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.     

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.     

Base-line and postthermal injury plasma histamine in rats

Although plasma histamine concentration has been reported to increase after thermal injury in the rat to as much as 100-fold over normal human plasma levels, the pathophysiological significance and relevance to human disease is questionable. Lack of confidence in the rat as a model of histamine-mediated disease is based on reports that normal rat base-line plasma histamine concentration exceeds that of human plasma by 20- to 70-fold. The present study confirms that high concentrations of histamine (20-68.9 ng/ml) are found in rat plasma obtained in an uncontrolled manner; but concentrations are lower (1.17 +/- 0.49 ng/ml) or undetectable in a sensitive radioenzymatic assay when sampling technique and plasma isolation are controlled. The primary cause for falsely elevated values for plasma histamine concentration appeared to be due to manipulation of the rat. Plasma histamine concentration increased within 1 min after thermal injury and the increase was proportional to extent of surface area injured. In contrast to the finding of a single time-related peak of plasma histamine concentration after partial-thickness burn, a biphasic elevation was found after full-thickness injury. Thus the data indicate that normal rat plasma histamine concentration is similar to that of the human and below the reported threshold for modulation of a variety of immune responses. Furthermore, the data support a role for histamine and other mast-cell mediators in the local and systemic responses to injury.     

Analysis of histamine and N tau-methylhistamine in plasma by gas chromatography-negative ion-chemical ionization mass spectrometry

Current methods of quantitation of histamine and its major metabolite N tau-methylhistamine are inaccurate and insensitive to the very low concentrations that exist in plasma samples. Therefore, an accurate and sensitive method for quantification in plasma has been developed using the stable isotope dilution assay with negative ion-chemical ionization mass spectrometry. For histamine, after the addition of [2H4]histamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH2C6F5)3-histamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z 430/434. For N tau-methylhistamine, after the addition of N tau-[2H3]methylhistamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the heptafluorobutyryl derivative (C3F7CO2)2-N tau-methylhistamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z 497/500. The precision of the histamine assay is 3.1% and the accuracy is 95.5 +/- 2.5% while the precision of the N tau-methylhistamine assay is 1.9% and the accuracy is 106.8 +/- 1.9%. The lower limits of sensitivity are 1 pg for histamine and 6 pg for N tau-methylhistamine injected on column. Using the assay in three normal human volunteers, plasma concentrations of histamine were 130, 92, and 85 pg/ml, and of N tau-methylhistamine were 229, 228, and 216 pg/ml. This assay provides a very sensitive and accurate method of quantitation of histamine and N tau-methylhistamine in plasma samples.     

Two polymorphic forms of human histamine methyltransferase: structural, thermal, and kinetic comparisons

BACKGROUND: Histamine plays important biological roles in cell-to-cell communication; it is a mediator in allergic responses, a regulator of gastric acid secretion, a messenger in bronchial asthma, and a neurotransmitter in the central nervous system. Histamine acts by binding to histamine receptors, and its local action is terminated primarily by methylation. Human histamine N-methyltransferase (HNMT) has a common polymorphism at residue 105 that correlates with the high- (Thr) and low- (Ile) activity phenotypes. RESULTS: Two ternary structures of human HNMT have been determined: the Thr105 variant complexed with its substrate histamine and reaction product AdoHcy and the Ile105 variant complexed with an inhibitor (quinacrine) and AdoHcy. Our steady-state kinetic data indicate that the recombinant Ile105 variant shows 1.8- and 1.3-fold increases in the apparent K(M) for AdoMet and histamine, respectively, and slightly (16%) but consistently lower specific activity as compared to that of the Thr105 variant. These differences hold over a temperature range of 25 degrees C-45 degrees C in vitro. Only at a temperature of 50 degrees C or higher is the Ile105 variant more thermolabile than the Thr105 enzyme. CONCLUSIONS: HNMT has a 2 domain structure including a consensus AdoMet binding domain, where the residue 105 is located on the surface, consistent with the kinetic data that the polymorphism does not affect overall protein stability at physiological temperatures but lowers K(M) values for AdoMet and histamine. The interactions between HNMT and quinacrine provide the first structural insights into a large group of pharmacologic HNMT inhibitors and their mechanisms of inhibition.     

A sensitive and specific radiometric method for the measurement of plasma histamine in normal individuals

A radioenzymatic method for assaying histamine using histamine-N-methyltransferase from guinea pig brain was modified to increase its sensitivity, precision, and specificity. This was achieved by incorporation of a thin-layer chromatography step and use of N_α-methyl histamine as an internal standard in each sample. No prior extraction of the plasma samples is required, permitting the assay of 30 samples in 1 working day. Histamine was detected in the plasma of 17 normal volunteers, at a level of 3.4 ± 0.69 nmol/liter (range 0.82 to 4.7 nmol/liter). In 19 asthmatics, a low-peak expiratory flow rate was found to be associated with a plasma histamine concentration above this “normal” range.     

Increased sensitivity of the enzymatic isotopic assay of histamine: measurement of histamine in plasma and serum.

