The subsynaptosomal localization of histamine, histidine decarboxylase and histamine methyltransferase in rat hypothalamus

Abstract— We have examined the subcellular localization of histamine, histamine methyltransferase (EC 2.1.1.8) (HMT) and histidine decarboxylase (EC 4.1.1.22) in rat hypothalamus after osmotic lysis of synaptosome-containing primary particulate fractions. When crude mitochondrial fractions are subjected to osmotic lysis, histamine is retained within particulate structures, while HMT is released into the supernatant fluid. The majority of histidine decarboxylase activity is also recovered in the supernatant fluid, although more histidine decarboxylase than HMT is retained in particulate fractions. After sucrose gradient fractionation of osmotically lysed crude mitochondrial or microsomal pellets, histamine is also retained in particulate structures, with the greatest amount occurring in a fraction enriched in synaptic vesicles. In these sucrose gradients histidine decarboxylase activity shows a greater particulate localization than does HMT activity.     

Imidazole derivatives as a novel class of hybrid compounds with inhibitory histamine N-methyltransferase potencies and histamine hH3 receptor affinities

In this study, a novel series of imidazole-containing compounds with dual properties, that is, inhibitory potency at the enzyme histamine N(tau)-methyltransferase (HMT) and antagonist potency at histamine H(3) receptors was designed and synthesized. Pharmacologically, these new hybrid drugs were evaluated in functional assays for their inhibitory potencies at rat kidney HMT and for their antagonist activities on synaptosomes of rat cerebral cortex. For selected compounds, binding affinities at recombinant human histamine H(3) receptors were determined. The first compounds (1-10) of the series proved to be H(3) receptor ligands of high potency at rat synaptosomes or of high binding affinity at human H(3) receptors, respectively, but of only moderate activity as inhibitors of rat kidney HMT. In contrast, aminoquinoline- or tetrahydroacridine-containing derivatives 11-17 also displayed HMT inhibitory potency in the nanomolar concentration range. Preliminary data from molecular modeling investigations showed that the imidazole derivative 15 and the HMT inhibitor quinacrine possess identical binding areas. The most interesting compound (14) is simultaneously a highly potent H(3) receptor ligand (K(i)=4.1nM) and a highly potent HMT inhibitor (IC(50)=24nM), which makes this derivative a valuable pharmacological tool for further development.     

Peroxidation-induced changes of histamine metabolism and transport of its precursor histidine in rat brain synaptosomes

The metabolism of histamine and transport of its precursor histidine were investigated in rat brain synaptosomes which underwent the ADP-Fe/ascorbate-induced peroxidation. Peroxidation impaired histidine uptake by 40%, and veratridine induced release of it by 25% of maximal uptake. Simultaneously, marked decrease of synaptosomal histamine (HA) content, to about 30% of control value, was found (p less than 0.01). Activity of the two histamine-metabolizing enzymes, histidine decarboxylase (HD) and histamine N-methyl-transferase (HMT), were drastically lowered, by 40% (p less than 0.02) and 60% of control (p less than 0.05), respectively. Pretreatment of rats with glucocorticoid analog, dexamethasone (DMX), 1 mg/kg of body weight, given twice, 20 and 2 h before decapitation, did not influence significantly the effects invoked by peroxidation on HA levels and the activity of HD and HMT, but impaired histidine transport. These results indicate that iron-dependent peroxidation decreases both neuronal pool of histamine and its turnover, which may affect the function of central nervous system. Short pretreatment with dexamethasone does not seem to influence this effect.     

Immunohistochemical localization of histamine N-methyltransferase in guinea pig tissues.

Histamine plays important roles in gastric acid secretion, inflammation, and allergic response. Histamine N-methyltransferase (HMT; EC 2.1.1.8) is crucial to the inactivation of histamine in tissues. In this study we investigated the immunohistochemical localization of this enzyme in guinea pig tissues using a rabbit polyclonal antibody against bovine HMT. The specificity of the antibody for guinea pig HMT was confirmed by Western blotting and the lack of any staining using antiserum preabsorbed with purified HMT. There was strong HMT-like immunoreactivity (HMT-LI) in the epithelial cells in the gastrointestinal tract, especially in the gastric body, duodenum, and jejunum. The columnar epithelium in the gallbladder was also strongly positive. Almost all the myenteric plexus from the stomach to the colon was stained whereas the submucous plexus was not. Other strongly immunoreactive cells included the ciliated cells in the trachea and the transitional epithelium of the bladder. Intermediately immunoreactive cells included islets of Langerhans, epidermal cells of the skin, alveolar cells in the lung, urinary tubules in the kidney, and epithelium of semiferous tubules. HMT-LI was present in specific structures in the guinea pig tissues. The widespread distribution of HMT-LI suggests that histamine has several roles in different tissues.     

