Studies on L-arginase in developing rat small intestine, brain, and kidney. II. Effect of hydrocortisone and thyroxine

The influences of hydrocortisone and thyroxine on the developmental changes of arginase activity in intestine, kidney, and brain of suckling rats were studied. A single injection of hydrocortisone (50 mg/kg) into rats aged 9 days evoked premature increase of jejunal arginase activity due to precocious formation of arginase A4. Arginase A4 can be detected about 48 hr after hydrocortisone injection, whereas in intact rats the enzyme appears in the intestinal mucosa on the 19th-21st days of postnatal life. After hydrocortisone administration to rats aged 6 days, a similar pattern of arginase activity in jejunum was observed. Under the same conditions, the influence of hydrocortisone on kidney arginase was weaker. The hormone did not have any influence on the activity of brain arginase. Daily injection of thyroxine (2 mg/kg) to 6-day-old rats (for 6 consecutive days) caused a precocious increase of the arginase activity in intestine. Under the same conditions, only a slight increase of the arginase activity was observed in kidney, whereas in brain the activity was unaffected.     

beta-Galactosidases in mouse duodenal mucosa.

beta-Galactosidase activity, in fetal mice, first appears at 16 days of gestation and has a pH optimum of 4. In postnatal development the enzyme activity of cell homogenates tends to show bimodal pH at 4 and 5.6. There are two molecular forms of the enzyme, separable both by molecular-sieve chromatography and electrophoresis. One of the molecular forms of the enzyme is active over a wider range of pH (3.2-6.2) and has half as much activity at 5.6 as it does at 4. This isoenzyme is continuously present in both fetal and postnatal stages. The second isoenzyme first appears at birth, is active over a narrower range of pH (4.6-6.2) and inactive at PH 4. The bimodal pH optima observed in postnatal stages in the cell homogenates, appears to be due to the combined activity of the two molecular forms. In isolated brush border membranes, isoenzyme 2 is the only one present. The other organelles (mitochondria, microsomes, lysosomes, nuclei and cytoplasm) have variable proportions of both isoenzymes, as indicated by the activity ratio at pH 4/5.6.     

Hexokinase isoenzymes in tissues of the adult and developing guinea pig

Changes in the activities and isoenzyme distribution of hexokinase were determined in a number of tissues during the development of the guinea pig. The total activity in the fetal liver showed a large fall during the second half of gestation to reach adult values by term. With normal diet the fetal, neonatal, and adult livers had isoenzymes I and III but little or no detectable IV (glucokinase). The fetal liver had predominantly type I, but the proportion of type III increased during development. The kinetics of the guinea pig isoenzymes were similar to those reported for the rat. Two additional isoenzymes with mobility between I and II were detected in the fetal liver and blood. They appear to have kinetic properties similar to type I. Detectable liver glucokinase activity was induced by glucose administration to adult guinea pigs. The total activity in kidney, brain and skeletal muscle showed a postnatal rise while in the fetal heart it was high and declined after birth. These tissues contained predominantly type I with varying proportions of type III hexokinase. The ratio of particulate-bound to soluble hexokinase varied from tissue to tissue. All except the liver showed a significant increase in binding after birth. The changes are discussed in relation to the control of glucose utilization in the fetal and neonatal periods.     

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.     

Adenylate kinase isoenzyme patterns in normal and neoplastic human lung, and in various adult as compared to fetal rat tissues

The isozyme pattern and total activity of adenylate kinase were studied in normal adult and fetal human and rat tissues using starch gel electrophoresis. Three adenylate kinase isoenzymes were identified in human tissues. Although normal adult lung exhibited higher adenylate kinase activity than did its fetal or neoplastic variant, isozyme patterns in the three types of tissues were indistinguishable from each other and from that in fetal human liver. The pattern of these three isozymes in rat lung (as in spleen) also did not change between fetal and adult life. However, adult kidney and heart of this species did appear to contain isozymes not present in fetal life. Brain (both adult and fetal) was striking different from all the other tissues in that it contained only one adenylate kinase isozyme. The total adenylate kinase activity per gram of adult rat liver, kidney and lung was significantly higher than in the cognate fetal organs, whereas that in brain or spleen did not change with age. The activity in adult heart (similar to the fetal one) was higher than in any other tissue examined.     