The use of rat kidney instead of guinea pig brain as the source of histamine-N-methyltransferase for the enzymatic assay of histamine was found to improve the sensitivity of the assay. A partially purified preparation (ammonium sulfate fractionation) of the kidney enzyme was 20- to 50-fold more active than the guinea pig preparation, and sufficient enzyme for 14,000 assays could be prepared from six rats. The kidney enzyme, unlike the guinea pig brain enzyme, was free of interfering enzyme activities and gave low values for assay blanks. The two enzymes otherwise had similar properties. The low blank values permitted direct measurement of histamine in normal plasma without the need to isolate and concentrate histamine from the sample. Plasma histamine levels in normal individuals ranged from 0.2-1.4 (mean 0.6, n = 19) ng/ml.     

Purification and kinetic properties of ox brain histamine N-methyltransferase.

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.     

Characterization of a new mRNA species from the human histamine N-methyltransferase gene

Histamine N-methyltransferase (HNMT), a cytosolic histamine-metabolizing enzyme, is the only known product of the 50-kb human HNMT. Here, a detailed investigation of HNMT products revealed the existence of a new brain mRNA product of HNMT. This species, named HNMT-Short (HNMT-S), encodes a 126-amino-acid protein. Northern blot analysis detected HNMT-S mRNA (1.0 kb) in placenta, but not in several other human tissues. In addition, unlike the known HNMT cDNA, HNMT-S cDNA did not result in histamine-methylating activity after transfection into COS-7 cells. These studies show that HNMT-S is a new mRNA species and putative protein product from HNMT. The physiological role of HNMT-S remains to be investigated.     

Ontogenesis of histamine in the chick nervous system

Tissues from the central and peripheral nervous systems of the chick were analyzed for concentration of histamine (Hm) during development. Of the three CNS organs examined, cerebral hemispheres had the highest Hm content. Expressed on the bases of wet weight, protein, and DNA concentrations, sciatic nerve and the pineal gland had the highest levels of this biogenic amine of the five tissues investigated. The concentration of Hm was higher in the cerebellum, cerebral hemispheres, and thalamus of adult animals than in the 15 to 17-day-old embryos. The level of Hm rose markedly in the sciatic nerve and pineal gland after the 15th day of embryonic development. These data might indicate a possible involvement of Hm in controlling the course of maturation of certain organs in the nervous system.     

Aplysamine-1 and related analogs as histamine H3 receptor antagonists

Aplysamine-1 (1), a marine natural product, was synthesized and screened for in vitro activity at the human and rat histamine H3 receptors. Aplysamine-1 (1) was found to possess a high binding affinity for the human H3 receptor (Ki = 30+/-4 nM). Synthetic analogs of 1, including des-bromoaplysamine-1 (10) and dimethyl-{2-[4-(3-piperidin-1-yl-propoxy)-phenyl]-ethyl}-amine (13), were potent H3 antagonists.     

Low density lipoprotein (LDL) inhibits histamine release from human mast cells

We examined the effect of low density lipoprotein (LDL) on histamine release from purified human lung mast cells. LDL inhibited anti-IgE- induced histamine release in a dose-dependent manner, with 100 micrograms/ml LDL-protein inhibiting histamine release by 53 +/- 8% (mean +/- SEM); half-maximal inhibition occurred at 40-80 micrograms/ml. LDL also inhibited calcium ionophore A23187-induced histamine release in a dose-dependent manner, with 1 mg/ml of LDL inhibiting histamine release by 83 +/- 9%; half maximal inhibition occurred at 220-280 micrograms/ml. Inhibition by LDL was time-dependent: half-maximal inhibition of anti-IgE- induced histamine release by LDL occurred at 30-50 minutes of incubation. The inhibitory effect of LDL was independent of buffer calcium concentrations (0-5 mM) or temperature (0-37 degrees C). These data are consistent with a newly defined immunoregulatory role for LDL.     

Measurement of plasma histamine by stable isotope dilution gas chromatography-mass spectrometry: methodology and normal values

A new method for the determination of histamine by stable isotope dilution mass fragmentography is described. The method is specific, sensitive, and accurate, resulting in a within-day coefficient of variation of 4.1% and a day-to-day variation of 7.9%. It was shown that the first blood sample after a venipuncture can contain an artificially elevated plasma histamine concentration. Platelets contain about 7 pmol histamine/10(9) cells. Serum histamine was elevated about four times in comparison with plasma histamine. This phenomenon was mainly ascribed to degranulation of basophilic leukocytes by complement activation during blood clotting. Normal values for plasma histamine were (n = 25) 2.07 +/- 0.75 nmol/liter (mean +/- 1 SD), which is one of the lowest values reported up to now.     

Radioimmunoassay of secretin in plasma.

