Analysis of nitrate,nitrite, and [15N]nitrate in biological fluids

A new automated system for the analysis of nitrate via reduction with a high-pressure cadmium column is described. Samples of urine, saliva, deproteinized plasma, gastric juice, and milk can be analyzed for nitrate, nitrite, or both with a lower limit of detection of 1.0 nmol NO₃^? or NO₂^?/ml. The system allows quantitative reduction of nitrate and automatically eliminates interference from other compounds normally present in urine and other biological fluids. Analysis rate is 30 samples per hour, with preparation for most samples limited to simple dilution with distilled water. The application of gas chromatography/mass spectrometry for the analysis of ¹⁵NO₃^? in urine after derivatization to ¹⁵NO₂-benzene is also described.     

Presence of prostaglandins E2 and A2 in canine gastric secretions

The presence of prostaglandins (PGs) was determined in gastric juice obtained from 3 conscious dogs, provided with a chronic gastric fistula. Outputs of acid (mequiv min^?1) and PGs (pg min^?1) were measured in gastric secretions stimulated by pentagastrin (100 or 200 ng kg^?1min^?1). Prostaglandin activity was estimated, after extraction and thin layer chromatography, by radioimmuno-assay of the PGB formed by treatment with alkali. Tritiated PGs were added to gastric juice for the purpose of correcting for PGs recovery. Using this method, the minimum mass of PGB which could be satisfactorily distinguished from zero was 25 pg. Prostaglandins A₂ and E₂ were present in pentagastrin-activated gastric secretions and averaged (mean ± SE, n = 8) 200.7 ± 18.1 and 260.1 ± 18.0 pg min^?1 respectively. The identity of PGA₂ and PGE₂ was confirmed by gas liquid chromatography combined with mass spectrometry. The amount of PGE₂ converted to PGA₂ during extraction, separation and conversion procedures was estimated from the amount of [³H] PGA₂ found when only [³H] PGE₂ had been added to a sample of gastric juice and averaged 14.5% ± 2.0. Our preliminary results support the possibility that PGE₂ and PGA₂ may be of physiological importance in the regulation of canine gastric secretions.     

ICI 125,211: a new gastric antisecretory agent acting on histamine H2-receptors.

$\text{In}$$\text{vitro}$, ICI 125,211 competitively antagonized the action of dimaprit on guinea pig atrium with an apparent dissociation constant of 1.5 × 10^?8M (pA₂ = 7.8). $\text{In}$$\text{vivo}$, the histamine dose-response curve in conscious gastric fistula beagles was shifted rightward in parallel without change in the maximal response by intravenous infusions of ICI 125,211 at doses of 0.01 and 0.03 umol/kg/hr (estimated pA₂ = 7.3). Our data show that this new drug is at least 10x more potent than cimetidine as an inhibitor of gastric secretion in the dog. ICI 125,211, which is an orally effective antisecretory agent in man and devoid of antiandrogenic activity, is the most potent selective H₂-blocker described to date.     

Effect of caffeine on mucus secretion and agonist-dependent Ca2+ mobilization in human gastric mucus secreting cells

Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca²⁺]_i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca²⁺]_i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [³H]glucosamine. Caffeine by itself failed to increase [Ca²⁺]_i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca²+]_i rise, resulting in inhibiting activation of Ca²⁺-dependent K⁺ current (I_K.Ca) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca²⁺]_i rise and activation of I_K.Ca induced by A23187 or inositol trisphosphate (IP₃). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca²⁺]_i rise in human gastric epithelial cells, probably through the blockade of receptor-IP₃ signaling pathway, which may affect the mucin secretion. © 1997 Elsevier Science B.V. All rights reserved.     

Specific modification of gastric K+-stimulated ATPase activity by thimerosal

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K⁺-ATPase and the K⁺-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K⁺-stimulated ATPase, while K⁺-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K⁺-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K⁺-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K⁺-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; K_m for p-nitrophenylphosphate unchanged for K_a for K⁺ increased. ATP, which is a potent inhibitor of K⁺-p-nitrophenylphosphatase activity in native membranes (K_I ≈ 200 μM). These data suggest that there are multiple SH groups which differentially influence the gastric K⁺-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K⁺-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H⁺-transport reaction.     