Properties and prenatal ontogeny of beta-D-mannosidase in selected goat tissues.

beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.     

PHOSPHOCHOLINE DIGLYCERIDE TRANSFERASE ACTIVITY DURING DEVELOPMENT OF THE CHICKEN BRAIN

Abstract— The activity of chicken brain phosphocholine diglyceride transferase was followed during pre- and postnatal development. The specific activity of this enzyme increases from the 10th day of embryonic life, reaches a maximum at hatching and decreases thereafter. Total brain activity increases in parallel with the increase of brain lecithins. The apparent K _m of the enzyme for CDP choline is 1.5 × 10^-4 m before the 10th day of embryonic life, 2.5 × 10^-5 m between the 13th day of the embryo and the 10th day after hatching, and finally 1.3 × 10^-4 m after the 38th day of postnatal life. These data suggest the existence of isoenzymes, one of which appears at the beginning of myelination.     

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.     

Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acid binding protein

The rat contains at least three homologous cytosolic proteins that bind long chain fatty acids, termed liver (L-), intestinal (I-), and heart (H-) fatty acid binding protein (FABP). I-FABP mRNA is confined to the gastrointestinal tract while L-FABP mRNA is abundantly represented in hepatocytes as well as enterocytes. We have isolated a rat heart FABP cDNA clone and determined the pattern of H-FABP mRNA accumulation in a wide variety of tissues harvested from late fetal, suckling, weaning, and adult rats. RNA blot hybridizations and primer extension analysis disclosed that the distribution of H-FABP mRNA in adult rat tissues is different from that of I- or L-FABP mRNA. H-FABP mRNA is most abundant in adult heart. This mRNA was also present in an adult slow twitch (type I) skeletal muscle (soleus, 63% of the concentration in heart), testes (28%), a fast twitch skeletal muscle (psoas, 17%), brain (10%), kidney (5%), and adrenal gland (5%). H-FABP mRNA was not detected in adult small intestine, colon, spleen, lung, or liver RNA. Distinct patterns of developmental change in H-FABP mRNA accumulation were documented in heart, placenta, brain, kidney, and testes. Myocardial H-FABP mRNA levels rise rapidly during the 48 h prior to and after birth, reaching peak levels by the early weaning period. The postnatal increase in myocardial H-FABP mRNA concentration and its relative distribution in adult fast and slow twitch skeletal muscle are consistent with its previously proposed function in facilitating mitochondrial beta-oxidation of fatty acids. However, the presence of H-FABP mRNA in brain, a tissue which does not normally significantly oxidize fatty acids in late postnatal life, suggests that H-FABP may play a wider role in fatty acid metabolism than previously realized. Mouse-hamster somatic cell hybrids were utilized to map H-FABP. Using stringencies which did not produce cross-hybridization between L-, I-, and H-FABP DNA sequences, we found at least three loci in the mouse genome, each located on different chromosomes, which reacted with our cloned H-FABP cDNA. None of these H-FABP-related loci were linked to the gene which specifies a highly homologous adipocyte-specific protein termed aP2 or to genes encoding two other members of this protein family, cellular retinol binding protein and cellular retinol binding protein II.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of arginase II and related enzymes in the rat small intestine and kidney

Arginase, which catalyzes the conversion of arginine to urea and ornithine, and consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Arginine is also used for the synthesis of nitric oxide and creatine phosphate, while ornithine is used for the synthesis of polyamines and proline, and thus collagen. Arginase II mRNA and protein are abundant in the intestine (most abundant in the jejunum and less abundant in the ileum, duodenum, and colon) and kidney of the rat. In the kidney, the levels of arginase II mRNA do not change appreciably from 0 to 8 weeks of age. In contrast, arginase II mRNA and protein in the small intestine are not detectable at birth, appear at 3 weeks of age, the weaning period, and their levels increase up to 8 weeks. On the other hand, mRNAs for ornithine aminotransferase (OAT), ornithine decarboxylase, and ornithine carbamoyltransferase (OCT) are present at birth and their levels do not change much during development. Arginase II is elevated in response to a combination of bacterial lipopolysaccharide, dibutyryl cAMP, and dexamethasone in the kidney, but is not affected by these treatments in the small intestine. Immunohistochemical analysis of arginase II, OAT, and OCT in the jejunum revealed their co-localization in absorptive epithelial cells. These results show that the arginase II gene is regulated differentially in the small intestine and kidney, and suggest different roles of the enzyme in these two tissues. The co-localization of arginase II and the three ornithine-utilizing enzymes in the small intestine suggests that the enzyme is involved in the synthesis of proline, polyamines, and/or citrulline in this tissue.     

