Histidine decarboxylase of Lactobacillus 30a: inactivation and active-site labeling by L-histidine methyl ester

Histidine decarboxylase from Lactobacillus 30a is rapidly and irreversibly inactivated upon incubation with L-histidine methyl ester. The rate of inactivation is first-order with respect to remaining active enzyme and exhibits saturation kinetics with a kinact of 1.2 mM and an apparent first-order rate constant of 0.346 min-1 at pH 4.8 and 25 degrees C. On complete inactivation, 3 mol of [14C]histidine (from L-[14C]histidine methyl ester) and 2 mol of 14C (from L-histidine [14C]methyl ester) are bound in nondialyzable form per mol (190 000 g) of protein inactivated with a corresponding loss of three of the five DTNB-titratable--SH groups that are essential for activity of the native enzyme. Imidazole propionate, a competitive inhibitor of the enzyme, protects against inactivation, loss of --SH groups, and incorporation of radioactivity from both the histidine and the methyl ester moieties of the labeled inhibitor, and kinetic evidence indicates that imidazole propionate and histidine methyl ester compete for binding at the active site of histidine decarboxylase in a mutually exclusive manner. Treatment of the labeled protein with either alkali or hydroxylamine results in the quantitative release of radioactivity. These data suggest that inactivation of histidine decarboxylase by L-histidine methyl ester results from two different modes of interaction between the inhibitor and the active site of histidine decarboxylase; the major interaction involves an essential -SH group.     

Intracranial hypertension in the infant: from its physiopathology to its therapeutic management

Histidine decarboxylase catalyses the formation of histamine, an important biological messenger. In spite of the essential biological functions exerted by histamine the knowledge about the mechanisms involved in the regulation of histidine decarboxylase is rather limited. This is most likely due to the limited supply of suitable tools, including highly specific antibodies. In the present study we describe the production and characterisation of specific antisera against rat histidine decarboxylase using recombinant protein synthesised in a bacterial expression system. The antisera were shown to effectively immunoprecipitate histidine decarboxylase activity in extracts of fetal rat liver as well as to detect the histidine decarboxylase protein by Western blot analysis of COS-7 cells expressing recombinant rat histidine decarboxylase. The results demonstrate the successful production of highly specific antisera to histidine decarboxylase which may become valuable tools in future studies of the structure and function of this enzyme.     

Absorption, distribution, and excretion of [14C]patulin by rats

Adult rats of both sexes were given a single oral dose of [14C] patulin and were sacrificed at various time intervals from 4 hr to 7 days following administration of the mycotoxin. Two groups of rats were employed; the treated group had been exposed to daily oral doses of unlabeled patulin (dissolved in pH 5.0 citrate buffer) in utero and for 41-66 wk after weaning, while the controls were given the buffer only throughout gestation and for 38-81 wk after weaning. Approximately 49% of the administered 14C radioactivity was recovered from feces and 36% from urine within 7 days after dosing. Most of the excretion of labeled material occurred within the first 24 hr. All of the 14C activity detected in the urine samples was either metabolites and/or conjugates of the original [14C]patulin. About 1-2% of the total radioactivity was recovered as 14CO2 from expired air. Carbon-14 radioactivity in various tissues and organs was determined throughout the 7 day period; the most significant retention site was the red blood cells.     

Functional impairment of the individual rat stomach ECL cell in response to sustained hypergastrinemia

ECL cells in the oxyntic mucosa secrete histamine and pancreastatin in response to gastrin. The present study examined gastrin-evoked ECL-cell responses over a 10-week time span in terms of individual ECL cells and unit ECL cell volume. Rats were treated with omeprazole (400 micromol/kg per day orally). The concentrations of gastrin and pancreastatin in serum and of histamine and pancreastatin in the oxyntic mucosa were measured as was the activity of the oxyntic mucosal histidine decarboxylase (HDC). The ECL cells were visualized by immunostaining of histamine and examined by electron microscopy. The total ECL cell number and volume, and the mean ECL cell diameter and volume were determined. The HDC, chromogranin A (CGA) and cholecystokinin-B (CCK-B) receptor mRNA concentrations were determined. In terms of individual ECL cells and unit ECL cell volume, the serum pancreastatin concentration, the oxyntic mucosal histamine content, HDC activity, and HDC, CGA and CCK-B receptor mRNA contents increased slowly at first and then leveled off or started to decline after 2 weeks. After 10 weeks all ECL-cell parameters (expressed per unit ECL cell volume) were back to or approaching the starting value. In conclusion, sustained hypergastrinemia first activates each individual ECL cell (with a peak after 1-2 weeks) and then causes gradual functional impairment, the activity returning towards the pre-stimulation level.     