A simple and sensitive assay for dopamine-beta-hydroxylase

A simple, rapid, and highly sensitive radiochemical assay for measuring the activity of dopamine-β-hydroxylase in tissues and serum is described. Enzyme activity is detected by converting [1-¹⁴C]tyramine to [1-¹⁴C]octopamine which is then subjected to periodate cleavage to form [¹⁴C]form-amide. This radiolabeled product is oxidized to ¹⁴CO₂ by addition of permanganate and the ¹⁴CO₂ is trapped and counted. The assay is simple and sensitive, it can linearly detect enzyme in all tissues with a wide range of activity, it uses maximal concentration of substrate, and it requires the addition of only one concentration of EMI to block endogenous inhibitor(s) in different tissues or enzyme concentrations.     

Effect of menthol in experimentally induced ulcers: Pathways of gastroprotection

Based on ethnopharmacological indications that Mentha species may be used in the treatment of gastrointestinal diseases, this study aimed to characterize the gastroprotective mechanisms of menthol (ME), the major compound of the essential oil from species of the genus Mentha. The gastroprotective action of ME was analyzed in gastric ulcers that were induced by ethanol or indomethacin in Wistar male rats. The mechanisms responsible for the gastroprotective effect were assessed by analyzing the amount of mucus secreted, involvement of non-protein sulfhydryl (NP-SH) compounds, involvement of calcium ion channels and NO/cGMP/K⁺_ATP pathway, gastric antisecretory activity and the prostaglandin E₂ (PGE₂) production. The anti-diarrheal activity and acute toxicity of ME were also evaluated. Oral treatment with ME (50 mg/kg) offered 88.62% and 72.62% of gastroprotection against ethanol and indomethacin, respectively. There was an increased amount of mucus and PGE₂ production. The gastroprotective activity of ME involved NP-SH compounds and the stimulation of K⁺_ATP channels, but not the activation of calcium ion channels or the production of NO. The oral administration of ME induced an antisecretory effect as it decreased the H⁺ concentration in gastric juice. ME displayed anti-diarrheal and antiperistaltic activity. There were no signs of toxicity in the biochemical analyses performed in the rats’ serum. These results demonstrated that ME provides gastroprotective and anti-diarrheal activities with no toxicity in rats.     

K+-stimulated phosphatase of microsomes from gastric mucosa

The light microsomal fraction was isolated from homogenates of rabbit and bullfrog gastric mucosa. On examination with the electron microscope, the light microsomes appear as tubular membranous structures with morphology and dimensions similar to the elements of the smooth-surfaced endoplasmic reticulum seen in intact oxyntic cells. A K⁺-stimulated, Mg⁺⁺-requiring p-nitrophenylphosphatase has been demonstrated in the gastric microsomes. Neither Na⁺ nor ouabain (10^?6–10^?3 M) altered the K⁺-stimulated phosphatase. SCN^? was not very effective as an inhibitor of the gastric microsomal phosphatase, in contrast to the effect of this anion on the ATPase activity; however, the gastric phosphatase as well as the ATPase are destroyed by phospholipase C, thus showing the lipoprotein nature of these enzymes. Kinetics of the K⁺ activation of the microsomal phosphatase suggest that the K⁺-PNPP complex is the active substrate for the enzymic reaction. Rb⁺, NH₄ ⁺ and Cs⁺ will substitute to some degree for K⁺ as an activator of the microsomal phosphatase. It is pointed out that K⁺ is an essential requirement for HCl secretion in intact gastric mucosa and the replacement of K⁺ with Rb⁺, Cs⁺ and NH₄⁺ is discussed. The K⁺-stimulated phosphatase presented in this paper may play a role in the H⁺ secretion process.     

Measurement of CO(2) Assimilation in Soils: an Experiment for the Biological Exploration of Mars

A method is described for the measurement of ¹⁴CO₂ assimilation by microorganisms in soils. A determination involves exposing soil to ¹⁴CO₂, pyrolyzing the exposed soil, trapping the organic pyrolysis products on a column of firebrick coated with CuO, combusting the trapped organics by heating, and measuring the radioactivity in the CO₂ produced in the combustion. The detection of significant levels of ¹⁴C in the trapped organic fraction appears to be an unambiguous indication of biological activity. The ¹⁴CO₂ which is adsorbed or exchanged into soils by nonbiological processes does not interfere. The method easily detects the ¹⁴CO₂ fixed by 10² to 10³ algae after light exposure for 3 to 24 hr. Assimilation of ¹⁴C is also demonstrable in dark-exposed soils containing 10⁵ to 10⁶ heterotrophic bacteria. Possible applications of the method in the biological exploration of Mars are discussed.     

