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.     

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.     

Studies on inositolphosphatase in rat small intestine

The possibility that inositolphosphatase differs from other intestinal phosphatases was tested by comparing several enzymatic characteristics of phosphatase activities of rat intestinal homogenate acting on various specific substrates. Optimum pH and temperature, Km, Vmax, heat stability, inhibition and metal ion requirement studies suggest that inositolphosphatase differs from phytase and p-nitrophenylphosphatase. Furthermore, we found that inositolphosphatase activity was about 2 times higher in duodenum and jejunum than ileum. It sedimented (90-100%) with a high-speed particulate fraction of mucosal homogenate; 42% of the activity was separated with the brush border membrane isolated from mucosal homogenate. Partial separation by gel filtration on Sephadex G200 and chromatography on phenyl Sepharose CL 4B provided additional evidence to suggest that inositolphosphatase and phytase are different enzymes.     

Studies on the immunoglobulin-G Fc-fragment receptor from neonatal rat small intestine.

1. A method for preparing the small-intestinal brush-border membrane of neonatal rats is described in which enzymic methods are used to remove associated polysaccharide and cell nuclei. 2. 125I-labelled IgG (immunoglobulin G) and 125I-labelled IgG Fc fragment have high specific binding and low non-specific binding to brush borders prepared in this way. F(ab)'2 fragment however, does not bind, indicating the existence of a specific receptor for the Fc fragment of IgG. The receptor system is saturable, and the affinity (KA) for the binding of rat IgG was determined by both equilibrium and kinetic methods. 3. The binding of heterologous IgG species (human and bovine) was compared and demonstrated a close similarity between human IgG and rat IgG in their receptor affinities. 4. Kinetic results are presented that are consistent with previously proposed models of ligand-induced receptor aggregation.     

Phosphatidylcholine synthesis in the developing small intestine.

1. Phosphatidylcholine synthesis in the foetal, newborn and adult small intestine of rats was studied by determination of cytidine diphosphocholine-1,2-diacylglycerocholine phosphotransferase (cholinephosphotransferase) and acyl-CoA-1-acyl-sn-glycerol-3-phosphocholine acyltransferase (lysophosphatidylcholine acyltransferase) activities and the incorporation of [1-14C]oleic acid into phosphatidylcholine. 2. Cholinephosphotransferase activity was low in foetal jejunum and ileum, increased 3-4 fold in the ileum by 6 days of age and by 12 days in the jejunum. Jejunal activity remained constant throughout weaning; ileal activity gradually decreased to values 25% of that of the jejunum. 3. Lysophosphatidylcholine acyltransferase activity was high in foetal jejunum and ileum, decreased 70% immediately after birth in the jejunum and increased to adult values by 12 days of age. Ileal activity decreased by 20% after birth, but decreased more rapidly at weaning to 30% of the activity in jejunum. 4. Initial rates and steady-state incorporation of [1-14C]oleic acid into phosphatidylcholine by jejunal rings of 10 day-old rats exceeded that observed in jejunal rings from adult rats by 2-4-fold. 5. In the postnatal jejunum, neither cholinephosphotransferase and lysophosphatidylcholine acyltransferase activities nor oleic acid incorporation were stimulated by cortisone administration in vivo.     

Studies on molecular species of choline glycerophospholipids of developing rat brain.

The chronological changes in molecular species of choline glycerophospholipids were studied for cerebra of 17-, 19- and 21-day-old rat fetuses, and 3-, 6-, 12-, 24- and 90-day-old rats. The molecular species found by gas chromatography-mass spectrometry and selected ion retrieval technique were phosphatidylcholines of '30 : 0, 32 : 0, 32 : 1, 34 : 0, 34 : 1, 34 : 2, 36 : 0, 36 : 1, 36 : 2, 36 : 3, and 36 : 4' where the larger number indicates the sum of chain lengths on positions C-1 and C-2; the smaller number is the total number of double bonds. Of these molecular species, '32 : 0' (mainly 16 : 0/16 : 0, dipalmitoyl glycerophosphorylcholine), '34 : 1' (mainly 16 : 0/18 : 1, palmitoyloleoyl glycerophosphorylcholine), '34 : 0' (16 : 0/18 : 0, palmitoylstearoyl glycerophosphorylcholine), '32 : 1' (mainly 16 : 0/16 : 1, palmitoylpalmitoleoyl glycerophosphorylcholine and '30 : 0' (14 : 0/16 : 0, myristoylpalmitoyl glycerophosphorylcholine) were main species. The '32 : 0' species increased to about 44% at around the 10th day and thereafter remained nearly constant. '34 : 1' and '34 : 0' decreased to about 17 and 6% at that time and then increased to about 30 and 14%, respectively. '30 : 0' increased from last stage of gestation to the 6th day and then decreased. '32 : 1' was about 16% for 17-day-old fetus and decreased grandually. '36 : 1' (18 : 0/18 : 1, stearoyloleoyl glycerophosphorylcholine) increased at the latter part of development.     

