Rat mast cell phospholipase A2: activity toward exogenous phosphatidylserine and inhibiton by N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylserine

The presence of phospholipase A2 in intact rat peritoneal mast cells was investigated by using two synthetic radiolabeled phosphatidylserine (PS) substrates. Incubation of intact cells with 1-oleoyl-2-[3H]oleoyl-PS resulted in the release of a considerable quantity of [3H]oleic acid from the substrate. To establish that [3H]oleic acid release was mediated via direct enzymatic attack at the sn-2 position, we measured release of the [3H]serine moiety from the glycerol backbone of 1,2-dimyristoylphosphatidyl[3H]serine. This activity, which represents the combined actions of phospholipases C and D, was 10-fold lower than [3H]oleic acid release, indicating that neither of these enzymes is required for the release of the preponderance of [3H]oleic acid. These results establish the existence in intact rat mast cells of a phospholipase A2 active toward exogenous PS. Over the concentration range at which exogenous PS activates mast cell secretion, intact mast cells and broken cells possessed nearly equal levels of phospholipase A2 activity, and enzyme activity was 3--4-fold higher toward PS than phosphatidylcholine. Several agents were tested for their ability to inhibit phospholipase A2 in intact mast cells. Of the agents tested, an N-substituted derivative of PS previously identified as an inhibitor of mast cell secretion was shown to be a particularly potent and efficacious inhibitor of mast cell phospholipase A2. The concentration dependence of enzyme inhibition paralleled inhibition of histamine secretion, providing a strong positive correlation between the level of phospholipase A2 in mast cells and the capacity for secretion.     

An In Vitro Characterization of γ-Glutamylhistamine Synthetase: A Novel Enzyme Catalyzing Histamine Metabolism in the Central Nervous System of the Marine Mollusk, Aplysia californica

The properties of the histamine metabolizing enzyme, gamma-glutamylhistamine synthetase (gamma-GHA synthetase) were studied in Aplysia ganglia in vitro. This enzyme catalyzes the incorporation of histamine into peptide linkage with L-glutamate to form a peptidoamine, gamma-glutamylhistamine (gamma-GHA). gamma-GHA synthetase is a soluble enzyme with an apparent Km of 653 microM for histamine and 10.6 mM for L-glutamate. Synthesis of gamma-GHA is energy-dependent, having an absolute requirement for ATP. Magnesium ions and dithiothreitol are also essential for activity. Of a variety of gamma-glutamyl compounds and glutamate analogs tested, only L-glutamate was effectively incorporated into peptide linkage with histamine. Similarly, the enzyme has a higher affinity for histamine than for numerous imidazole analogs. In addition, 3,4-dihydroxyphenylethylamine (dopamine), 5-hydroxytryptamine, octopamine, and several other amines tested are effective inhibitors of gamma-GHA synthesis. Ganglia, nerve trunks, and the capsule surrounding the ganglion had the highest synthetase activity. The specific activity of the enzyme in muscle, heart, and hemolymph was less than 10% of that in ganglia. Differences in substrate specificity and effect of inhibitors distinguish gamma-GHA synthetase from gamma-glutamyl transpeptidase, glutamine synthetase, and carnosine synthetase.     

Inhibitory effects of GMCHA-OPhBut on phospholipid methylation and histamine release in mast cells activated by concanavalin A, anti-IgE, and antigen

[3H]Methyl group incorporation and histamine secretion in rat mast cells induced by anti-IgE and con A were strongly inhibited by trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut), a strong and specific inhibitor for pH 7 tryptase (Muramatsu et al. (1988) Biol. Chem. Hoppe-Seyler 369, 617-625) which is present in rat mast cells. The IC50s for these events were of the order of 10(-6) M. Addition of GMCHA-OPhBut after the maximal increase in [3H]methyl group incorporation in rat mast cells activated by con A and anti-IgE induced rapid reduction of the methylated phospholipid, and the later histamine release was strongly suppressed. Mast cells were prepared with Mg2+-free Tyrode-HEPES solution, and challenged with anti-IgE with or without Mg2+. With Mg2+, [3H]methyl group incorporation was enhanced, and histamine was secreted time-dependently. Without Mg2+, [3H]methyl group incorporation fell to one-third, whereas histamine secretion was not affected. These results were incompatible with the above results. From these results it was strongly suggested that a trypsin-like protease, probably pH 7 tryptase, is involved not only in the early events, such as activation of phosphatidylethanolamine methyltransferase I and/or II, but also in the late events such as histamine release, and phospholipid methylation is not associated with histamine secretion.     