Survival of Probiotic Lactobacilli in Acidic Environments Is Enhanced in the Presence of Metabolizable Sugars

Lactobacillus rhamnosus GG is an industrially significant probiotic strain with proven health benefits. In this study, the effect of glucose on L. rhamnosus GG survival was analyzed in simulated gastric juice at pH 2.0. It was found that the presence of 19.4 mM glucose resulted in up to 6-log₁₀-enhanced survival following 90 min of exposure. Further work with dilute HCl confirmed that glucose was the sole component responsible. Comparative analysis with other Lactobacillus strains revealed that enhanced survival was apparent in all strains, but at different pH values. The presence of glucose at concentrations from 1 to 19.4 mM enhanced L. rhamnosus GG survival from 6.4 to 8 log₁₀ CFU ml⁻¹ in simulated gastric juice. The mechanisms behind the protective effect of glucose were investigated. Addition of N′,N′-dicyclohexylcarbodiimide to simulated gastric juice caused survival to collapse, which was indicative of a prominent role in inhibition of F₀F₁-ATPase. Further work with neomycin-resistant mutants that exhibited 38% to 48% of the F₀F₁-ATPase activity of the parent confirmed this, as the survival in the presence of glucose of these mutants decreased 3 × 10⁶-fold compared with the survival of the wild type (which had a viability of 8.02 log₁₀ CFU ml⁻¹). L. rhamnosus GG survival in acidic conditions occurred only in the presence of sugars that it could metabolize efficiently. To confirm the involvement of glycolysis in the glucose effect, iodoacetic acid was used to inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. The reduction in GAPDH activity caused survival to decrease by 8.30 log₁₀ CFU ml⁻¹ in the presence of glucose. The data indicate that glucose provides ATP to F₀F₁-ATPase via glycolysis, enabling proton exclusion and thereby enhancing survival during gastric transit.     

Ferroxidase activity of recombinant Desulfovibrio vulgaris rubrerythrin

 Rubrerythrin (Rr) is the trivial name given to a non-heme iron protein of unknown function which has been found in anaerobic sulfate-reducing bacteria. Rr is unique in containing both rubredoxin-type FeS₄ and diiron-oxo sites in the same protein. The results described here demonstrate for the recombinant protein that: (a) Rr catalyzes oxidation of Fe²⁺ to Fe³⁺ by O₂, i.e., Rr has ferroxidase activity, (b) both FeS₄ and diiron domains of the Rr protein are required for ferroxidase activity, (c) with excess Fe²⁺ and O₂ the initial rate of this oxidation appears to be first order in [Rr] and independent of starting [Fe²⁺] above 30 μM, (d) the Fe³⁺ is produced in a form which is capable of rapid incorporation into the iron-binding site of ovotransferrin, and (e) the ferroxidase activity of Rr is comparable to that of published ferroxidase activities of apoferritins on a subunit basis. Ferroxidase activity of Rr was monitored either by the rate of increase in absorbance at 315 nm (which lies near an isosbestic point for oxidized and reduced Rr) or by using apoovotransferrin as Fe³⁺ acceptor, and measuring the rate and extent of diferric transferrin formation at 460 nm. No polyironoxyhydroxide aggregates appeared to associate with Rr after the ferroxidase reaction. A truncated form of Rr containing only the diiron domain had little or no ferroxidase activity. Rr could function as one component of a set of enzymes which channels the reaction products of O₂ and Fe²⁺ onto a non-toxic pathway during transient exposure of the bacteria to air.     

The NH2-terminus of K-Cl cotransporter 3a is essential for up-regulation of Na,K-ATPase activity

K⁺-Cl⁻ cotransporter-3 has two major amino terminal variants, KCC3a and KCC3b. In LLC-PK1 cells, exogenously expressed KCC3a co-immunoprecipitated with endogenous Na⁺,K⁺-ATPase α1-subunit (α1NaK), accompanying significant increases of the Na⁺,K⁺-ATPase activity. Exogenously expressed KCC3b did not co-immunoprecipitate with endogenous α1NaK inducing no change of the Na⁺,K⁺-ATPase activity. A KCC inhibitor attenuated the Na⁺,K⁺-ATPase activity in rat gastric mucosa in which KCC3a is predominantly expressed, while it had no effects on the Na⁺,K⁺-ATPase activity in rat kidney in which KCC3b is predominantly expressed. In these tissue samples, KCC3a co-immunoprecipitated with α1NaK, while KCC3b did not. Our results suggest that the NH₂-terminus of KCC3a is a key region for association with α1NaK, and that KCC3a but not KCC3b can regulate the Na⁺,K⁺-ATPase activity.     