Inhibitors of Histamine Methylation in Brain Promote Formation of Imidazoleacetic Acid, Which Interacts with GABA Receptors

Abstract: In brain, the precursor of imidazoleacetic acid (IAA), a GABA_A agonist but a GABA_C antagonist, is not known. In the periphery, IAA derives from oxidation of histamine. But in brain, histamine is thought to be metabolized solely by histamine methyltransferase (HMT), forming tele -methylhistamine (t-MH) and tele -methylimidazoleacetic acid (t-MIAA). We showed that [³H]histamine (intracerebroventricularly) could be converted to IAA in brains of rats, a process increased by inhibition of HMT. This demonstrated that brain can oxidize histamine and suggested that endogenous histamine might also be oxidized if HMT activity were reduced. We examined, in rat cerebral cortex, effects of the following HMT inhibitors (mg/kg i.p.): metoprine (10), tacrine (10), velnacrine (10, 30), and physostigmine (1, 2). Tacrine was a potent inhibitor ( K _i∼ 22 n M ). To measure histamine in tissue that contained HMT inhibitors, we developed a gas chromatography-mass spectrometry method. After 2 h, all drugs reduced endogenous levels of t-MH and t-MIAA and increased levels of histamine and IAA. Our results show that inhibition of HMT promotes oxidation of histamine in brain, probably by shunting histamine to an alternative metabolic pathway. Formation of IAA provides a novel interaction between histaminergic and GABAergic systems in brain. Accumulation of IAA should be considered when inhibitors of HMT are used to probe brain histamine function.     

Pathogenesis of lupus dermatoses in autoimmune mice. X. Evaluation of histamine-N-methyltransferase activity in the skin of autoimmune

We measured histamine concentration and its metabolizing enzymes in the skin of MRL/Mp-lpr/lpr (MRL/l) and BXSB mice to clarify the contribution of histamine metabolism to the mechanisms of the development of lupus dermatoses. The concentration of histamine seemed to differ with the mouse strain. The activity of histamine-N-methyltransferase (HMT), one of two major metabolizing enzymes, was significantly lower in the tail and back skin of MRL/l mice at the age of 5 months than in the control MRL/Mp-+/+(MRL/n) mice, although there were no characteristic differences among several mouse strains of 1 mo of age. In the back skin of MRL/l mice, an age-dependent decrease of HMT activity was observed along with a corresponding decrease in histamine concentration, whereas an age-dependent increase of both HMT activity and histamine concentration was demonstrated in BXSB mice and other control mouse strains. Autoimmune-prone male BXSB mice and non-autoimmune female BXSB mice at 5 mo of age showed similar HMT activity. Corticosteroid treatment restored HMT activity in the skin of MRL/l mice but not in MRL/n mice. In addition, the change in HMT activity in MRL/l mice treated with corticosteroid appeared earlier than changes in clinicopathological examinations including skin eruptions, dermatopathology and proteinuria. Diamine oxidase (DAO) activity, another major metabolizing enzyme, was not detected in the skin of any autoimmune or control mouse strains. These findings suggest that the low activity of HMT in the skin of MRL/l mice plays a significant pathological role in the development of spontaneous lupus-like eruption. In other mouse strains, it is assumed that HMT activity is regulated by genetic factors.     

