Arginase activity patterns and their regulation during embryonic development in liver and kidney

The developmental patterns for mouse liver and kidney arginase were measured by a sensitive radioactive assay from day 8 of gestation until adulthood. On day 8 high arginase activity is generally distributed throughout early embryos. Then, as development proceeds, the arginase activity drops rapidly in liver and kidney, apparently because of mass increase unaccompanied by net arginase synthesis. Suddenly, on day 12 of gestation in liver and on day 16 in kidney, arginase activity begins to accelerate toward adult values.In order to study the mechanisms controlling arginase acceleration, 12- and 13-day fetal livers were explanted to organ cultures containing various exogenous chemicals, and subsequently assayed for arginase. Physiological concentrations of hydrocortisone causes the arginase activity to rise more than 100-fold to adult levels within 4 days in culture. Glucagon, thyroxine, and dibutyryl adenosine-3′-5′-cyclic phosphate have no effect in this system. Experiments with cycloheximide, actinomycin D, and 5-fluorodeoxyuridine suggest that the hydrocortisone response is dependent upon protein and RNA synthesis but independent of DNA synthesis.     

Studies on L-arginase in developing rat small intestine, brain, and kidney. I. Ontogenic evolution of arginase isoenzymes

The adult patterns of arginase isoenzymes in rat intestine, kidney, and brain are nearly identical and consist of two forms, cationic A1 and anionic A4. In this paper, the organ-specific maturation of the enzyme equipment in these tissues is reported. The activity of arginase in all tissues studied could be detected on the 13th to 16th days of gestation. In fetal intestine and kidney the arginase activity is low, and persists up to the weaning time when the rapid, 10-fold rise of the enzyme activity occurs. However, the adult pattern of arginase isoenzymes in these tissues is accomplished in different ways. In the intestine, arginase A1 appears in fetal life and is the only form of the enzyme till the 19th to 21st days of postnatal life when the second form of arginase, A4, appears and rapidly accumulates, being exclusively responsible for the rise of the total enzyme activity at the time of weaning. In kidney, arginase A1 alone is present in the early fetal period. Arginase A4 appears 3-4 days before birth and its activity persists unchanged within the first 2 weeks of postnatal life. The intensive rise in total specific activity of kidney arginase at weaning is due to the accumulation of preexisting arginase A4. In brain, the adult pattern of arginase isoenzymes is achieved earlier than in other tissues. Both forms, A1 and A4, occur on Days 13-14 of gestation.     

Maltase-glucoamylase and trehalase in the rabbit small intestine and kidney brush border membranes during postnatal development, the effects of hydrocortisone

Kidney and intestinal brush border membranes were isolated from 14-day-old rabbits and papa?n solubilized maltase-glucoamylase was purified to almost homogeneity from both membranes. Maltase-glucoamylase from kidney and intestine have the same molecular weight (669,000 daltons by AcA 22 gel filtration) and the same Km (4 mM, for maltose). Tris (Ki = 12.5 mM, for maltose) is a non-competitive inhibitor for both enzymes. In intestine, maltase and glucoamylase have low activity during the first two postnatal weeks and then undergo a sharp increase during the next 2 weeks. In contrast, for trehalase, adult levels are reached about 6 days after birth. Hydrocortisone injection to 10 days rabbits causes precocious increases in the specific activities of trehalase (3.6 x), maltase (5.2 x) and glucoamylase (7.4 x). Conversely, kidney maltase, glucoamylase and trehalase activities rise gradually from birth, reaching adult levels by the end of the third week. Administration of hydrocortisone to suckling rabbit does not affect either trehalase or maltase and glucoamylase in kidney brush border membrane.     