ISOLATION AND PARTIAL CHARACTERIZATION OF NITRATE ASSIMILATION MUTANTS OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

Fifteen nitrate assimilation-deficient mutants of the euryhaline green alga, Dunaliella tertiolecta Butcher were selected by their chlorate resistance. Ten mutants, unable to grow on NO₃^? but able to grow on NO₂^?, had no detectable nitrate reductase activity. Five mutants, unable to grow on either NO₃^? or NO₂^?, had depressed levels of both nitrate and nitrite reductase. A method for assaying methyl viologen-nitrate reductase in the presence of nitrite reductase is described.     

Distribution of prostaglandin E2 binding sites in the porcine gastrointestinal tract

Binding of biologically active ³H-PGE₂ to particulate fractions of porcine gastrointestinal mucosa and muscle was investigated. Specific binding activity was detected in the 2500 xg and 30,000 xg sedimentation fractions of mucosa from esophagus, fundus, antrum, duodenum, ileum and colon, as well as in serosal muscle taken from the antrum, ileum, and colon. Optimal binding (> 40 fmol/mg protein) was observed in the 30,000 xg fraction of fundic mucosa incubated at pH 5.0. The characteristics of ³H-PGE₂ binding were variable in the remainder of the gastrointestinal tract although binding in these tissues was significantly less (0.2 to 15 fmol/mg protein) than that observed in the fundic mucosa. These data suggest that the cellular and/or subcellular site of PG binding is not uniform throughout the gastrointestinal tract. In fundic mucosa removal of the surface epithelial layer by scraping did not significantly alter the total binding activity for PGE. This result suggests that in gastric secretory mucosa optimal binding activity for PGE₂ occurs within the gastric pits deep to the surface epithelium.     

The estimation of phenylalanine ammonia-lyase (PAL)-activity in intact cells of higher plant tissue

Summary A procedure is described which permits the estimation of the relative activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5.) in intact plant cells, exemplified by buckwheat hypocotyls. Hypocotyl segments are incubated at pH 5.5 with L-[3-³H]phenylalanine. N³HH₂, which is liberated from phenylalanine by the action of phenylalanine ammonia-lyase, equilibrates with tissue water to yield ³HOH, which is recovered by sublimation. Participation of phenylalanine transaminase in the reactions leading to ³HOH formation is excluded, and it is conclusively shown that ³HOH is formed intracellularly and not by enzymatic activity leaking out of wounded tissue.Abbreviation PAL phenylalanine ammonia-lyase (E.C. 4.3.1.5.)     

Radiorespirometer for continuous monitoring of chick embryo development.

A radiorespirometer is described that is capable of continuous monitoring of O₂ utilization, CO₂ and/or ¹⁴CO₂ production per minute, heart rate, and body activity of an embryonated egg while it develops for several days in a closed chamber with near normal pO₂ or other desired pO₂. Oxygen is supplied to the embryo by an electrolytic cell, and CO₂ is removed by a KOH solution flowing through a diffusion cell separated from the embryo chamber by a CO₂ permeable rubber membrane. The instrument permits selecting embryos for viability and developmental stage, according to their O₂ utilization per minute and respiratory quotient, without breaking the eggshell. Inoculation of the embryonated egg with ¹⁴C-labeled substrates, drugs, or toxins can occur without interfering with continuous recording of metabolic activity.     

Enzymatic sulfation of cholesterol by rat gastric mucosa

A sulfotransferase which catalyzes transfer of the sulfate group from 3'-phosphoadenosine-5'phosphosulfate to cholesterol has been demonstrated in the rat gastric mucosa. The product of the reaction was characterized as cholesterol sulfate by two-dimensional thin-layer Chromatographic behavior, and gas-liquid chromatography of cholesterol after acid solvolysis. The bulk of enzyme activity was found in the cytosol fraction. Sulfation of cholesterol did not require added Mg⁺², Mn⁺², or Ca⁺², and was unaffected by ethylenedia-minetetraacetate. Triton X-100 moderately enhanced the enzyme activity. A broad pH optimum from pH 6.0–9.0 was exhibited with a maximum at pH 7.0–7.5. The apparent Km for PAPS was 0.8 × 10^?6M. The possible function of cholesterol sulfate in gastric mucosa is discussed.     