Cartilage fucoproteins with sites for cross-linking by transglutaminase

Slices of various types of cartilage were incubated with either L-[6-3H]fucose or [1,4-3H(N)]putrescine. Homogenization of the slices and fractionation of the homogenates showed for both labels that an insoluble collagenase-resistant fraction had the highest specific activity (dpm/mg dry weight). Examination of an exhaustive proteolytic digest of this insoluble fraction by ion-exchange high performance liquid chromatography showed the presence of gamma-glutamyl[3H]putrescine. Chromatography of solubilized [3H]fucoprotein fractions showed the presence of several low molecular weight peaks, as well as high molecular weight material. Incubation of [3H]fucoprotein extracts with transglutaminase increased the high molecular weight peaks and decreased the low molecular weight ones. Incubation of the cartilage slices with L-[3H]fucose plus 0.5 mM dansylcadaverine, an inhibitor of transglutaminase, caused a decrease in the insoluble and high molecular weight fraction relative to the low molecular weight peaks. It is hypothesized that this is due to inhibition of cross-link formation between fucoprotein components of the cartilage which are transglutaminase substrates. One major low molecular weight peak, which labels with both fucose and putrescine, corresponds in size with the 15,000 subunit of collagen III aminopropeptide, which is known to be a substrate for transglutaminase.     

Activation of transglutaminase during embryonic development

Incorporation of [3H]putrescine into proteins was shown to increase markedly in sea urchin eggs upon fertilization. Emetine, an inhibitor of protein synthesis, had no effect on the rate of protein labeling. However, the reaction could be prevented by the addition of 2-[3-(diallylamino)-propionyl]benzothiophene, a noncompetitive inhibitor of transglutaminase, and also by dansylcadaverine, which is a substrate for transglutaminase. The inert N alpha-dimethyl analogue of dansylcadaverine had no influence. Considering the complexity of the incorporation of the [3H]putrescine tracer in this system, it was deemed essential to prove by rigorous analytical methods that the reaction was, indeed, consistent with a transglutaminase mechanism. gamma-Glutamyl[3H]putrescine could be recovered in 80-90% yield from the proteolytic digest of proteins from the 20-min fertilized cell. Another sign of the in vivo activity of transglutaminase was the isolation of substantial amounts of epsilon-(gamma-glutamyl)lysine from proteins of sea urchin embryo, yielding a frequency value for this cross-link as high as 1 mol/400 000 g of protein in the 32-cell-stage material.     

Stimulation of histamine release and arachidonic acid metabolism in rat peritoneal mast cells by thapsigargin, a non-TPA-type tumor promoter

Thapsigargin, a non-TPA-type tumor promoter, releases histamine and stimulates arachidonic acid metabolism in rat peritoneal mast cells. In order to clarify the relationship between the histamine-releasing activity and the arachidonic acid metabolism-stimulating activity of thapsigargin in mast cells, the effects of cyclooxygenase inhibitors, indomethacin and ibuprofen, a lipoxygenase inhibitor, AA861, and dual inhibitors for cyclooxygenase and lipoxygenase, nordihydroguaiaretic acid and BW755C, on histamine release and arachidonic acid metabolism were examined. High-performance liquid chromatography analysis revealed that the peritoneal mast cells preferentially produce prostaglandin D2 by thapsigargin treatment. These inhibitors suppressed thapsigargin-induced prostaglandin D2 production in a dose-dependent manner, but failed to inhibit histamine release, suggesting that the mechanisms for stimulation of histamine release by thapsigargin is not dependent on increased arachidonic acid metabolism. Time-course experiments of histamine release and the release of radioactivity from [3H]arachidonic acid-labeled mast cells also provide evidence for a difference in mechanism.     

Inhibitory effect of diethylstilbestrol on histamine release by rat mast cells and its relation to the cellular ATP content

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.     