Rat and mouse brain histamine N-methyltransferase: modulation by methylated indoleamines

A purification procedure for rat and mouse brain histamine N-methyltransferase (HMT, EC 2.1.1.8) is described which achieves the preparation of 87-fold purified rat brain and 166-fold purified mouse brain enzyme. The purified HMT (MW 29,000) is inhibited by a number of physiologically and pharmacologically active amines, among them several methylated indoleamines, at concentrations above 5 ± 10^-6M. At concentrations below 1 ± 10^-7M, most of the methylated indoleamines stimulate HMT , provided histamine is maintained at, or close to, its optimal concentration as an HMT substrate, namely 1 ± 10^-5M. A study of the nature of the inhibitory process revealed a non-competitive inhibition of HMT by dopamine as against a competitive inhibition of the enzyme by most methylated indoleamines. Increasing the concentration of histamine beyond the optimal value, i.e. to inhibitory levels, resulted in less stimulation. The findings support the notion that MSO elicits the formation in selected brain cells of supranormal amounts of several methylated indoleamines which are able to stimulate HMT (and possibly other methyltransferases, see Salas et al., 1977), thereby causing the depletion of the cerebral levels of S-adenosyl-L-methionine, reported previously (Schatz & Sellinger , 1975b).     

Enzymatic histamine catabolism in vertebrate ontogenesis. A comparative study

1. The synthesizing and degrading activities of histamine were determined in the liver and small intestine of developing guinea pig and chick embryos. 2. Though increasing with age, HDC values were always 2-3 orders of magnitude lower than those of degrading enzymes. 3. DAO activity on the other hand was 10-100 fold higher than HMT at all ages studied, suggesting a decisive role for oxidative deamination in control of tissue histamine levels. 4. Generally histamine levels were higher in tissues of developing guinea pig than chick embryo, however, in the laying hen intestine histamine concentration was approximately 5 times greater than in the adult guinea pig intestine.     

Purification of a histamine H3 receptor negatively coupled to phosphoinositide turnover in the human gastric cell line HGT1.

The histamine H3 receptor agonist (R)alpha-methylhistamine (MeHA) inhibited, in a nanomolar range, basal and carbachol-stimulated inositol phosphate formation in the human gastric tumoral cell line HGT1-clone 6. The inhibition was reversed by micromolar concentrations of the histamine H3 receptor antagonist thioperamide and was sensitive to cholera or pertussis toxin treatment. Using [3H]N alpha-MeHA as specific tracer, high affinity binding sites were demonstrated with a Bmax of 54 +/- 3 fmol/mg of protein and a KD of either 0.61 +/- 0.04 or 2.2 +/- 0.4 nM, in the absence or presence of 50 microM GTP[gamma]S, respectively. The binding sites were solubilized by Triton X-100 and prepurified by gel chromatography. They were separated from the histamine H2 receptor sites by filtration through Sepharose-famotidine and finally retained on Sepharose-thioperamide. The purified sites concentrated in one single silver-stained protein band of 70 kDa in SDS-polyacrylamide gel electrophoresis. They specifically bound [3H]N alpha-MeHA with a KD of 1.6 +/- 0.1 nM and a Bmax of 12,000 +/- 750 pmol/mg of protein. This corresponds to a 90,225-fold purification over cell lysate and a purity degree of 84%. Binding was competitively displaced by N alpha-MeHA (IC50 = 5.8 +/- 0.7 nM), (R) alpha-MeHA (IC50 = 9 +/- 1 nM), and thioperamide (IC50 = 85 +/- 10 nM), but not by famotidine (H2 antagonist) or by mepyramine (H1 antagonist). These findings provide the first evidence for solubilization, purification, and molecular mass characterization of the histamine H3 receptor protein and for the negative coupling of this receptor phosphatidylinositol turnover through a so far unidentified G protein.     

Histamine N-methyltransferase Modulates Human Bronchial Smooth Muscle Contraction

To elucidate the modulatory role of histamine-degrading enzymes in airway constrictor responses, human bronchial strips were studied under isometric conditions in vitro. Pretreatment of tissues with the histamine N-methyltransferase (HMT) inhibitor SKF 91488 specifically potentiated the contractile responses to histamine, causing a leftward displacement of the concentration response curves, whereas the diamine oxidase inhibitor aminoguanidine had no effect. This potentiation was attenuated by mechanical removal of the epithelium. The HMT activity was detected in the human bronchi, which was less in the epithelium-denuded tissues than the epithelium-intact tissues. These results suggest that HMT localized to the airway epithelium may play a protective role against histamine-mediated bronchoconstriction in humans.     