Precocious induction of pepsinogen in the stomach of suckling mice by hormones

Effects of hormones on pepsinogen activity in mouse stomach were investigated by enzyme assay and electron microscopy. Administration of hydrocortisone alone to mice on days 5–10 increased the enzyme activity in the stomach to as much as 4.5-fold that of untreated mice and the increase was dose dependent. Thyroxine also evoked precocious differentiation of the stomach. The effects of thyroxine and hydrocortisone were additive. Injections of insulin had little effect when given alone, or in combination with other hormones. Injection of hydrocortisone alone or plus thyroxine also caused morphological differentiation of the chief cells in the stomach mucosa. Administration of thyroxine to mice on days 15–20 induced as much enzyme activity as that induced by hydrocortisone, but neither of these hormones had any effect when injected after day 23.These results suggest that besides hydrocortisone, thyroxine is also involved in differentiation of the stomach in mice for the first 20 days after birth and that the normal increase of pepsinogen activity in the stomach of mice during the late suckling period is brought about by serum glucocorticoids, possibly with thyroxine.     

The regulation of phosphate-activated glutaminase activity and glutamine metabolism in the streptozotocin-diabetic rat.

The activity of phosphate-activated glutaminase was increased in the kidney, liver and small intestine of rats made diabetic for 6 days with injection of streptozotocin (75 mg/kg body wt.). Insulin prevented this increase in all three tissues. Treatment with NaHCO3, to correct the acidosis that accompanies diabetes, prevented the increase in renal glutaminase activity, but not that in liver or small intestine. Chemically induced acidosis (NH4Cl solution as drinking water) or alkalosis (NaHCO3 solution as drinking water) increased and decreased, respectively, glutaminase activity in the kidney, but were without significant effect on the activity in liver and small intestine. The increase in glutaminase activity in the small intestine during diabetes was due to an overall increase in the size of this organ, and was only detectable when activity was expressed in terms of whole organ, not mucosal scrapings or isolated enterocytes. Prolonged diabetes (40 days) resulted in an even greater increase in the size and glutaminase activity of the small intestine. Despite this marked increase in capacity for glutamine catabolism, arteriovenous-difference measurements showed a complete suppression of plasma glutamine utilization by the small intestine during diabetes, confirming the report by Brosnan, Man, Hall, Colbourne & Brosnan [(1983) Am. J. Physiol. 235, E261-E265].     

Premature induction of pepsinogen in developing rat gastric mucosa by hormones

Daily injection of ACTH into rats from the age of 9 days caused precocious development of peptic activity in the gastric mucosa. A single injection of hydrocortisone into rats aged 2 to 9 days also evoked premature development of peptic activity in the gastric mucosa. The patterns on polyacrylamide gel electrophoresis of pepsinogen from adults and from infants treated with hormone were quite similar but differed from that of untreated infants. Enzyme from the gastric mucosa of adult rats and enzyme evoked with hormones were more stable than that from infant rats.     

The sedimentation of rat skeletal-muscle ribosomes. Effect of hydrocortisone, insulin and diet

1. The influence of hydrocortisone, insulin and diet on the size distribution of ribosomes in a post-mitochondrial supernatant prepared from rat skeletal muscle was studied by sedimentation analysis with a linear 15-40% (w/v) sucrose gradient. 2. Within 4hr. after the injection of 5mg. of hydrocortisone to well-nourished rats, a decrease in the yield per g. of muscle and proportion of total RNA due to polyribosomes was observed. Similar results were obtained in rats given a protein-free diet for 3 days before administration of the hormone. 3. Insulin injection increased the yield and proportion of polyribosomes within 2hr. and decreased the proportion of the lighter ribosomal aggregates. Similar results were noted in rats given a protein-free diet for 3 days before injection. A protein-free diet given for 3 days decreased the yield and proportion of polyribosomes. Insulin did not increase the yield of polyribosomes if rats were starved for 52hr. before injection, but decreased the yield and proportion of the lighter ribosome species. 4. A 52hr. period of starvation or 2,4-dinitrophenol (15mg./kg. body wt.) given 1hr. before the rats were killed resulted in a decreased yield and proportion of polyribosomes, and, within 6hr. of re-feeding the rats with protein-free diets, an increased concentration of polyribosomes was noted. 5. The effects of a protein-free diet, hydrocortisone and insulin on the sedimentation of muscle ribosomes were found to be in accord with their net effects on muscle protein synthesis.     