Glucose inhibits insulin release when not promoting the entry of calcium into the beta-cells

The effect of antigen on the metabolism of polyphosphoinositides was investigated in sensitized rat peritoneal mast cells. Addition of antigen to rat peritoneal mast cells prelabelled with [3H]arachidonic acid resulted in a very rapid decrease in the level of phosphatidylinositol 4-phosphate (DPI) within 5 sec, which appeared to precede the breakdown of phosphatidylinositol (PI), while there was no significant decline of PI 4,5-bisphosphate (TPI). The reduced levels of these phosphoinositides returned almost to control or even slightly higher values by 300 sec in parallel with the antigen-stimulated [32P]phosphate incorporation into these lipids. This early and transient disappearance in DPI prior to that in PI was also observed in [3H]glycerol-prelabelled cells. These data suggest that DPI degradation upon stimulation by antigen in mast cells may be an initial step in the histamine release process.     

Effects of histamine H1 antagonist dithiaden on acetic acid-induced colitis in rats.

To assess the possible involvement of mast cells and/or their mediators in inflammatory bowel diseases, the effect of the histamine H1 antagonist Dithiaden was studied on a model of acetic acid-induced colitis in rats. Dithiaden pretreatment by intracolonic administration was found to reduce the extent of acute inflammatory colonic injury. This was manifested by a decrease in the score of gross mucosal injury, by lowered colonic wet weight and by diminished myeloperoxidase activity reflecting reduced leukocyte infiltration. Vascular permeability and gamma-glutamyl transpeptidase activity, elevated by acetic acid exposure, were decreased after Dithiaden pretreatment. The results indicate that locally administered Dithiaden may protect the colonic mucosa against an acute inflammatory attack by interfering with the action of the major mast cell mediator histamine.     

Reduced transglutaminase-catalyzed cross-linking of exogenous amines to membrane proteins in sickle erythrocytes

In order to determine the capacity of sickle cells to undergo transglutaminase-catalyzed cross-linking of membrane proteins, human normal and sickle erythrocytes were incubated with [ring-2-14C]histamine in the presence of Ca2+ and ionophore A23187. The [14C]histamine incorporation into membrane components was observed in freshly prepared erythrocytes. Incorporation of radioactivity into spectrin and Band 3 membrane components was significantly (P less than 0.001) less in sickle erythrocytes than in normal cells. Transglutaminase deficiency was excluded by the finding of increased activity of this enzyme in sickle cells from patients with reticulocytosis. The incorporation of [3H]spermine into red cell membranes was also less in sickle erythrocytes than in normal cells under the same conditions of incubation used for [ring-2-14C]histamine. Sickle erythrocytes were more permeable to these amines than normal cells. It is proposed that the gamma-glutamyl sites of membrane proteins in sickle erythrocytes are less accessible for transglutaminase-catalyzed cross-linking to histamine and polyamines in vitro, perhaps due to prior in vivo activation of this enzyme by the increased calcium in sickle cells and/or shielding secondary to altered membrane organization.     

Antigen-and ionophore-stimulated synthesis of platelet-activating factor by the cloned mast cell line, MC9

MC9 mast cells stimulated by a soluble (calcium ionophore A23187) or by an Fc epsilon-receptor agonist (IgE plus hapten) produce platelet activating factor (PAF). MC9 cells incorporate either exogenous [3H]acetic acid or [3H]lyso-PAF into PAF. PAF was identified by mobility on thin layer chromatography, platelet aggregatory activity inhibitable by known PAF antagonists, and by enzymatic modification. Quantified by aggregation of rabbit platelets, MC9 cells produce 6 pmoles PAF/10(6) cells. MC9 cells express acetyltransferase activity of 0.19 nmole/5 min-mg protein. Analysis of MC9 phospholipids by HPLC showed that MC9 cells contain large amounts of phosphatidylcholine (82 nmoles/10(7) cells) but contain little ether-linked phosphatidylcholine (4 nmoles/10(7) cells).     