Evaluation of biologic gastrin activity of compound CI-988 in the isolated, vascularly perfused rat stomach

BACKGROUND: The peptoid CI-988 has previously been shown to have high affinity for the cholecystokinin (CCK)-B/gastrin receptor and has been reported to be a powerful CCK antagonist in many systems, although it has agonist activity on histidine decarboxylase in the rat. METHODS: In the present study the effect of CI-988 on acid secretion and histamine release in the totally isolated, vascularly perfused rat stomach was assessed. RESULTS: CI-988 was found to be a gastrin agonist with regard to the stimulation of both histamine release and acid secretion. CONCLUSION: Thus, in this stomach model CI-988 behaved as a CCKB/gastrin agonist. The present study underlines the importance of testing the biologic activity of ligands in models with sufficient sensitivity.     

Regulation of mevalonate 5-pyrophosphate decarboxylase in HeLa cells. Inhibition of enzymatic protein synthesis by serum lipoproteins

Mevalonate 5-pyrophosphate decarboxylase (EC 4.1.1.33) has been considered as a secondary site of regulation of cholesterogenesis. Because of this, we have studied the regulation of decarboxylase in HeLa cells by serum lipoproteins in the cell culture medium. A first group of experiments was performed with cells grown in Eagle's medium with 10% foetal calf serum. The specific activity of decarboxylase was increased when whole foetal calf serum was replaced with lipoprotein-poor serum. This increase was clearly reduced in the presence of cycloheximide. Addition of serum lipoproteins to a medium containing lipoprotein-poor serum led to a clear decrease in the decarboxylase activity. An identical decrease was observed after the addition of lipoproteins alone or in combination with cycloheximide. These results suggest for the first time that the effect of serum lipoproteins on decarboxylase activity should be a decrease in the rate of enzymatic protein synthesis, and corroborate the important role of reactions other than those catalysed by 3-hydroxy-3-methylglutaryl-CoA reductase in the regulation of cholesterogenesis.     

Increase in histamine synthesis by liver macrophages in CCl4-injured mast cell-deficient W/Wv mice

This study set out to examine the possible role of liver macrophages in histamine synthesis in the injured liver. The effects of the hepatotoxins Escherichia coli lipopolysaccharide (LPS) and CCl4 on histamine synthesis in the liver of mice were evaluated. C3H/HeJ mice were resistant to LPS in including histidine decarboxylase (HDC) in the liver compared with C3H/HeN mice and mast cell-deficient W/Wv mice. However, C3H/HeJ mice did respond strongly to another hepatotoxin, CCl4, leading to a significant increase in HDC activity. CCl4 also caused a marked increase in HDC activity and histamine levels in the liver of W/Wv mice. In addition, injection of CCl4 produced a large increase in the activity of HDC in the spleen and lung of W/Wv mice. HDC activity was confined to the nonparenchymal cells, with parenchymal cells expressing essentially no HDC activity. The CCl4-induced increase in HDC activity was confined, at least in part, to the liver macrophages. These results indicate that the macrophages are responsible for the increase in HDC-dependent histamine production in the liver caused by the injection of hepatotoxins. The possible role of histamine in liver regeneration after injury is discussed.     

Histamine synthesis by cells of the macrophage lineage in bone marrow of mice

An injection of Escherichia coli lipopolysaccharide (LPS) increased the activity of histidine decarboxylase (HDC) in bone marrow (BM) cells of C3H/HeN mice much more than in C3H/HeJ mice, which are resistant to various effects of LPS. In WBB6/F1 (W/Wv) mice, which are genetically deficient in mast cells, HDC activity increased more than in C3H/HeN mice. Cultured BM cells of W/Wv mice spontaneously synthesized histamine in a HDC-dependent way. LPS caused a slight increase in HDC-associated histamine synthesis by these cells. Treatment of the BM cells with murine recombinant granulocyte-macrophage colony stimulating factor (mrGM-CSF) increased the histamine synthesis. In addition, treatment with mrGM-CSF made the cells respond to LPS by a dose-dependent increase in HDC activity and histamine synthesis. Most dish-adherent BM cells that had been treated with both mrGM-CSF and LPS for 48 h were stained for nonspecific esterase and not for chloroacetate esterase, and had twice as much HDC activity as the nonadherent cells had. Immunocytochemical analysis of the BM cells of W/Wv mice treated with both mrGM-CSF and LPS showed that HDC was in the cytoplasm of cells having Mac-1, a macrophage-differentiation antigen. These results suggest that cells of the macrophage lineage in the BM of mice synthesize histamine.     