Effect of caffeine on ornithine metabolism in rat brain, liver and kidney

Prolonged treatment with caffeine promotes in rats an increase of liver ornithine carbamyltransferase activity (14-day treatment). In contrast, arginase activity is already reduced in brain and kidney after 10 days, and in the liver much later (17 days). Ornithine transaminase activity was increased in both liver and kidney, while in the brain it was reduced (17 days). Ornithine decarboxylase activity showed only minor modifications in kidney, while it was unchanged in brain. Of the polyamines, only spermidine was significantly modified, being increased in brain, decreased in liver and kidney. Although these results do not explain the mechanism of the modification of brain arginine and ornithine concentration promoted by caffeine, they point to further marked effects, i.e. on OAT activity and on spermidine concentration, which could have a relevant metabolic role.     

Essential role for polyamine biosynthesis in thyroxine stimulated pancreatic development in neonatal rats.

Administration of thyroxine to rat pups leads to precocious development of the pancreas. The role of ornithine decarboxylase (ODC) and polyamines in thyroxine-induced pancreatic maturation was examined. Rat pups (aged 5 days) were given daily subcutaneous injection of thyroxine (0.1 micrograms/g body wt.) until the day before death. Serial ODC activities were measured in pancreatic homogenates after 1, 2, 3, 4, 5, 6, 7 and 10 days of thyroxine treatment. There was a biphasic induction of ODC activities by thyroxine: an early peak appeared on day 2 of treatment followed by a decrease on day 4; a second peak was evident on day 5 and then a decrease to control values by day 7. Significant increases in tissue concentrations of putrescine and spermidine were observed concomitant with two peaks of ODC activity. Pancreatic amylase concentration, DNA and protein also showed a significant increase after thyroxine treatment. Difluoromethyl ornithine (DFMO), a specific ODC inhibitor, given orally (8% in drinking water) to nursing dams at postnatal day 5 for 5 days caused an 83% inhibition of pancreatic ODC activity in thyroxine-treated pups when compared to thyroxine-treated pups not exposed to DFMO. Concomitantly, the thyroxine-induced increases in pancreatic weight, protein and amylase activity were suppressed. Our results suggest that increases in ODC activities and polyamine levels are critical intermediary steps in the precocious induction of pancreatic development by thyroxine.     

Ontogenesis of Monoamine-Synthesizing Enzyme Activities and Biopterin Levels in Rat Brain or Salivary Glands, and the Effect of Thyroxine Administration

Neonatal changes in the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TrpH) and in the content of the co-factor, biopterin, were studied in rat midbrain for the first 20 days after birth. Changes in TH activity in the parotid and submandibular glands were also examined. Changes in TH activity per unit weight in the developing rat brain were briefly similar to those in the salivary glands; the activity increased from day 2 or 4 to day 9 after birth, and remained constant or slightly decreased at day 12, then rapidly increased on day 16. TrpH activity in the midbrain increased about twofold up to day 16. The biopterin concentration in the brain increased, reached a maximum level on day 12 after birth, and thereafter decreased. The effect of hyperthyroidism in rats given 0.2 mg/kg i.p. of thyroxine every 2 days postnatally was studied on the activity of TH in rat salivary glands at 12-day-old rats. In parotid or submandibular gland of hyperthyroid rats, TH activity increased at day 12 postnatally. In comparison with the effect on TH activity in the salivary glands, TH activity in the midbrain on day 20 postnatally was not induced by hyperthyroidism. Furthermore, increase of the TrpH activity and biopterin and catecholamine levels in the midbrain of hyperthyroid rats was not found on day 20 after birth in comparison with the corresponding controls. From these data, we suppose that postnatal hyperthyroidism may cause precocious induction of TH in rat salivary gland, but may not increase the activity of TH or TrpH, and the level of their co-factor, biopterin, in rat midbrain.     

