batrachus, perfused with 5 and 10 mM NH(4)Cl. The level of the various non-essential FAAs increased significantly, with a total increase of about 150%, which was accompanied by a significant increase of both ammonia and urea-N in the perfused liver both with 5 and 10 mM NH(4)Cl. The rate of efflux of these non-essential FAAs from the perfused liver also increased significantly with a total increase of about 115% and 160% at 5 and 10 mM NH(4)Cl, respectively. The activity of the mentioned amino acid metabolism-related enzymes in the perfused liver also got stimulated, except for GDH in the ammonia forming direction and ALT, under a higher ammonia load. The activity (both tissue and specific) of GDH in the glutamate forming direction increased maximally, followed by AST and GS in a decreasing order. Owing to these physiological adaptive strategies related to amino acid metabolism along with the presence of a functional and regulatory urea cycle (reported earlier), it is believed that this catfish is able to survive in very high ambient ammonia or in the air or in the mud during habitat drying.

Carbamyl Phosphate Synthetases in an Air-Breathing Teleost, Heteropneustes fossilis

Abstract The Indian air-breathing teleost fish Heteropneustes fossilis has been shown to have a functional urea cycle and to be able to switch from ammoniotelic to ureotelic nitrogen metabolism when exposed to high levels of ammonia or air. The objective of this study was to identify the type of carbamyl phosphate synthetase (CPS) catalyzing the first step of the urea cycle in H. fossilis . Mitochondrial CPS III [glutamine- and N-acetyl-L-glutamate (NAG)-dependent] and cytosolic CPS II (glutamine-dependent) activities were found to be present in liver, analogous to that described for two other teleosts that have CPS III activity. The same activities and subcellar localization were found in kidney. Unexpectedly, a CPS I-like activity (ammonia- and NAG-dependent) was found to be present at levels higher than the CPS III activity in the mitochondrial fraction of both liver and kidney. The urea cycle-related CPS III found in invertebrates and fish is considered to be the evolutionary precursor of the urea cycle-related CPS I in ureotelic mammalian and amphibian species. Whether or not this CPS I-like activity 1) is due to the presence of a separate CPS I gene in addition to a CPS III gene or 2) represents an adapted CPS III activity in H. fossilis , these results suggest that the presence of both CPS I-like and CPS III activities may play an important physiological adaptive role in the tolerance of these fish to high concentrations of external ammonia.

Effect of alkalinity (pH 10) on ureogenesis in the air-breathing walking catfish, Clarias batrachus

Exposure of fish to alkaline conditions inhibits the rate of ammonia excretion, leading to ammonia accumulation and toxicity. The purpose of this study was to determine the role of ureogenesis via the urea cycle, to avoid the accumulation of ammonia to a toxic level during chronic exposure to alkaline conditions, for the air-breathing walking catfish, Clarias batrachus, where a full complement of urea cycle enzyme activity has been documented. The walking catfish can survive in water with a pH up to 10. At a pH of 10 the ammonia excretion rate by the walking catfish decreased by approximately 75% within 6 h. Although there was a gradual improvement of ammonia excretion rate by the alkaline-exposed fish, the rate remained 50% lower, even after 7 days. This decrease of ammonia excretion was accompanied by a significant accumulation of ammonia in plasma and body tissues (except in the brain). Urea-N excretion for alkaline-exposed fish increased 2.5-fold within the first day, which was maintained until day 3 and was then followed by a slight decrease to maintain a 2-fold increase in the urea-N excretion rate, even after 7 days. There was also a higher accumulation of urea in plasma and other body tissues (liver, kidney, muscle and brain). The activity of glutamine synthetase and three enzymes operating in the urea cycle (carbamyl phosphate synthetase, argininosuccinate synthetase, argininosuccinate lyase) increased significantly in hepatic and extra-hepatic tissue, such as the kidney and muscle in C. batrachus, during exposure to alkaline water. A significant increase in plasma lactate concentration noticed during alkaline exposure possibly helped in the maintenance of the acid-base balance. It is apparent that the stimulation of ureogenesis via the induced urea cycle is one of the major physiological strategies adopted by the walking catfish (C. batrachus) during chronic exposure to alkaline water, to avoid the in vivo accumulation of ammonia to a toxic level in body tissues and for the maintenance of pH homeostasis.