Enzymes of ornithine metabolism in adult and developing rat intestine.

The levels of 11 enzymes, most of them involved in the metabolism of ornithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, ornithine aminotransferase, and ornithine transcarbamylase were compared with those in liver. Changes with age (late gestation of adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggest that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotransferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the ornithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

A comparative polyacrylamide gel electrophoretic study of arginase in vertebrate tissues

The electrophoretic behaviour of arginase in the tissue extracts of rat, beef, lizard and frog was studied by bidirectional polyacrylamide gel electrophoresis. The enzyme from rat liver and submaxillary gland migrated to the cathode with the activity concentrated in a single peak. Arginase from beef liver emerged as a single peak of anodal migration with a significant shoulder in the sample gel. Frog liver and kidney enzymes also appeared as single peaks with a distinct anodal movement. The activity in mammalian kidney and lizard liver and kidney resolved into two peaks of anodal migration suggesting the presence of two isoenzymes of arginase in these tissues.     

Types of arginase in rat tissues.

Significant amounts of arginase activity were found in homogenates of submaxillary salivary gland and epididymis, as well as of liver, kidney, mammary gland, and small intestine. The isoelectric point of arginase solubilized from kidney was at pH 7.0 in contrast to that of pH 9.4 characteristic of hepatic arginase in rat. The isozymic variants of arginase in the different tissues were identified by their electrophoretic migration on polyacrylamide gels and by titration of the enzymes against antibody prepared against purified rat liver arginase. Antibody titrations confirmed the indications obtained by electrophoresis that one type of arginase is limited to hepatic tissues (and possibly submaxillary gland) while the other type is found in all other tissues. The physiological role of arginase in hepatic tissues has been previously associated with the urea cycle; the possible function of arginase in proline synthesis in other tissues remains to substantiated.     

Widespread expression of arginase I in mouse tissues. Biochemical and physiological implications.

Arginase I (AI), the fifth and final enzyme of the urea cycle, detoxifies ammonia as part of the urea cycle. In previous studies from others, AI was not found in extrahepatic tissues except in primate blood cells, and its roles outside the urea cycle have not been well recognized. In this study we undertook an extensive analysis of arginase expression in postnatal mouse tissues by in situ hybridization (ISH) and RT-PCR. We also compared arginase expression patterns with those of ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). We found that, outside of liver, AI was expressed in many tissues and cells such as the salivary gland, esophagus, stomach, pancreas, thymus, leukocytes, skin, preputial gland, uterus and sympathetic ganglia. The expression was much wider than that of arginase II, which was highly expressed only in the intestine and kidney. Several co-localization patterns of AI, ODC, and OAT have been found: (a) AI was co-localized with ODC alone in some tissues; (b) AI was co-localized with both OAT and ODC in a few tissues; (c) AI was not co-localized with OAT alone in any of the tissues examined; and (d) AI was not co-localized with either ODC or OAT in some tissues. In contrast, AII was not co-localized with either ODC or OAT alone in any of the tissues studied, and co-localization of AII with ODC and OAT was found only in the small intestine. The co-localization patterns of arginase, ODC, and OAT suggested that AI plays different roles in different tissues. The main roles of AI are regulation of arginine concentration by degrading arginine and production of ornithine for polyamine biosynthesis, but AI may not be the principal enzyme for regulating glutamate biosynthesis in tissues and cells.     