Differences in the methylation of brain histamine in vivo between audiogenic seizure-sensitive and -resistant deermice

We have investigated the catabolism of [³H] histamine (HA), after intraventricular (i.vt.) administration, in brains of the audiogenic seizure susceptible (SS) and resistant (SR) deermouse $\text{Peromyscus}$. Brains of SS mice had lower endogenous HA levels and contained less [³H]-HA 20, 60 and 300 sec after i.vt. [³H]-HA than did brains of SR deermice. Twenty sec after [³H]-HA, brain [³H] methylhistamine (MeHA) levels and the resulting MeHA conversion index were found to be increased in the SS animals while later, at 60 and 300 sec, these parameters were found to be decreased. There were no SS-SR differences in the levels of brain [³H] methylimidazoleacetic acid. The data indicate that SS deermice catabolize exogenous HA, at least initially, more rapidly than their SR counterparts, confirming a like result noted immediately prior to seizure activity elicited by the administration of L-methionine-dl-sulfoximine in $\text{Mus}$.     

Membrane-bound histamine N-methyltransferase in mouse brain: possible role in the synaptic inactivation of neuronal histamine

In the CNS, histamine is a neurotransmitter that is inactivated by histamine N-methyltransferase (HNMT), a soluble enzyme localized to the cytosol of neurons and endothelial cells. However, it has not been established how extracellular histamine, a charged molecule at physiological pH, reaches intracellular HNMT. Present studies investigated two potential routes of histamine inactivation in mouse brain nerve terminal fractions (synaptosomes): (i) histamine uptake and (ii) histamine metabolism by HNMT. Intact synaptosomes demonstrated a weak temperature-dependent histamine uptake (0.098 pmol/min-mg protein), but contained a much greater capacity to metabolize histamine by HNMT (1.4 pmol/min-mg protein). Determination of the distribution of HNMT within synaptosomes revealed that synaptosomal membranes (devoid of soluble HNMT) contribute HNMT activity equivalent to intact synaptosomes (14.3 +/- 2.2 and 18.2 +/- 4.3 pmol/min-tube, respectively) and suggested that histamine-methylating activity is associated with the membrane fraction. Additional experimental findings that support this hypothesis include: (i) the histamine metabolite tele-methylhistamine (tMH) was found exclusively in the supernatant fraction following an HNMT assay with intact synaptosomes; (ii) the membrane-bound HNMT activity was shown to increase 6.5-fold upon the solubilization of the membranes with 0.1% Triton X-100; and (iii) HNMT activity from the S2 fraction, ruptured synaptosomes, and synaptosomal membranes displayed different stability profiles when stored over 23 days at - 20 degrees C. Taken together, these studies demonstrate functional evidence for the existence of membrane-bound HNMT. Although molecular studies have not yet identified the nature of this activity, the present work suggests that levels of biologically active histamine may be controlled by an extracellular process.     

THE ELEVATION OF CEREBRAL HISTAMINE-N-AND CATECHOL-O-METHYL TRANSFERASE ACTIVITIES BY l-METHIONINE-dl- SULFOXIMINE1

Abstract— The administration of the convulsant, l -methionine-dl-sulfoximine (MSO), increased histamine N-methyl transferase (E.C. 2.1.1.8) (HMT) activity in rat and mouse brain and, to a lesser extent, catechol-O-methyl transferase (E.C. 2.1.1.6) (COMT) activity in rat brain. The duration of this effect was shortened by co-administration of l -methionine. The increased HMT activity was seen in 5 or 7 rat brain regions tested. l -Methionine administration had no effect on the activity of either enzyme. Partially purified HMT preparations from rat or guinea-pig brain exhibited no alterations in activity after the in vitro addition of MSO or l -methionine over a wide range of histamine and S-adenosyl-l -methionine concentrations. Rat brain COMT was equally unaffected by MSO and l -methionine. The in vitro inhibition of HMT and COMT by S-adenosyl-l -homocysteine was the same whether tested on preparations derived from MSO-treated or control animals. The data are discussed with respect to the possible involvement of aberrant methylation processes in the MSO-induced seizure.     

