Significance of gamma-glutamyltranspeptidase in exocrine pancreatic amino acid transport.

The exocrine pancreas is rich in gamma-glutamyltranspeptidase (GGT, EC 2.3.2.2) and exhibits high rates of amino acid transport and protein synthesis. The role of the gamma-glutamyl cycle in mediating neutral amino acid transport in the isolated perfused rat pancreas was investigated using acivicin, an inhibitor of GGT, and a rapid dual isotope dilution technique. When treatment in vivo with acivicin (50 mg/kg) was followed 1 h later by continuous perfusion of the isolated pancreas with 10 microM acivicin, GGT levels decreased from 53 +/- 3 IU/g to 4.9 +/- 1.5 IU/g. This marked inhibition of GGT activity was not associated with decreased uptake for either L-alanine or L-glutamine, suggesting that the gamma-glutamyl cycle plays a negligible role in amino acid transport across the basolateral membrane of the pancreatic epithelium.     

Hepatic and intestinal gamma-glutamyltranspeptidase activity: its activation by chronic ethanol administration.

To study the effect of chronic ethanol administration on the activity of gamma-glutamyltranspeptidase (GGTP) in various tissues, female rats were pair-fed liquid diets with 36% of total calories either as ethanol or isocaloric carbohydrate (controls). Six weeks of ethanol feeding in an increase of cytochrome P450 content by 70%. Hepatic microsomal GGTP activity was more than doubled after ethanol feeding whether expressed per gram of liver or per mg of microsomal protein. Furthermore intestinal GGTP activity was significantly enhanced after ethanol, whereas there was no change in the enzyme activity in either kidney or pancreas. Phenobarbital administration to rats also resulted in an enahancement of GGTP activity in the liver but not in the intestine. These results suggest that enhanced hepatic and intestinal GGTP activities may contribute, at least partly, to increased serum GGTP activity frequently seen in alcoholics.     

Determination of rates of protein synthesis, gain and degradation in intact hind-limb muscle of lambs.

A model of leucine metabolism in the hind-limb muscles of the milk-fed lamb was developed which permitted simultaneous estimation of the rates of protein synthesis (Ks, days-1), degradation (Kd) and therefore gain (Kg) of muscle in vivo. The conclusions drawn from the model were: the rate of protein synthesis in muscle was related to uptake of leucine; the rate of degradation of protein was related to leucine output, as leucine, or its corresponding oxo acid, 4-methyl-2-oxopentanoic acid, or CO2. These findings support findings drawn from a wide range of studies in vitro. There was no correlation between rate of protein synthesis and rate of protein degradation, which suggests that the method can allow independent estimates of each. Estimates of protein synthesis obtained from the model (of leucine metabolism in muscle) were compared with those obtained simultaneously by constant infusion of radioisotope and analysis of incorporation into tissue. There were no significant differences between the mean values obtained for synthesis (Ks), gain (Kg) and degradation (Kd) by either method (Ks 0.051 +/- 0.002, 0.046 +/- 0.007; Kg 0.016 +/- 0.002, 0.004 +/- 0.008; Kd 0.035 +/- 0.004, 0.041 +/- 0.008 day-1, respectively, for tissue analysis and the model). However, Ks obtained from the model was significantly and positively correlated with uptake of leucine from plasma, whereas Ks obtained from tissue analysis was not.     

Protein turnover and proliferation. Turnover kinetics associated with the elevation of 3T3-cell acid-proteinase activity and cessation of net protein gain.

1. At least 95% of the total protein of A31-3T3 cell cultures undergoes turnover. 2. First-order exponential kinetics were used to provide a crude approximation of averaged protein synthesis, Ks, degradation, Kd, and net accumulation, Ka, as cells ceased growth at near-confluent density in unchanged Dulbecco's medium containing 10% serum. The values of the relationship Ka = Ks - Kd were : 5%/h = 6%/h - 1%/h in growing cells, and 0%/h = 3%/h - 3%/h in steady-state resting cells. 3. As determined by comparison of the progress of protein synthesis and net protein accumulation, the time course of increase in protein degradation coincided with the onset of an increase in lysosomal proteinase activity and decrease in thymidine incorporation after approx. 2 days of exponential growth. 4. After acute serum deprivation, rapid increases in protein degradation of less than 1%/h could be superimposed on the prevailing degradation rate in either growing or resting cells. The results indicate that two proteolytic mechanisms can be distinguished on the basis of the kinetics of their alterations. A slow mechanism changes in relation to proliferative status and lysosomal enzyme elevation. A prompt mechanism, previously described by others, changes before changes in cell-cycle distribution or lysosomal proteinase activity. 5. When the serum concentration of growing cultures was decreased to 1% or 0.25%, then cessation of growth was accompanied by a lower steady-state protein turnover rate of 2.0%/h or 1.5%/h respectively. When growth ceased under conditions of overcrowded cultures, or severe nutrient insufficiency, protein turnover did not attain a final steady state, but declined continually into the death of the culture.     