Influenza virus hemagglutinin: hydrodynamic properties and molecular weight

Following acute alcohol administration (80-160 mg/100 g body weight) histamine levels of rat brain cortex and thalamus were elevated and histidine decarboxylase activity was decreased. The effect was less pronounced after chronic alcohol treatment (15% v/v in drinking water for 4 weeks). In the striatum there was no change in the metabolic pattern of histamine. Histamine-N-methyltransferase was unaffected in either case. Depolarisation-induced release of histamine was inhibited by alcohol in the hypothalamus, thalamus and cortex. The results indicate that ethanol affects the histamine metabolism and release processes in the histaminergic pathway of the brain.     

Adenosine deaminase activity in human erythrocytes and lymphocytes

A radiochromatographic method is described for measuring enzymatic activity of adenosine deaminase in human erythrocytes and lymphocytes. [8-14C]-adenosine is converted into inosine and hypoxanthine; after chromatographic separation of the products, the radioactivity is determined. The kinetic properties of the enzyme have been studied. The Km values for the erythrocyte and lymphocyte enzymes are higher as compared with purified deaminase. Optimum conditions for substrate concentration for assay were established. The mean normal activity (+/- S.E. of mean) is: for erythrocytes, 494 +/- 61; nmol min-1 ml-1; for lymphocytes- 147 +/- 0.18 nmol min-1 10(6) cellules. The mean values are higher than that given by other methods working at a lower (non-staurating) substrate concentration.     

Histamine synthesis and content in benign and malignant breast tumours. Its effects on other host tissues

We studied 100 patients: 40 with breast cancer, 41 with benign breast tumours and 19 non-cancer-bearing cholecystectomy patients, in order to measure the histidine decarboxylase activity and histamine content in benign and malignant breast tumours, and to determine whether the histamine metabolism affected skin and muscle tissue distant from the tumour. The HDC-activity of cancerous tissue was significantly higher (P < 0.01) than that registered in the healthy mammary gland tissue of the same patient, being even more pronounced in benign tumour tissue (P < 0.001). However, the histamine content was found to be significantly lower (P < 0.01) in malignant tumours, but higher in benign tumoural tissue as compared with the healthy tissue of the same patient. We also found that the histamine content in muscle tissue was significantly higher in cancer patients than in non-cancer patients. These findings highlight the fact that intracellular histamine metabolism varies in benign and malignant tumours, and that high histamine synthesis of malignant tumours affects other host tissue.     

Noninvasive detection of intracranial vascular lesions by recording blood flow sounds

The actions of S-alpha-fluoromethylhistidine (FMH), an irreversible inhibitor of the histamine biosynthetic enzyme histidine decarboxylase (HD), were studied on rat brain HD, as measured by a recently developed CO2-trapping enzymatic method. As expected, FMH induced a virtually complete inhibition of HD in the hypothalamus both in vivo and in vitro. In the frontal cortex, however, maximal doses of FMH did not maximally inhibit HD, suggesting the existence of an FMH-resistant form of HD. Careful studies of the conditions under which the assays were performed (homogenate dilution, preincubation times, incubation times, temperatures), as well as experiments with inhibitors of other decarboxylases, were unable to provide an explanation for this. When comparable studies of the effects of FMH in these brain regions were performed by alternative methods for measuring HD activity, no evidence for the existence of an FMH-resistant form of HD could be found. Thus, even though the CO2-trapping method appears to be accurate for measuring HD activity in rat hypothalamic homogenates, the present results show that this method may not be specific when studying brain regions other than the hypothalamus.     