Disposition of Histamine, Its Metabolites, and pros-Methylimidazoleacetic Acid in Brain Regions of Rats Chronically Infused with α-Fluoromethylhistidine

Abstract: In mammalian brain, histamine is known to be metabolized solely by histamine methyltransferase (HMT), forming tele -methylhistamine (t-MH), then tele -methylimidazoleacetic acid (t-MIAA). We previously showed that imidazoleacetic acid (IAA), a GABA agonist, and histamine's metabolite in the periphery, is present in brain where its concentration increased after inhibition of HMT. Also, when [³H]histamine was given intracerebroventricularly to rats, a portion was converted to IAA, a process increased by inhibition of HMT. These results indicated that brain has the capacity to oxidize histamine but did not show whether this pathway is operative under physiological conditions. To address this question, rats were infused for >4 weeks with α-fluoromethylhistidine (α-FMHis), an irreversible inhibitor of histamine's synthetic enzyme, l -histidine decarboxylase. Compared with controls (untreated and saline-treated rats), brain levels of histamine, t-MH, and t-MIAA in all regions were markedly reduced in treated rats. As a percentage of controls, depletion of t-MIAA > t-MH > histamine in all regions, and regional depletions of histamine corresponded to its turnover rates in regions of rat brain. In contrast, levels of IAA were unchanged as were levels of pros -methylimidazoleacetic acid, an isomer of t-MIAA unrelated to histamine metabolism. Results suggest that in brains of rats, unlike in the periphery, most IAA may not normally derive from histamine. Because histamine in brain can be converted to IAA under certain conditions, direct oxidation of histamine may be a conditional phenomenon. Our results also support the existence of a very slow turnover pool of brain histamine and use of chronic α-FMHis infusion as a model to probe the histaminergic system in brain.     

Kinetic analysis of the histamine N-methyltransferase reaction as used in the histamine radioenzymatic assay: Optimization of assay specificity

The specificity of the histamine N-methyltransferase (HNMT) based radioenzymatic assay for histamine has been questioned since N-α-methylhistamine is also a substrate for this enzyme. Purification of HNMT for use in the radioenzymatic assay improves sensitivity and specificity of this procedure. In this investigation, further improvements in specificity, with respect to other HNMT substrates, were attained by optimization of reaction conditions based on the evaluation of HNMT kinetic parameters. These studies demonstrate that appropriate control of reaction temperature and concentration of both the enzyme and the radiolabeled methyl donor improve the specificity of this assay for histamine.     

Rat kidney histamine N-methyltransferase: purification and partial characterization

Histamine N-methyltransferase (HMT, EC 2.1.1.8) was purified 8,420-fold in 44% yield from rat kidney. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE cellulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous as determined by gel electrophoresis and was stable for at least five months at -80 degrees C. The apparent molecular weight of the enzyme was found to be 31,500 as determined by gel filtration through Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was determined to be 5.4. The Km's for histamine and S-adenosyl-L-methionine were 12.4 +/- 1.3 microM and 10.2 +/- 0.5 microM, respectively. When S-adenosyl-L-methionine was the variable substrate, the Ki's for S-adenosyl-L-homocysteine and S-adenosyl-D-homocysteine were 31.9 +/- 3.4 microM and 32.0 +/- 3.5 microM, respectively. When histamine was the variable substrate, the Ki for S-adenosyl-L-homocysteine was 11.8 +/- 0.6 microM. Comparison of physico-chemical and catalytic properties of the rat kidney and the guinea pig enzymes suggest that these proteins have similar structural and catalytic characteristics.     

Purification and characterization of histamine dehydrogenase from Nocardioides simplex IFO 12069

Histamine dehydrogenase from Nocardioides simplex IFO 12069 was purified to homogeneity. The enzyme had a molecular mass of 170 kDa and was suggested to be a dimer of subunits that had a molecular mass of 84 kDa. The enzyme showed highest activity toward histamine and produced ammonia in its oxidative deamination to imidazole acetaldehyde. The K(m) and V(max) values for histamine were 0.075 mM and 4.76 micromol min(-1) mg(-1), respectively. The enzyme was sensitive to the carbonyl reagent iproniazid and a structurally similar compound, tryptophan. The enzyme showed absorption maxima at 442 and 280 nm. Reduction with histamine under anaerobic conditions resulted in a different absorption maximum at 360 nm instead of 442 nm. The enzyme was most active at pH 8.5 in Tris-HCl buffer and most stable at pH 7.0 in potassium phosphate buffer. The E(1%) value of the enzyme was 8.6 at 280 nm.     