Pentose phosphate pathway,glutathione-dependent enzymes and antioxidant defense during oxidative stress in diabetic rodent brain and peripheral organs: effects of stobadine and vitamin E

The aim of the present study was to investigate the effects of treatment with antioxidant stobadine (ST) on the activities of enzymes related with pentose phosphate pathway and glutathione-dependent metabolism and the other markers of oxidative stress in brain and peripheral organs of diabetic rats, and to compare the effects of ST treatment alone with the effects of treatments with another antioxidant vitamin E and ST plus vitamin E. Rats were made diabetic by the injection of streptozotocin (STZ; 55 mg/kg IP), and, 2 days later, some control and diabetic rats were left untreated or treated with ST (24.7 mg/kg/day, orally), vitamin E (400–500 U/kg/day, orally), or both substances together. In the brain, although 6-phosphogluconate dehydrogenase activity (6-PGD) did not change, glucose-6-phosphate dehydrogenase activity (G-6PD) was markedly increased in diabetic rats compared with controls; only combined treatment with ST and vitamin E produced a partial prevention on this alteration. The aorta G-6PD and 6-PGD of diabetic rats were 52% and 36% of control values, respectively. Neither single treatments with each antioxidant nor their combination altered the G-6PD and 6-PGD in aorta of diabetic rats. Glutathione peroxidase (GSHPx) activity was increased by STZ-diabetes in brain, heart, and kidney. In diabetic brain, vitamin E alone or combination with ST kept GSHPx at normal levels. Diabetes-induced stimulation in GSHPx did not decrease in response to the treatment with vitamin E in heart and kidney, but was greatly prevented by ST alone. The activity of glutathione reductase (GR) was decreased in brain and heart of diabetic rats. The treatment with each antioxidant or with a combination of both agents completely prevented this deficiency and resulted in further activation of GR in diabetic tissues. Glutathione S-transferase (GST) activity did not significantly change in diabetic brain and aorta. GST was stimulated by all treatment protocols in the brain of diabetic rats and was depressed in aorta of control rats. Catalase (CAT) was activated in diabetic heart but depressed in diabetic kidney. Diabetes-induced abnormalities in CAT activity did not respond to vitamin E alone in heart, was moderately ameliorated by the treatment with this vitamin in kidney, and was completely prevented by ST alone in both tissues. Superoxide dismutase (SOD) activity of brain and heart was unchanged by the diabetes but inhibited in diabetic kidney after the treatment ST alone or ST plus vitamin E. The lipid peroxidation (MDA) was increased in diabetic brain and heart. ST or vitamin E alone partly prevented diabetes-induced increase in MDA in brain and heart; however, antioxidant combination achieved a completely amelioration in MDA of these tissues of diabetic rats. Kidney MDA levels were similar in control and untreated diabetic animals. ST and vitamin E treatments, when applied separately or together, significantly reduced kidney MDA in both control and diabetic rats; and the combined effect of antioxidants was greater than that of each alone. These results are consistent with the degenerative role of hyperglycemia on cellular reducing equivalent homeostasis and antioxidant defense, and provide further evidence that pharmacological intervention of different antioxidants may have significant implications in the prevention of the prooxidant feature of diabetes and protects redox status of the cells.     

Increased thyroxine turnover after 3,3',4,4',5,5'-hexabromobiphenyl injection and lack of effect on peripheral triiodothyronine production

Juvenile male Wistar rats were injected i.p. with 0, 20, or 40 mg/kg 3,3',4,4',5,5'-hexabromobiphenyl and blood samples collected periodically up to 28 days. A dose-dependent depression of the serum thyroxine level was detected, while the circulating triiodothyronine concentration was not affected by the biphenyl congener. Thyroxine turnover in vivo 7 days after injection of the 20 mg/kg dose revealed significant increases of various clearance parameters relative to controls. The fractional clearance rate (day-1) increased by 84%, the daily metabolic clearance rate (mL.kg-1.day-1) increased by 128%, and the daily thyroxine disposal rate (ng.kg-1.day-1) increased by 41%. Also, the thyroxine distribution space (mL/kg) increased by 21%. These results indicated greater thyroxine binding in major organs as well as a marked increase in the peripheral metabolism of thyroxine. The increased thyroxine metabolism is explained by a 4.8-fold induction of uridine 5'-diphosphoglucuronyltransferase activity in liver microsomes. The type I 5'-deiodinase activity in liver homogenates and endogenous concentrations of the cofactor for this reaction, glutathione, were not affected by the biphenyl. This result means that homeostatic mechanisms involving thyroxine conversion to triiodothyronine do not explain the maintenance of serum T3 under these conditions.     