Granule-associated serine neutral proteases of the mouse bone marrow-derived mast cell that degrade fibronectin: their increase after sodium butyrate treatment of the cells

Mouse bone marrow-derived mast cells, differentiated in vitro with concanavalin A splenocyte-conditioned medium and sensitized with monoclonal IgE, release neutral serine proteases after activation with specific antigen. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the supernatants from immunologically activated mast cells revealed the presence of four prominent proteins of 27,000, 29,000, 30,000 and 31,000 m.w. When the supernatants and sonicated residual cells from antigen-challenged or nonactivated IgE-sensitized mast cells were incubated with [3H]diisopropylfluorophosphate ([3H]DFP) and the proteins were subjected to SDS-PAGE followed by autoradiography, proteins of 27,000 to 31,000 m.w. were labeled with [3H]DFP. The antigen-dependent release of labeled proteins was accompanied by a corresponding depletion of similarly sized [3H]DFP-labeled proteins from these cell pellets relative to unactivated cells. The SDS gels were also stained with Coomassie Blue and were sectioned to separate the individual proteins for measurement of their incorporated radioactivity; the net percent antigen-dependent release of all four [3H]DFP-labeled proteins ranged from 64 to 68% and was comparable to that of the secretory granule markers, beta-hexosaminidase and histamine. That the [3H]DFP-labeled proteins were derived from the secretory granules of the cells was supported by studies in which mast cells were cultured for 4 days in the presence of 1 mM sodium butyrate. This treatment produced a differential increase in their cellular content of histamine (10-fold), [3H]DFP binding proteins (two- to fourfold), and beta-hexosaminidase (minimally), while the net percent antigen-dependent release of each of these constituents was unchanged. After sensitization and antigen activation, the net percent release of histamine, beta-hexosaminidase, and the four [3H]DFP-labeled proteins was 51, 59, and 53 to 61%, respectively, for sodium butyrate-treated cells, and 53, 60, and 64 to 68%, respectively, for cells not exposed to sodium butyrate. Human plasma fibronectin was used as a substrate to demonstrate that the exocytosed proteins possessed proteolytic activity. As assessed by optical density scanning of stained SDS-PAGE gels of the substrate, the proteases present in the supernatants of antigen-activated cells, but not of sensitized unchallenged cells, rapidly degraded native fibronectin at pH 7.0. This degradation was prevented by pretreatment of the exocytosed proteins from immunologically activated cells for 90 min at 37 degrees C with 2 mM DFP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Redistribution of phospholipid/Ca2+-dependent protein kinase in mast cells activated by various agonists

Three types of agonists; receptor-mediated concanavalin A), direct (phorbol ester), and membrane-perturbing (compound 48/80), elicit histamine secretion from rat peritoneal mast cells. We tested whether activation of the mast cells by these agents is accompanied by subcellular redistribution of protein kinase C. Phorbol ester treatment predictably caused a profound decrease of phospholipid/Ca2+-dependent histone kinase activity in the cytosol and a concomitant increase of [3H]PMA-binding capacity in the membrane fraction, in a time- and concentration-dependent manner. Similar, but less marked effects were observed with stimulations by concanavalin A and compound 48/80. When mast cells labeled with [32P] and then stimulated with the agents, phosphorylation of a 50,000-Dalton protein was enhanced in the membrane fraction. These results suggest that protein kinase C may play a role in mast cell activation through phosphorylation of the membrane protein.     

Evidence for de novo synthesis of phosphatidylinositol coupled with histamine release in activated rat mast cells

Phospholipid metabolisms in rat mast cells activated by ionophore A23187 and compound 48/80 were examined with reference to 'phosphatidylinositol (PI) cycle'. The addition of A23187 to [3H]glycerol-prelabeled mast cells induced a marked accumulation of the radioactivity in 1,2-diacylglycerol(DG) and phosphatidic acid(PA) within 10 to 30 sec. A great enhancement of [3H]glycerol incorporation into PA and PI was also detected during histamine release. On the other hand, 48/80 was far less effective than A23187 both in producing 1,2- DG and PA and in accerelating [3H]glycerol incorporation into PA and PI, despite the comparable ability of histamine release. The activity of Ca2+ uptake into mast cells, as measured by pulse-labeling with 45Ca2+, was increased when exposed to both of two agents. These data provide circumstantial evidence that phospholipid metabolisms, mainly de novo PI synthesis, may be a part of the triggering events for Ca2+ mobilization and secretory process. The PI metabolism induced by two different stimulants appears to behave in a different manner.     