Studies on distribution and metabolism of valproate in rat brain,liver, and kidney

Time-course studies on the distribution and metabolism of valproate (VPA) in rat brain, liver, and kidney, after intraperitoneal injection of a mixture of [¹⁴C]VPA and [³H]VPA, showed that: (1) maximal amount of radioactivity in the various tissues was observed after 30 min from the time the drug was administered; (2) at 30 min the distribution of labeled VPA in brain, liver, and kidney was 17%, 64%, and 19% of the total radioactivity, respectively; (3) at 24 hr more than 97% of the total radioactivity was lost from the tissues and the¹⁴C/³H ratios increased significantly with time. Studies on the regional distribution of the drug showed that it is relatively homogeneously distributed. Studies on the subcellular distribution of the drug showed that it is associated mostly with the soluble and mitochondrial fractions, with little radioactivity in the myelin and synaptosomal fractions. Radiochromatography of VPA metabolites in perchloric acid extracts from brain, liver, and kidney revealed the presence of four metabolites. VPA was not incorporated into phospholipids of the neuronal membranes. Furthermore, it had no significant effects on Mg²⁺-ATPase and (Na⁺+K⁺)-ATPase in synaptosomes and microsomes obtained either from control or from rats injected with VPA. It was concluded that this antiepileptic drug does not appear to act through its incorporation into neuronal membrane or through its action on the Na⁺ pump.Contribution No. 0601 from the Department of Cell and Molecular Biology, the Medical College of Georgia, Augusta, Georgia 30912.     

Hybridization of subunits of rat liver arginase A1 and rat kidney arginase A4

Recombination of subunits of rat liver arginase A1 and rat kidney arginase A4 yielded a product which in polyacrylamide gel electrophoresis and DEAE-cellulose chromatography separated into five proteins with arginase activity. Proteins I and V corresponded in polyacrylamide gel-electrophoresis, DEAE-cellulose chromatography and immunological properties to the parental forms A1 and A4, respectively. Formation of five arginase hybrids proved the tetrameric structure of native arginases.     

Purification and properties of arginase of rat kidney

l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn(2+). Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.     

Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney.

Studies on proton and Na+ transport by isolated intestinal and renal brush-border-membrane vesicles were carried out to test for the presence of an Na+/H+-exchange system. Proton transport was evaluated as proton transfer from the intravesicular space to the incubation medium by monitoring pH changes in the membrane suspension induced by sudden addition of cations. Na+ transport was determined as Na+ uptake into the vesicles by filtration technique. A sudden addition of sodium salts (but not choline) to the membrane suspension provokes an acidification of the incubation medium which is abolished by the addition of 0.5% Triton X-100. Pretreatment of the membranes with Triton X-100 prevents the acidification. The acidification is also not observed if the [K+] and proton conductance of the membranes have been increased by the simultaneous addition of valinomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the K+-rich incubation medium. Either valinomycin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone when added alone do not alter the response of the membranes to the addition of Na+. Na+ uptake by brush-border microvilli is enhanced in the presence of a proton gradient directed from the intravesicular space to the incubation medium. Under these conditions a transient accumulation of Na+ inside the vesicles is observed. It is concluded that intestinal and renal brush-border membranes contain a NA+/H+ antiport system which catalyses an electroneutral exchange of Na+ against protons and consequently can produce a proton gradient in the presence of a concentration difference for Na+. This system might be involved in the active proton secretion of the small intestine and the proximal tubule of the kidney.     

Serotonin increases the cAMP concentration and the phosphoenolpyruvate carboxykinase mRNA in rat kidney, small intestine, and liver.

Within 60 min of the administration of serotonin to fasted-refed rats, there was a 5-, 16-, and 20-fold stimulation of the mRNA coding for the cytosolic form of P-enolpyruvate carboxykinase in the kidney, small intestine and liver, respectively. This stimulation was 5-, 1.3-, and 2-fold higher than noted in the same tissue after 24 h of starvation. Dose- and time-response curves to serotonin in the three tissues were similar. The level of PEPCK mRNA in the liver was significantly elevated within 30 min of serotonin administration, whereas 60 min was required in the small intestine and the kidney. The direct effect of serotonin on PEPCK mRNA was also assessed in hepatocytes maintained in primary culture. Serotonin (10(-8) M to 10(-4) M) caused a dose-dependent increase in the level of PEPCK mRNA and a transient increase in cAMP concentration. Within the first min of serotonin (10(-6) M) addition to cells, cAMP concentration increased 4-fold and returned after 10 min to basal level. Therefore, these results provide functional evidence of serotonin action in the rat peripheric tissues and suggest that cAMP is involved in its intracellular signalling.