Contrasting effects of lipopolysaccharides (endotoxins) from oral black-pigmented bacteria and Enterobacteriaceae on platelets, a major source of serotonin, and on histamine-forming enzyme in mice

By measurement of serotonin levels, the translocation of platelets to various tissues was examined following intravenous injection of a lipopolysaccharide (LPS) into C3H/HeN mice. There was a rapid platelet accumulation (within 5 min and particularly in the lung), followed by a slower accumulation in the liver, which reached its plateau 3-5 h later. The severity of the anaphylactoid shock corresponded well with the magnitude of the rapid response. LPSs from the oral black-pigmented bacteria, Porphyromonas gingivalis and Prevotella intermedia, were much more potent in inducing the rapid platelet response than were those from the Enterobacteriaceae Escherichia coli and Salmonella typhimurium. However, LPSs from these Enterobacteriaceae were significantly more potent than those from black-pigmented bacteria in inducing the slow platelet response. There was also a contrast between their abilities to induce histidine decarboxylase, which forms histamine from histidine: LPSs from the Enterobacteriaceae were much more potent than those from black-pigmented bacteria.     

Deletional and mutational analyses of the human CD4 gene promoter: characterization of a minimal tissue-specific promoter

Chloroanilines (CA) are widely used chemical intermediates which induce numerous toxicities including hematotoxicity, splenotoxicity, hepatotoxicity and nephrotoxicity. Although chloroaniline-induced hematotoxicity has been studied in detail, little information is available on the organ-directed toxicity seen following exposure to these agents. The purpose of this study was to examine and compare the excretion and distribution of two nephrotoxicant and hepatotoxicant chloroanilines (2- and 4-chloroaniline) to liver, kidney, spleen, plasma and erythrocytes. Subcellular distribution and covalent binding in kidney and liver were also determined. Male Fischer 344 rats (four per group) were administered [14C]-2-chloroaniline or [14C]-4-chloroaniline (0.5 or 1.0 mmol/kg; approximately 50 microCi/rat) intraperitoneally (i.p.). Urine, feces, blood and tissues were collected at 3 and 24 h. Both 2- and 4-chloroaniline-derived radioactivity were primarily renally excreted with < 1% excretion in the feces by 24 h post-treatment. Both chloroanilines accumulated mainly in liver (percentage of administered dose/total tissue), but kidney generally had similar or higher equivalent concentrations (micromol/g tissue) compared to liver. Subcellular distribution revealed that for both chloroanilines, the cytosolic fraction generally had the highest level of radioactivity independent of time or dose. Covalent binding was detected in both liver and kidney, with the highest concentration (pmol/mg protein) of binding observed in the hepatic microsomal fraction regardless of compound, dose or time studied. In general, 2-chloroaniline derived radioactivity was excreted faster, reached peak tissue concentrations earlier, disappeared from tissues faster and had less covalent binding in target tissue at 24 h than 4-chloroaniline-derived radioactivity. These results suggest that the increased toxic potential of 4-chloroaniline as compared to 2-chloroaniline may be due in part to a more prolonged and persistent accumulation of 4-chloroaniline and/or its metabolites in target tissue.     

Generation and subcellular distribution of histamine in human blood monocytes and monocyte subsets

OBJECTIVE AND DESIGN: This study was designed to establish the sites of formation and storage of histamine and histidine decarboxylase (HDC) in human monocytes and two of their subsets. MATERIALS AND METHODS: The experiments were carried out using monocytes from buffy coats of healthy blood donors. Histamine was quantitated by RIA, HDC activity by the formation of histamine. RESULTS: The monocyte subtype RM3/1 contained significantly more histamine than the subset 27E10 (0.041+/-0.025 vs. 0.005+/-0.004 pg/cell, p < 0.05) and also more HDC activity and HDC mRNA. After fractionation of monocyte homogenates in a discontinuous Percoll gradient or by differential centrifugation more than 80% of both, HDC activity and histamine, were recovered from the cytosolic fractions. About 50% of this histamine was found to be bound to proteins. CONCLUSIONS: In monocytes histamine and HDC are colocalized in the cytoplasm indicating a subcellular distribution different from mast cells or basophils. The data also show that histamine is synthesized by the monocytes themselves.     