Differential activation of membrane phospholipid turnover by compound 48/80 and ionophore A23187 in rat mast cells

Membrane phospholipid turnover was investigated during histamine release from rat mast cells. Addition of calcium ionophore A23187 (0.5 microgram/ml) to mast cells prelabeled with [3H]glycerol induced the rapid and progressive increase in phosphatidic acid (PA) and 1,2-diacylglycerol (DG), which was concomitant with the small rise in phosphatidylinositol (PI). Loss of the level in triacylglycerol (TG) was very marked. Polyamine compound 48/80 (5 micrograms/ml) was shown to cause rises in PA, 1,2-DG, and PI without any significant changes in TG. Both stimuli increased incorporation of exogenous [3H]glycerol into phospholipids, indicating the involvement of de novo synthesis in phospholipid metabolism. Studies with [3H]arachidonic acid-labeled mast cells showed an enhanced liberation of radioactive arachidonate and metabolites upon histamine release. There were associated decreases of radioactivity in phosphatidylcholine (PC) and TG when exposed to A23187, while phosphatidylethanolamine (PE) was degraded as a result of 48/80 activation. The transient increases of [3H]arachidonoyl-1,2-DG and PA were caused by 48/80, while A23187 showed a gradual rise in the radioactivity in these two lipid fractions. These findings reflect activation of phospholipase C. When mast cells were activated by low concentrations of A23187 (0.1 microgram/ml) and 48/80 (0.5 microgram/ml), different behaviors of PI metabolism were observed. An early degradation of PI and a subsequent formation of 1,2-DG and PA suggest that the lower concentrations of these agents stimulate the PI cycle initiated by PI breakdown rather than de novo synthesis. These results demonstrate that marked and selective changes in membrane phospholipid metabolism occur during histamine release from mast cells, and that these reactions seem to be controlled by the coordination of degradation and biosynthesis, depending on the type and the concentration of stimulants. A23187 stimulates arachidonate release perhaps via the cleavages of PC and TG, whereas 48/80 liberates arachidonate from PE.     

Signalling mechanism in the lysophosphatidylserine-induced activation of mouse mast cells.

Lysophosphatidylserine (0.1-1 microM) elicits histamine release in isolated mouse peritoneal mast cells. The effect becomes manifest after a lag of 30 s and reaches completion in 5 min. Maximal activity is observed when serine is in L-configuration. As shown by the activity of a lysophosphatidylserine analogue lacking the OH group in C2 position of glycerol, conversion into phosphatidylserine is not required. When 32PO4-labeled mast cells are challenged 2-5 min with lysophosphatidylserine, the labeling of phosphatidate, phosphatidylinositol and phosphatidylcholine is increased. When [3H]arachidonate-labeled mast cells are used, lysophosphatidylserine increases the appearance of isotopic diacylglycerol and phosphatidate. Like the secretory response, these effects are independent of the presence of extracellular Ca2+. Incubations in the presence of [3H]glycerol show that lysophosphatidylserine does not activate the de novo synthesis of phospholipids. In agreement with a participation of phosphoinositidase C in the action of lysophosphatidylserine, we observe accumulation of inositol phosphates in [3H]inositol labeled mast cells incubated in the presence of Li+. The results suggest that lysophosphatidylserine delivers its stimulus to mast cells, by the activation of phosphoinositide-dependent signalling mechanism.     

Metabolism of histamine in the CNS of Aplysia californica: cellular distribution of gamma-glutamylhistamine synthetase

1. The cellular distribution of the histamine-metabolizing enzyme, gamma-glutamylhistamine synthetase, was studied in the CNS of Aplysia californica. 2. Enzyme activity was assayed in single, re-identifiable neuronal cell bodies, clusters of nerve cells and neuropil and capsule tissue surrounding the ganglia. 3. The "histaminergic" C-2 cells and all other single nerve cell bodies contained measurable gamma-glutamylhistamine synthetase activity. 4. The cerebral E cluster, which houses the C-2 cells and several of its post-synaptic neurons, had an apparently higher specific enzyme activity than other neuronal clusters. 5. The finding of measurable enzyme activity in the histamine-rich C-2 cell bodies and in clusters of cells responsive to this imidazoleamine supports the hypothesis that one function for gamma-glutamylhistamine synthetase is in the disposal of neuronally released histamine. 6. The average specific activity of gamma-glutamylhistamine synthetase in single cells was 3.64 +/- 0.32 mumol g protein hr. This represented only 5% of the enzyme activity measured in the whole ganglion. 7. The bulk of synthetase activity was found to reside in the capsule tissue.     