Prenatal treatment of mosaic mice (Atp7a mo-ms) mouse model for Menkes disease, with copper combined by dimethyldithiocarbamate (DMDTC)

Menkes disease is a fatal neurodegenerative disorder in infants caused by mutations in the gene ATP7A which encodes a copper (Cu) transporter. Defects in ATP7A lead to accumulated copper in the small intestine and kidneys and to copper deficiencies in the brain and the liver. The copper level in the kidney in postnatal copper-treated Menkes patients may reach toxic levels. The mouse model, mosaic Atp7a (mo-ms) recapitulates the Menkes phenotype and die about 15.75±1.5 days of age. In the present study we found that prenatal treatment of mosaic murine fetuses throughout gestation days 7, 11, 15 and 18 with a combination of CuCl(2) (50 mg/kg) and dimethyldithiocarbamate (DMDTC) (280 mg/kg) leads to an increase in survival to about 76±25.3 days, whereas treatment with CuCl(2) alone (50 mg/kg) only leads to survival for about 21 days ±5 days. These copper-DMDTC treated mutants showed an improved locomotor activity performance and a gain in body mass. In contrast to treatment with CuCl(2) alone, a significant increase in the amount of copper was observed in the brain after prenatal copper-DMDTC treatment as well as a decrease in the amount of accumulated copper in the kidney, both leading towards a normalization of the copper level. Although copper-DMDTC prenatal treatment only leads to a small increase in the sub-normal copper concentration in the liver and to an increase of copper in the already overloaded small intestine, the combined results suggest that prenatal copper-DMDTC treatment also should be considered for humans.     

Comparative study on the inhibition of acetylcholinesterase activity in the freshwater fish Cyprinus carpio by mercury and zinc.

The effects of sublethal concentrations of mercury (0.1mg/l) and zinc (6 mg/l) on acetylcholinesterase activity and acetylcholine content of gill, kidney, intestine, brain, liver and muscle of the freshwater fish Cyprinus carpio at 1, 15 and 30 days of exposure were studied. A significant suppression in acetylcholinesterase activity was recorded in all the organs from both mercury and zinc intoxicated fish at all the exposure periods. Concurrently, a significant increase in the content of acetylcholine in the organs was observed. These changes observed in the organs of mercury treated fish in different exposure periods were in the order 1 greater than 15 less than 30 days and in zinc treated fish 1 greater than 15 greater than 30 days. Further, these changes were greater in magnitude in the brain, liver and muscle (non-osmoregulatory organs) than in the gill, kidney and intestine (osmoregulatory organs) in both metal media.     

Cortisone and thyroxine modulate intestinal lactase and sucrase mRNA levels and activities in the suckling rat.

Glucocorticoids and thyroxine modulate postnatal intestinal sucrase and lactase activities. Whether changes in enzyme activity are accompanied by changes in enzyme mRNA levels were determined in day 6 rats given thyroxine, cortisone, or thyroxine plus cortisone and killed 3 days later. Cortisone induced precocious expression of jejunal sucrase activity which was enhanced when cortisone plus thyroxine was administered; sucrase mRNA changed in parallel. Jejunal lactase activity was unaffected by thyroxine and was increased after cortisone, but not after thyroxine plus cortisone. Jejunal lactase mRNA levels increased equally after cortisone or after cortisone plus thyroxine. Thus, cortisone induces coordinated increases in sucrase and lactase activities and in corresponding mRNA levels. Thyroxine only enhances cortisone induced sucrase expression and antagonizes cortisone by depressing lactase activity post-translationally.     