In vivo and in vitro biotransformation of the lithium salt of gamma-linolenic acid by three human carcinomas

Lipid metabolism has been considered recently as a novel target for cancer therapy. In this field, lithium gamma-linolenate (LiGLA) is a promising experimental compound for use in the treatment of human tumours. In vivo and in vitro studies allowed us to assess the metabolism of radiolabelled LiGLA by tumour tissue and different organs of the host. In vitro studies demonstrated that human pancreatic (AsPC-1), prostatic (PC-3) and mammary carcinoma (ZR-75-1) cells were capable of elongating GLA from LiGLA to dihomo-gamma-linolenic acid (DGLA) and further desaturating it to arachidonic acid (AA). AsPC-1 cells showed the lowest delta5-desaturase activity on DGLA. In the in vivo studies, nude mice bearing the human carcinomas were given Li[1-(14)C]GLA (2.5 mg kg(-1)) by intravenous injection for 30 min. Mice were either sacrificed after infusion or left for up to 96 h recovery before sacrifice. In general, the organs showed a maximum uptake of radioactivity 30 min after the infusion started (t = 0). Thereafter, in major organs the percentage of injected radioactivity per g of tissue declined below 1% 96 h after infusion. In kidney, brain, testes/ovaries and all three tumour tissues, labelling remained constant throughout the experiment. The ratio of radioactivity in liver to tumour tissues ranged between 16- and 24-fold at t = 0 and between 3.1- and 3.7-fold at 96 h. All tissues showed a progressive increase in the proportion of radioactivity associated with AA with a concomitant decrease in radiolabelled GLA as the time after infusion increased. DGLA declined rapidly in liver and plasma, but at a much slower rate in brain and malignant tissue. Seventy-two hours after the infusion, GLA was only detected in plasma and tumour tissue. The sum of GLA + DGLA varied among tumour tissues, but it remained 2-4 times higher than in liver and plasma. In brain, DGLA is the major contributor to the sum of these fatty acids. Data showed that cytotoxic GLA and DGLA, the latter provided either by the host or by endogenous synthesis, remained in human tumours for at least 4 days.     

Plasma insulin and growth hormone concentrations in pregnant sheep II: post-absorptive levels in mid- and late pregnancy

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Gastrin-producing endocrine cells: a novel source of histamine in the rat stomach

Gastrin and histamine both potently stimulate secretion of acid into the gastric lumen. How these agents interact and how their release is controlled is poorly understood. Therefore, we decided to look for histamine in the antral portion of the rat stomach where the gastrin-producing G cells are located. We used immunocytochemical methods to visualize histamine, histidine decarboxylase (HDC, the enzyme that converts histidine to histamine), and the type 1 vesicular monoamine transporter (VMAT1, the protein responsible for moving histamine into vesicles for storage and release). We were surprised to find that histamine, HDC, and VMAT1 were all present in G cells. Our results suggest that G cells synthesize and secrete gastrin and histamine. Whether histamine acts in concert with gastrin to stimulate acid secretion, or functions as an autocrine inhibitor of gastrin release remains to be seen.     

A comparison of mass balance, pharmacokinetics and disposition of [14C(U)]- and [125I]recombinant human interleukin-2 in cynomolgus monkeys

Recombinant human interleukin-2 (rHuIL-2) has been metabolically labeled with 14C amino acids in Escherichia coli and affinity purified on a rHuIL-2 receptor affinity column. The radiolabeled molecule had a specific radioactivity of 238 dpm/unit and the identical amino acid sequence and biological activity as unlabeled rHuIL-2. In this study, we used this labeled [14C(U)]rHuIL-2 and commercially available [125I]rHuIL-2 (identical in sequence to the [14C(U)]rHuIL-2) to compare the mass balance, pharmacokinetics, and disposition in cynomolgus monkeys. After a single intravenous bolus dose of 4 x 10(5) units/kg, serum samples were collected for 7 days and examined for biological activity, total radioactivity, and by molecular size exclusion chromatography. Urine and feces were analyzed for total radioactivity. When analyzed for biological activity, both [14C(U)]- and [125I]rHuIL-2 exhibited the following pharmacokinetic parameters: terminal elimination half-life of 1-2 hr, AUC0-infinity ranged from 2005 to 4659 units x hr/ml, clearance was 90-200 ml/hr/kg, and volume of distribution ranged from 103 to 163 ml/kg. Comparison of the pharmacokinetic profiles of the two radiolabels were very different from bioactivity, in that the elimination half-lives for radioactivity were approximately 8 days and 10 hr for [14C(U)]- and [125I]rHuIL-2, respectively. We conclude that the [14C(U)]rHuIL-2 was metabolized to constituent amino acids and recycled into newly synthesized proteins from our size exclusion chromatography studies.(ABSTRACT TRUNCATED AT 250 WORDS)