No direct involvement of plasma membrane divalent cation-activated adenosine triphosphatase in histamine release from rat mast cells

Showdomycin, a very slowly penetrating SH reagent, hardly affected the histamine release induced by any of secretagogues tested, suggesting no exposure of sulfhydryl groups involved in the granule secretion process on the cell surface. N-ethylmaleimide(NEM), a considerably penetrating SH reagent, almost completely inhibited histamine release induced by secretagogues such as compound 48/80, polymyxin B, concanavalin A or digitonin at 100 microM and by A23187 at 500 microM. However, (Ca2+-Mg2+)-ATPase activity was hardly inhibited by NEM modification at 500 microM. These findings suggest that plasma membrane divalent cation-activated ATPase is not involved directly in the granule secretion process of mast cells.     

Action of histamine on the rapidly adapting airway receptors in the dog

The effects of histamine on the activity of rapidly adapting receptors (RAR) of the airways were investigated in anesthetized dogs. With bolus injections given into the right atrium, the threshold dose of histamine required for the excitation of RAR (n = 7) was 0.82 microgram/kg (+1.33/-0.51, geometric mean). With increasing doses of histamine, a dose-response relationship was seen in the activity of RAR. Obstruction of the lymphatic drainage from the lungs reduced the threshold dose to histamine (i.e., shifted the dose-response curve to the left significantly). This change in the dose-response relationship was not accompanied by a corresponding change in the relationship of histamine dose to airway pressures recorded before and after lymphatic obstruction. Against a background of pulmonary venous congestion produced by partial obstruction of the mitral valve, subthreshold doses of histamine stimulated the RAR (n = 4). The excitatory effect of histamine on RAR was found to be abolished by the administration of the H1 receptor antagonist diphenhydramine but not by the H2 receptor antagonist cimetidine. Intravenous infusion of histamine (0.4 microgram.kg-1.min-1) for a period of 10 min increased the RAR activity (n = 6) significantly without producing detectable changes in airway mechanics. The results indicate that contraction of the smooth muscle of the airways may not be a prerequisite for the excitation of RAR, especially at low doses. It is suggested that some of the effects of histamine on RAR are mediated by a local expansion of the extravascular fluid caused by an increase in the permeability of the bronchial vasculature.     

Ischemia of rat stomach mobilizes ECL cell histamine

Microdialysis was used to study how ischemia-evoked gastric mucosal injury affects rat stomach histamine, which resides in enterochromaffin-like (ECL) cells and mast cells. A microdialysis probe was inserted into the gastric submucosa, and the celiac artery was clamped (30 min), followed by removal of the clamp. Microdialysate histamine was determined by enzyme-linked immunosorbent assay. In addition, we studied the long-term effects of ischemia on the oxyntic mucosal histidine decarboxylase activity in omeprazole-treated rats. Gastric mucosal lesions induced by the ischemia were enlarged on removal of the clamp. The microdialysate histamine concentration increased immediately on clamping (50-fold rise within 30 min) and declined promptly after the clamp was removed. In contrast, histidine decarboxylase activity of the ECL cells was lowered by the ischemia and returned to preischemic values 9 days later. Mast cell-deficient rats responded to ischemia-reperfusion much like wild-type rats with respect to histamine mobilization. Pretreatment with the irreversible inhibitor of histidine decarboxylase, alpha-fluoromethylhistidine, which is known to eliminate histamine from ECL cells, prevented the rise in microdialysate histamine. Pharmacological blockade of acid secretion (cimetidine or omeprazole) prevented the lesions induced by ischemia-reperfusion insult but not the mobilization of histamine. In conclusion, ischemia of the celiac artery mobilizes large amounts of histamine from ECL cells, which occurs independently of the gross mucosal lesions. The prompt reduction of the mucosal histidine decarboxylase activity in response to ischemia probably reflects ECL cell damage. The lesions develop not because of mobilization of histamine per se but because of ischemia plus reperfusion plus gastric acid.