A major role for phospholipase A2 in antigen-induced arachidonic acid release in rat mast cells.

Cross-linking of IgE receptors by antigen stimulation leads to histamine release and arachidonic acid release in rat peritoneal mast cells. Investigators have reported a diverse distribution of [3H]arachidonate that is dependent on labelling conditions. Mast cells from rat peritoneal cavity were labelled with [3H]arachidonic acid for different periods of time at either 30 or 37 degrees C. Optimum labelling was found to be after 4 h incubation with [3H]arachidonate at 30 degrees C, as judged by cell viability (Trypan Blue uptake), responsiveness (histamine release) and distribution of radioactivity. Alterations in 3H-radioactivity distribution in mast cells labelled to equilibrium were examined on stimulation with antigen (2,4-dinitrophenyl-conjugated Ascaris suum extract). The results indicated that [3H]arachidonic acid was lost mainly from phosphatidylcholine and, to a lesser extent, from phosphatidylinositol. A transient appearance of radiolabelled phosphatidic acid and diacylglycerol indicated phosphatidylinositol hydrolysis by phospholipase C. Pretreatment with a phospholipase A2 inhibitor, mepacrine, substantially prevented the antigen-induced liberation of [3H]arachidonic acid from phosphatidylcholine. It can be thus concluded that, in the release of arachidonic acid by antigen-stimulated mast cells, the phospholipase A2 pathway, in which phosphatidylcholine is hydrolysed, serves as the major one, the phospholipase C/diacylglycerol lipase pathway playing only a minor role.     

Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation

This study evaluated the effect of inhibitors of transmethylation on histamine release from rat mast cells and rat basophilic leukemia cells. IgE-mediated histamine release from rat basophilic leukemia cells (RBL-2H3 cells) was inhibited by 3-deazaadenosine (DZA) in the presence of L-homocysteine thiolactone (Hcy) or the combination of adenosine, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), and Hcy in a dose-dependent fashion. There were no significant changes in the cellular cAMP levels by these inhibitors. Histamine release induced by anti-IgE or dextran from normal rat mast cells was also blocked by DZA plus Hcy in a dose-dependent manner. DZA at 10(-3) M in the presence of 10(-4) M Hcy or the combination of 10(-3) M adenosine, 10(-4) M EHNA, and 10(-3) M Hcy inhibited lipid (perhaps phospholipid) methylation into RBL-2H3 cells without affecting choline incorporation. In the presence of 10(-3) M DZA plus 10(-4) M Hcy there was a 170-fold increase in [35S]AdoHcy with the concomitant appearance of 3-deaza-AdoHcy when the cells were incubated with [35S]methionine, thus indicating that these drugs inhibited methylation reaction(s) through the intracellular accumulation of AdoHcy and 3-deaza-AdoHcy. In contrast, histamine release from rat mast cells induced by the calcium ionophore A23187, compound 48/80, polymyxin B, or ATP was not inhibited by these compounds. These results suggest that IgE- or dextran-mediated histamine release involves methylation reactions(s), whereas the other secretagogues bypass this early step.     

Quantitative radioisotopic determination of histidine decarboxylase using high-performance liquid chromatography

We have developed a procedure for the accurate measurement of histidine decarboxylase in tissues expressing low levels of enzymatic activity. Briefly, histamine is enzymatically synthesized from [3H]-labeled histidine, followed by purification using high-performance liquid chromatography (HPLC) and quantitation by liquid scintillation counting. This method presents three advantages over previous techniques. First, prior to HPLC purification, excess precursor [3H]histidine is removed on an anion-exchange resin. Second, purification by HPLC is considerably more selective than that of classical cation-exchange gravity columns or organic solvent extractions. Finally, the accuracy of this method is improved by including non-radiolabeled histamine as internal standard, which is quantified by ultraviolet detection. This simple procedure allows highly sensitive and accurate determinations of histamine synthesis.