Estrone sulfatase activity in guinea pig tissues

Estrone sulfatase activity is widespread in guinea pig tissues. Whole homogenates of adult testis. uterus. lung, adrenal, amnion, ovary, chorion, small intestine, placenta, spleen, kidney and liver exhibit approximately descending order of specific activity. Certain properties, including pH requirement, lack of inhibition by inorganic sulfate and magnitude of estimated K_mvalues, are similar to that for arylsulfatase C of rat liver. Of the subcellular fractions prepared from guinea pig tissues, microsomes exhibit the highest specific activity although considerable enzyme activity remains associated with large cellular fragments sedimenting at 750 g. The sulfatase activity is readily inhibited by inorganic phosphate even when substrate concentration satisfies zero order kinetics. Rat liver arylsulfatase C is not inhibited under these conditions. Sensitivity of the guinea pig enzyme activity to inhibition by a variety of steroids and related compounds, is markedly less than for rat liver. Diethylstilbestrol (DES) strongly inhibits the rat liver enzyme but has little effect on the guinea pig liver system. Guinea pig testicular activity is suppressed to a degree intermediate between these extremes by increasing DES concentration. In guinea pig lung. kidney, and possibly liver, elevated fetal enzyme activities decrease from neonatal to adult life. Teslicular activity appears to follow the opposite trend. Uterine enzyme activity is not markedly affected by pregnancy.     

Uridine kinase activities in developing, adult and neoplastic rat tissues.

Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3--5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.     

Vitamin D-dependent calcium-binding protein. Changes during gestation, prenatal and postnatal development in rats

During the perinatal period, calcium metabolism is stressed. As intestinal Ca-binding protein is considered as a molecular expression of the hormonal effect of 1,25-dihydroxycholecalciferol (1,25(OH)2D3), Ca-binding protin measurements may document the vitamin D roles during this period. We describe the variations of Ca-binding protein concentrations in the rat during the last 5 days of gestation, in the maternal duodenum, placentas, fetal membranes and fetal intestines. We also report intestinal Ca-binding protein changes from birth until weaning. The evolution of the maternal intestinal Ca-binding protein, which increases on day 19.5 of gestation, is consistent with that of calcium intestinal absorption and may be explained by increased 1,25(OH)2D3 production. Placental Ca-binding protein rises from day 17.5 until the end of gestation, and may be related to the profile of calcium transfer from mother to fetuses. It is noteworthy that the placental Ca-binding protein is predominantly found in the fetal part of the organ where materno-fetal exchanges occur. The yolk sac synthesizes substantial amounts of Ca-binding protein. In the fetal membranes, Ca-binding protein plateaus from day 17.5 until day 20.5 and decreases on day 21.5. The Ca-binding protein presence in the fetal placenta and in the yolk sac may suggest that these tissues are also targets for vitamin D. In the fetus the intestinal Ca-binding protein s is detected as early as day 17.5 of gestation and increases markedly during the last day of gestation. From birth and during the first 3 weeks of postnatal life, the intestinal Ca-binding protein concentration does not change. It undergoes a sharp rise just at the time of weaning. We have also shown that the specific distribution of Ca-binding protein along the intestine is acquired during intrauterine life and does not change with sucking or weaning. The two main changes of intestinal Ca-binding protein, observed just before birth and at weaning, may reflect the intestinal maturation and/or variations in vitamin D metabolism.     

Studies on the mechanisms of histidase development in rat skin and liver. I. Basis for tissue specific developmental changes in catalytic activity.

Skin and liver histidases of the rat follow markedly differing developmental courses. In skin, the enzyme activity emerges in late fetal life, rises to a maximum during the first postnatal week and then declines; in liver, histidase activity initially appears in the neonatal period and progressively rises thereafter. The enzymes in both tissues, however, are immunologically identical, as demonstrated by Ouchterlony double diffusion, immunoelectrophoresis, and immunotitration analyses of histidase in adult animals. Furthermore, although crude preparations of skin and liver histidase differ slightly in electrophoretic mobility on acrylamide gels, the isoelectric points and Michaelis constants of the enzyme from both skin and liver are the same. During development neither dissociable activators nor inhibitors of the enzyme are evident, which might account for the divergent developmental patterns, nor are enzyme inhibitors found in tissues devoid of histidase activity. No immunologically cross-reacting materials are detectable in skin and liver at developmental stages prior to the emergence of enzyme activity, nor in tissues not expressing histidase catalytic activity. Furthermore, there is no evidence of elaboration of immunological variants of histidase in skin and liver during development. These data are compatible with the view that all developmental changes in catalytic activity are due to altered amounts of the same enzyme antigen.     

Enzymes of orithine metabolism in adult developing rat intestine

The levels of 11 enzymes, most of them involved in the metabolism of orithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, orithine aminotransferase, and orithine transcarbamylase were compared with those in liver. Changes with age (late gestation to adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described.The results suggests that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotraferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue.Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the orithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.