Gastric HCO3-stimulated ATPase: Evidence against its microsomal localization in rat fundus mucosa

Rat gastric mucosa was shown to contain a Mg²⁺-dependent ATPase which is stimulated by HCO₃⁻ at pH 8–9.Triton X-100 solubilizes this HCO₃⁻-stimulated, Mg²⁺-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3).The gastric mucosa was resolved into five subcellular fractions by differential centrifugation. A large granule fraction (Fraction M), 28 000 g · min, was characterized by cytochrome c oxidase (marker enzyme for mitochondria). A microsomal fraction (Fraction P), 2 760 000 g · min, was characterized by 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) (plasma membrane).The Mg²⁺-dependent ATPase was demonstrated to have a bimodal mitochondrial membranous localization: 24% of its activity is associated with cytochrome c oxidase, and 75% with 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) at pH 8.The HCO₃⁻ addition resulted in two opposite effects: (1) a strong stimulation (84%) in Fraction M; (2) a slight inhibition (12%) in Fraction P.Fraction M was subfractionated by equilibration on a sucrose gradient. It gave rise to a homogeneous mitochondrial (d, 1.17–1.21) Mg²⁺-dependent ATPase, closely associated with cytochrome c oxidase. This ATPase is strongly stimulated (×2) by HCO₃⁻. The subfractionation of Fraction P gave rise to two distinct ATPases: (1) the major one is associated with membranous (d, 1.10–1.15) material marked by 5′-nucleotidase and is slightly inhibited by HCO₃⁻; (2) the other is associated with denser (d, 1.17–1.21) material and is stimulated by HCO₃⁻.The bicarbonate-stimulated fraction of the Mg²⁺-dependent ATPase activity found in the gastric microsomal fraction is assumed to arise from mitochondrial cross-contamination. Further support comes from the optimal HCO₃⁻ concentration. In addition, SCN⁻ is shown to specifically inhibit the ATPase of Fraction M.From these results it appears that the implication of HCO₃⁻-stimulated ATPase in the gastric secretion of H⁺ is not as clear as had been suggested. However, in the view of an ATPase-supported model for H⁺ secretion, attention can be directed towards the Mg²⁺-dependent ATPase found to be associated with microsomes.     

A mutant of CHO-K1 cells deficient in two nonsequential steps of de novo purine biosynthesis

An unusual new purine-requiring mutant, Ade^?P_AB, of Chinese hamster ovary cells (CHO-K1) is described. Ade^?P_AB will grow in medium supplemented with hypoxanthine, adenine, or aminoimidazole carboxamide. Ade^?P_AB fails to show genetic complementation with either Ade^?A, defective in amidophosphoribosyltransferase (E.C. 2.4.2.14), or Ade^?B, defective in phosphoribosylformylglycinamidine FGAM) synthetase (E.C. 6.3.5.3.), but will complement all five of our other hypoxanthine-requiring Ade^? complementation groups. Analysis of purine synthesis in wild-type, mutant, and revertant cells and analysis of relevant enzyme activities in cell-free extracts prepared from these cells demonstrates that Ade^?P_AB is similar to Ade^?B in that it has lost FGAM synthetase activity, and is similar to Ade^?A in that it has lost glutamine-dependent amidophosphoribosyltransferase activity. Unlike Ade^?A, however, Ade^?P_AB retains the ability to synthesize phosphoribosylamine (PRA), the product of the amidophosphoribosyltransferase reaction, if NH₄Cl is substituted for glutamine as the nitrogen donor. Moreover, partial revertants of Ade^?P_AB can apparently synthesize sufficient purines for growth using the NH₄Cl-dependent reaction. The available evidence indicates that neither a double mutation nor a deletion is probable in Ade^?P_AB. We discuss the relevance of these observations for our understanding of both the regulation of purine biosynthesis in mammalian cells and the structural organization of the enzymes defective in Ade^?P_AB and the genes coding for these enzymes.