Role of ureogenesis in the mud-dwelled Singhi catfish (Heteropneustes fossilis) under condition of water shortage

The air-breathing Singhi catfish Heteropneustes fossilis was kept inside moist peat for 1 month mimicking their normal habitat in summer and the role of ureogenesis for their survival in a water-restricted condition was studied. The ammonia excretion rate by the mud-dwelled fish increased transiently between 6 and 12 h of re-immersion in water to approximately between eight and 10-fold, followed by a sharp decrease almost to the normal level at the later part of re-immersion. The urea-N excretion by the mud-dwelled fish increased to approximately 11-fold within 0-3 h of re-immersion, followed by a gradual decrease from 9 h onwards. The rate of urea-N excretion by the mud-dwelled fish, however, remained significantly higher (approx. threefold more) than the control fish even after 36-48 h of re-immersion. Although there was a significant increase of both ammonia and urea levels in the plasma and other tissues (except ammonia in the brain), the level of accumulation of urea was higher than ammonia in the mud-dwelled fish as indicated by the decrease in the ratio of ammonia: urea level in different tissues including the plasma. The activities (units/g tissue and /mg protein) of glutamine synthetase and three enzymes of the urea cycle, carbamyl phosphate synthetase, argininosuccinate synthetase and argininosuccinate lyase increased significantly in most of the tissues (except the brain) of the mud-dwelled fish as compared to the control fish. Higher accumulation of ammonia in vivo in the mud-dwelled Singhi catfish is suggested to be one of the major factors contributing to stimulation of ureogenesis. Due to this physiological adaptive strategy of ureogenesis, possibly along with other physiological adaptation(s), this air-breathing amphibious Singhi catfish is able to survive inside the moist peat for months in a water-restricted condition.

Functional ureogenesis and adaptation to ammonia metabolism in Indian freshwater air-breathing catfishes

The Indian freshwater air-breathing catfishes Heteropneustes fossilis and Clarias batrachus are hardy and capable of living in derelict water bodies and tolerating temporary water deprivation. Several studies have been made on their ureogenic adaptations, ureogenic metabolic machinery, and regulation under different physiological and environmental conditions. Both species are potentially ureogenic teleosts expressing the complete repertoire of ornithine-urea cycle (OUC) enzymes, not only in hepatic tissue but also in certain non-hepatic tissues. This review compiles the information available on the peculiarities of their ureogenic machinery and the induction of ureogenesis during adaptation to various stressful conditions such as exposure to high environmental ammonia, water deprivation, highly alkaline environment, etc. The biochemical profile and pattern of physiological adaptations, suggesting an intermediary status of these fishes in the evolution of ureotely in vertebrates, is discussed.

Anthelmintic efficacy of Flemingia vestita: genistein-induced effect on the activity of nitric oxide synthase and nitric oxide in the trematode parasite, Fasciolopsis buski

The root-tuber peel of Flemingia vestita, an indigenous leguminous plant of Meghalaya (Northeast India), has usage in local traditional medicine as curative against worm infections. The peel and its active component, genistein, have been shown to cause flaccid paralysis, deformity of tegumental architecture and alterations in the activity of several enzymes in platyhelminth parasites. To investigate further the mode of action and anthelmintic efficacy of the plant-derived components, the crude peel extract of F. vestita and genistein were tested, hitherto for the first time, in respect of the unique neuronal messenger nitric oxide (NO) and the enzyme nitric oxide synthase (NOS) in Fasciolopsis buski, the large intestinal fluke of swine and human host. NADPH-diaphorase histochemical staining (a selective marker for NOS in neuronal tissues), which was demonstrable in the neuronal cell bodies in the cerebral ganglia, the brain commissure, the main nerve cords and in the innervation of the pharynx, ventral sucker, terminal genitalia and genital parenchyma of the parasite, showed a stronger activity in the treated worms. In biochemical analysis also, the NOS activity showed a significant increase in the parasites treated with the test materials and reference drug, compared to the untreated controls. The increase in NOS activity in the treated parasites can be attributed to an inducing effect of the plant-derived components.