Viral RNA kinetics is associated with changes in trace elements in target organs of Coxsackie virus B3 infection

Trace elements are pivotal for the host defense, as well as potentially important for viral replication and virulence. Studies of sequential changes in viral replication in target organs of infection are sparse and a possible association with changes in specific trace elements is unknown. In this study Balb/c mice were infected with Coxsackie virus B3 (CVB3). Results indicated that sequential changes in viral replication (RT-PCR) were related to changes in trace element (arsenic, copper, iron, selenium and zinc) concentrations (as determined by ICP-MS) on days 3, 5 and 7 of the infection in serum, heart, lung, liver, pancreas, kidney, spleen, intestine and brain. After an initial viral peak on day 3, viral load drastically decreased in all organs, i.e. by >99% (serum), 97% (lung), 98% (liver), 60% (pancreas), 95% (kidney) and 93% (spleen), except in the heart, intestine and brain in which viral load increased after day 3. Selenium decreased in all organs except the heart while arsenic decreased in all organs except the kidney, spleen and brain. Moreover, selenium was negatively correlated to viral load in serum, liver, pancreas and intestine. To conclude, these findings give evidence that trace elements are directly involved in the replication of CVB3.     

Effects of vitamin E on serum enzymes and electrolytes in hypercholesterolemia

It is not known if vitamin E in hyperlipidemia and hypercholesterolemia of longer duration has any beneficial or adverse effects on electrolytes, and liver and kidney function. The objectives of this study are to determine (i) if long duration of mild hypercholesterolemia has any adverse effects on serum electrolytes, glucose and enzymes related to liver and kidney functions; (ii) if vitamin E has any effects on serum electrolytes, glucose and enzymes related to liver and kidney function in hypercholesterolemia. Blood samples were collected from the rabbits before and at various intervals during administration of a high cholesterol diet (0.25%) for 2 and 4 months, and while on a high cholesterol diet with vitamin E following a high cholesterol diet. Measurements of serum total cholesterol (TC), glucose, aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), albumin, creatinine, electrolytes [sodium (Na), potassium (K), chloride (Cl), and carbon dioxide (CO₂)] were made. High cholesterol diet for 2 months produced hypercholesterolemia which was associated with reductions in serum glucose, unaltered serum electrolytes, ALT, ALP, GGT, albumin and creatinine, and increased levels of AST. Hypercholesterolemia for 4 months had effects similar to hypercholesterolemia for 2 months except it lowered serum ALP. Vitamin E did not affect any of the parameters except serum glucose and Cl, which decreased compared to the values at month 2. Hypercholesterolemia for short and long term does not have adverse effects on liver or kidney function, and serum electrolytes. Vitamin E during hypercholesterolemia does not affect serum electrolytes or liver and kidney function.     

Acylase distribution in animal and human tissues

Distribution of acylase in different tissues of nine species of animals was studied. The following types of nitrogen elimination were distinguished: ammoniatelic (fish), uricotelic (birds) and uriotelic (amphibians, mammalians). The enzymic activity was estimated in the tissues of the brain, lung, muscle, liver, kidney, spleen, pancreas, small intestine and blood serum. The acylase activity was found in the kidney, liver and pancreas. Its level in the kidney increases with the animal weight growth, the enzyme activity being observed only in the cortical layer.     

Use of acivicin in the determination of rate constants for turnover of rat renal gamma-glutamyltranspeptidase

The specific enzymatic activity of renal gamma-glutamyltranspeptidase is decreased from control levels (0.86 unit-1 mg-1) to minimal values within 2 h postinjection of 100-g rats with acivicin, an irreversible inhibitor of the enzyme. The recovery of transpeptidase specific activity was followed from 2 to 24 h postinjection and the data were used to calculate the absolute rate constants for degradation (kd = 0.47 +/- 0.03 day-1) and synthesis (ks = 0.41 +/- 0.04 unit-1 mg-1 day-1). This corresponds to a half-life for the renal transpeptidase of 1.46 +/- 0.09 days and 99% recovery of the specific activity by 10 days postinjection. Recovery was followed for 14 days and closely approximates this theoretical curve. The data from control experiments designed to test for secondary effects of the drug, acivicin, show that neither the relative rate of synthesis nor apparent rate of degradation for either total protein or gamma-glutamyltranspeptidase is significantly altered by acivicin treatment of rats. The results also show that the acivicin-inhibited transpeptidase is not degraded differently than enzymatically active enzyme. The individual heterodimer subunits also exhibit similar apparent half-lives in both control and treated animals. Thus, recovery of renal gamma-glutamyltranspeptidase specific activity after acivicin treatment can be used in vivo to determine absolute values of ks and kd for this enzyme. These values have not been reported for any other constituent of the renal brush-border membrane.     