Alterations in the activities of subcellular fractions marker enzymes in rat liver and brain by hydrocortisone and corticosterone treatment

The effect of subcutaneous injection of hydrocortisone and corticosterone on the activity values of some subcellular fractions marker enzymes from rat liver and brain was investigated and compared with controls (without treatment with hormones). The following enzymes were studied (subcellular fraction are shown between parentheses): N-acetyl-beta-D-glucosaminidase and beta-glucuronidase (lysosomes); succinate dehydrogenase = SDH (mitochondria); glucose-6-phosphatase (endoplasmic reticulum); 5'-nucleotidase and Na+-K+-Mg2+ ATPase (plasma membrane). The specific activity of lysosomal enzymes from liver showed no change when rats were injected either with hydrocortisone or corticosterone. The same enzymes from brain showed significant increases in their activities with both hydrocortisone or corticosterone except beta-glucuronidase; this enzyme gave activity values remaining between the control levels, after treatment with corticosterone. The activity of mitochondrial SDH was increased after corticosterone injection either in liver or brain. After hydrocortisone injection, its activity rises significantly in brain (72%), but it falls in liver compared to the control values. Glucose-6-phosphatase behaves similarly in brain or liver fractions; its activity increases always after corticosterone treatment and decreases by hydrocortisone. The plasma membrane marker enzymes did not change practically in brain fractions, excepted Na+-K+-Mg2+ ATPase which tends to rise its activity after hydrocortisone injection. In liver fractions, both 5'-nucleotidase and Na+-K+-Mg2+ ATPase activities increase either by corticosterone or hydrocortisone treatment, except 5'-nucleotidase which specific activity decreases in liver after hydrocortisone treatment.     

Differential recovery of intestine, bone marrow, and thymus of rats with solid tumors following 5-fluorouracil administration.

Time relationships for recovery of several host organs from toxic effects of 5-fluorouracil were determined in ACI rats bearing Morris hepatoma 3924A. A single injection of 150 mg/kg body weight 5-fluorouracil (the LD10) resulted in loss of 90% of the tibial bone marrow, 60% of the intestinal mucosa, and 90% of the thymus as measured by total DNA content of the organs. Organ DNA contents following 150 mg/kg of the drug were minimal on day 3 for intestine and on day 5 for marrow and thymus. A return to pretreatment or higher levels of DNA was observed by day 4 for intestine, day 11 for tibial marrow, and day 19 for thymus. Incorporation of 3H-deoxyuridine into host organ DNA after 150 mg/kg 5-fluorouracil was inhibited 36 hrs for intestine, 3 days for thymus, and 5 days for tibial bone marrow. Inhibition of 3H-deoxyuridine incorporation into DNA was similar for 50, 100, and 150 mg/kg doses both in tumor and in host organs, but recovery of 3H-deoxyuridine incorporation and DNA content of host organs began later with the higher doses of 5-fluorouracil. Maximal incorporation of 3H-deoxyuridine into DNA was observed on day 4 for intestine, day 8 for marrow, and day 9 for thymus after treatment with 150 mg/kg 5-fluorouracil. Animal lethality following the second of two 150 mg/kg injections of 5-fluorouracil was related to the extent of recovery of intestinal mucosa and bone marrow at the time of the second injection. Survival decreased to 0% for normal rats when the interval between injections was 3-4 days, improved at 5 days and was 100% when the interval was 10-11 days.     

Tissue-specific differential modulation of arginase and ornithine aminotransferase by hydrocortisone during various developmental stages of the rat

The activities and regulatory patterns of arginase and ornithine aminotransferase (OAT) of the liver (a mitotic tissue) and kidney cortex (a post-mitotic tissue) of immature, adult, and senescent male rats were studied. The activities of the liver enzymes were highest in the immature rat and decreased gradually with age. However, in the kidney cortex, the activity of arginase was highest and decreased significantly thereafter while that of OAT shows no significant change throughout the life span of the rat. Further, the activity of kidney cortex arginase was approximately 1/20th of that of the liver enzyme. Adrenalectomy and hydrocortisone treatments altered the activity of arginase in both tissues and that of OAT in the liver only. However, the kidney cortex OAT was not responsive towards these treatments. Actinomycin D inhibited the hydrocortisone-mediated induction of arginase of both the liver and kidney cortex and that of the liver OAT.