Lipopolysaccharides induce p8 mRNA expression in vivo and in vitro.

Systemic LPS endotoxin is associated with acute pancreatic damage. Whether damage results from direct interaction of LPS with pancreatic cells is unknown. We addressed that question by monitoring p8 expression in reponse to LPS, in vivo and in vitro, because overexpression of the p8 protein is a sensitive marker of pancreatic agression. For in vivo studies, rats were sacrificed at different times after a single intraperitoneal injection of LPS, and pancreas, liver, kidney, lung, brain, and intestine were processed for RNA preparation. In vitro, pancreatic acinar AR4-2J cells were cultivated with 0.1, 1, or 10 micrograms/ml LPS for 6, 12, or 24 h. p8 mRNA expression was monitored by Northern blotting. In vivo, it was strongly increased in the pancreas after 12 h of treatment and remained elevated after 24 h. It was also induced in kidney and liver, with a maximum at 6 and 12 h, respectively, but not in lung, brain, or intestine. In AR4-2J cells, basal p8 mRNA expression was very low and increased in a time- and dose-dependent manner after treatment with LPS. LPS-induced overexpression of p8 mRNA in vivo confirmed the adverse effect of endotoxemia on pancreas and its overexpression in vitro demonstrated a direct interaction of LPS with pancreatic cells.     

Effect of zinc supplementation on metallothionein,copper, and zinc concentration in various tissues of copper-loaded rats

The present study was designed to investigate the effects of Zn administration on metallothionein concentrations in the liver, kidney, and intestine of copper-loaded rats. Male CD rats were fed a diet containing 12 mg Cu and 67 mg Zn/kg body wt. They were divided into either acute or chronic experimental protocols. Rats undergoing acute experiments received daily ip injections of either Cu (3 mg/kg body wt) or Zn (10 mg/kg body wt) for 3 d. Chronic experiments were carried out on rats receiving Cu ip injections on d 1, 2, 3, 10, 17, and 24, Cu injections plus a Zn-supplemented diet containing 5 g Zn/kg solid diet, or a Zn-supplemented diet alone. Rats injected Zn or Cu had increased MT concentrations in liver and kidney. Zn produced the most important effects and the liver was the most responsive organ. Rats fed a Zn-supplemented diet had significantly higher MT concentrations in liver and intestine with respect to controls. Increased MT synthesis in the liver may contribute to copper detoxification; the hypothesis of copper entrapment in enterocytes cannot be confirmed.     

The effect of alcohols on daily variations in aspartate amino- transferase activity in rat organs

The 7 day-long intragastric administration of ethanol and ethyleneglycol in a dose of 1/3 DL50 was studied for its effect on the circadian variations of the aspartate aminotransferase activity (AST, EC 2.6, 1.1) in the liver, brain, myocardium and kidney of male rats. The ethanol and ethylene glycol administration reduced the mean circadian enzymic activity in the above organs. Moreover, ethanol significantly reduced the amplitude of circadian variations of the AST activity in the liver, brain and kidney, while ethylene glycol--in the liver, myocardium and kidney.     

Gamma-glutamyl transferase distribution in the organs of Papio papio and Macaca fascicularis

Tissue distribution of non solubilized gamma-glutamyl transferase (GGT) was studied in eight baboons and ten macaques. In both, GGT is mainly in kidney, pancreas and liver. This tissue distribution of GGT is roughly similar to that observed in man.     

Prostaglandin synthesis and metabolism in the human uterus and midtrimester fetal tissues

Prostaglandin (PG) synthesis and metabolism was studied in human fetal kidney, lung, small intestine, heart, brain and liver (gestational ages: 10, 12, 14, 18 and 23 weeks) and pregnant uterus (4-40 weeks of pregnancy). PG synthesis was increased in the myometrium during pregnancy while the capacity of metabolism did not change. PG synthesis increased in lung and kidney (4-fold), brain (20-fold) and small intestine (2-fold) but not in heart or liver. Metabolic activity increased only in fetal kidney and lung.     

Inhibition of the hydrolytic and transpeptidase activities of rat kidney gamma-glutamyl transpeptidase by specific monoclonal antibodies.

Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.     

Biodistribution and pharmacokinetics of vanadium following intraperitoneal administration of vanadocene dichloride to mice

The biodistribution and pharmacokinetics of vanadium following i.p. administration of vanadocene dichloride (VDC), a representative of a new class of organometallic anticancer agents, is reported for Strain A mice. A convenient flameless atomic absorption spectroscopic assay is described and is used to determine kinetic profiles for vanadium in blood, kidney, liver, small intestine and brain tissue for times up to 24 h after administration. For a VDC dose of 80 mg/kg, vanadium concentration decreases rapidly from both the blood and small intestine, and the data can be fit to a phenomenological exponential function (blood: t1/2 = 118 +/- 43 min; small intestine: t1/2(alpha) = 18.10 +/- 0.14 min, t1/2(beta) = 341 +/- 45 min). In contrast, vanadium accumulates in both the kidney and liver up to a maximal concentration (1.12 +/- 0.06 mM and 0.56 +/- 0.06 mM after 12 and 8 h, respectively), and is then excreted with estimated half-lives of 7.9 +/- 0.7 and 12.1 +/- 0.1 h, respectively. No detectable levels of vanadium are found in the brain tissue over the temporal course of the experiment. These results are compared to previous mammalian studies with cis-dichlorodiammineplatinum(II) (CDDP) and related 'second generation' platinum derivatives; there are both qualitative similarities between the vanadium and platinum systems as well as important quantitative differences.     

Effect of exogenous cortisone on amino acid metabolism in rats

1. The effect of exogenous cortisone on concentration of free amino acids in serum, skeletal muscle, kidney, small intestine and liver was studied. 2. The amino acid pool in serum, skeletal muscle and small intestine decreased significantly. 3. Glutamine synthesis increased significantly in skeletal muscle. 4. Levels of branched amino acids increased in serum and small intestine. 5. Levels of alanine increased in kidney and liver.     

Glucocorticoids and the regulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in the rat.

The effect glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP)(EC4.1.1.32) in rat liver and kidney in vivo was studied immunochemically. The glucocorticoid analogue triamcinolone (9alpha-fluoro-11beta, 21-dihydroxy-16alpha,17alpha-isopropylidenedioxypregna-1,4-diene-3,20-dione) increased the synthesis rate of the kidney enzyme in starved animals. Both triamcinolone and cortisol decreased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP) in fed and starved rats, but were without effect on the degradation rate of the enzyme. This effect of triamcinolone in liver was reversed by injection of dibutyryl cyclic AMP. However, in diabetic animals glucocorticoids increased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP). Triamcinolone administration to starved rats in vivo is shown to cause an increase in the portal blood concentrations of insulin and glucose. Since the physiological de-inducer of liver phosphoenolpyruvate carboxykinase (GTP) is insulin, this is the probable cause of the decrease in the synthesis rate of the hepatic enzyme noted when glucocorticoids are administered to non-diabetic animals.     

Distribution of apolipoprotein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction of apolipoprotein E mRNA by cultures of human monocyte-macrophages

Recently developed molecular probes for human apolipoprotein (apo) genes have been used to study the specificity of human tissue expression of the apo A-I, apo C-II, apo C-III, and apo E genes. We have found that apo E mRNA was present in all tissues examined. On the basis of total RNA concentration the relative abundance of apo E mRNA expressed as a percentage of the liver value is as follows: adrenal gland and macrophages, 74-100%; gonads and kidney, 12-15%; spleen, brain, thymus, ovaries, intestine, and pancreas, 3-9%; heart, 1.5%; stomach, striated muscle, and lung, less than 1%. The relative concentration of apo E mRNA in cultures of human peripheral blood monocyte-macrophages increases dramatically as a function of time in culture, and after 5 days, it compares to that of liver. The human tissues shown to synthesize apo E mRNA were also examined for their ability to synthesize apo A-I, apo C-II, and apo C-III mRNA. The relative abundance of apo A-I, apo C-III, and apo C-II mRNA expressed as a percentage of the liver value is as follows: apo A-I, intestine, 50%; apo A-I, pancreas and gonads, 12%; apo A-I, kidney, 4%; apo A-I, adrenal, 2.5%; apo A-I, ovaries and heart, 1%; apo A-I, stomach and thymus, less than 1%; apo C-III, intestine, 62%; apo C-III, pancreas, 7%; apo C-II, intestine, 3%; apo C-II, pancreas, less than 1%. The knowledge of tissue specificities in the synthesis of apolipoproteins is important for our understanding of the regulation of apolipoproteins and lipoprotein metabolism.