A sensitive, specific and reproducible radioimmunoassay for secretin is described. Antibodies were readily produced against low microgram quantities of synthetic secretin. The secretin antibodies did not cross-react with the structurally similar G.I.P., V.I.P., or glucagon. Synthetic secretin was iodinated using Chloramine "T" and purified by a two-state procedure incorporating gel filtration and radient elution from a cation exchange column. Plasma samples were found to produce variable interference in the assay necessitating the incorporation of secretin-free "blands" for each patient's plasma. Production of secretin-free plasma was by incubation of plasma samples at 37 degrees C for 96 hours. The sensitivity of the assay was 12.5-25 pg/ml. Normal fasting secretin levels were 21 +/- S.E. 7 pg/ml. A mean rise in plasma secretin to 220 pg/ml was observed after intraduodenal acidification.     

Characterization of histamine H1 binding sites on human T lymphocytes by means of 125I-iodobolpyramine. Preferential expression of H1 receptors on CD8 T lymphocytes

The presence of histamine H1 receptors on lymphocytes has been indirectly suggested by the various effects of agonists or antagonists on the functionally distinct T lymphocyte subsets. Recently, a new H1 antagonist, 125I-iodobolpyramine, whose structure is similar to mepyramine, has become available for the detection of H1 receptors in guinea pig brain. When using 125I-iodobolpyramine on human T lymphocytes, the presence of a single highly specific H1 binding site was evidenced. The binding of 125I-iodobolpyramine to human T cells was reversible when using 1000-fold excess of the cold H1 antagonist, d-chlorpheniramine. Binding saturation was achieved at 0.60-0.65 nM of 125I-iodobolpyramine, the binding equilibrium was reached in 20-30 min at 27 degrees C. The dissociation constant was KD = 0.41 +/- 0.07 (mean +/- SE) and the number of receptors per T cell was 3407 +/- 592 (mean +/- SE) as deduced from saturation and kinetic curves. In competition experiments using a panel of H1 ligands, the T cell binding sites detected by 125I-iodobolpyramine showed a pharmacological behavior characteristic of histamine H1 receptors. It was of particular interest that 125I-iodobolpyramine binding displayed clearcut stereoselectivity as assessed by the higher affinity of the d-configuration of chlorpheniramine than the l form. Study of purified CD4 and CD8 T cells showed that twice as much H1 histamine receptors were expressed by CD8 T lymphocytes (6615 +/- 1125) as compared to CD4 T cells (3545 +/- 459). These results underline the need for studying the functional properties of such pharmacologically defined T lymphocyte H1 binding sites.     

Validation of the internal reference technique for microdialysis measurements of interstitial histamine in the rat

The internal reference technique (IRT) was compared with the no net flux method (NFM) as a microdialysis calibration technique for sampling of interstitial histamine in the rat. Microdialysis catheters (polyacrylonitrile, 50 kD cut off) were inserted in liver, muscle, subcutaneous tissue and in an induced adenocarcinoma. Estimated relative recovery with IRT ranged from 23+/-2% in liver to 30+/-3% in subcutaneous tissue with and without tumor (p<0.05). By using the NFM-technique we found similar recovery as compared to the IRT in all tissues studied. Interstitial histamine was up to 3-fold higher than the mean plasma histamine concentration (54+/-2 nmol/l). Subcutaneous tissue (177+/-39 nmol/l) and subcutaneous tumor (165+/-29 nmol/l) exhibited high histamine while liver (65+/-14 nmol/l) and liver tumor (75+/-7 nmol/l) had low interstitial histamine concentrations. In conclusion, the IRT was validated against the NFM as a rapid method for histamine measurements in situ in the rat.     

Allergen induced histamine release and immunoreactive-leukotriene C4 generation from leukocytes in mite sensitive asthmatic patients

Leukocytes from mite sensitive asthmatic patient were challenged with the allergen and the supernatant was assayed for histamine and immunoreactive-leukotriene C4 (i-LTC4). The release of histamine was quantitated by an automated fluorometric technique and i-LTC4 was determined using a commercial radioimmunoassay kit. The results of analysis of the supernatant by high speed liquid chromatography, together with observations of modulation of the formation by agents, indicated that i-LTC4 consisted of LTC4 with a little amount of LTD4. i-LTC4 was generated as a result of basophil activation but not derived from the other cells such as monocytes and eosinophils. Allergen induced a concentration-dependent release of histamine and i-LTC4 and the maximal release of histamine and i-LTC4 occurred at the same dose of the allergen. At optimal concentration of the allergen, basophils produced 20.4 +/- 17.9 ng of i-LTC4/10(6)-cells (mean +/- S.D., n = 39) and histamine release was 55.6 +/- 20.1% of total histamine. There was a significant correlation in the capacity of leukocytes to release histamine and i-LTC4 (r = 0.47, p less than 0.01). We found a correlation between maximal histamine release or cell sensitivity, allergen concentration for 50% histamine release, and a ratio of specific IgE to mite to total IgE in the serum, but the amount of i-LTC4 failed to correlate significantly with the ratio. The releasability and the cell sensitivity of asthmatic patients' cells to the allergen for histamine release paralleled the severity to symptoms, but this correlation was not significant in i-LTC4 generation.     

A more sensitive and specific radioenzymatic assay for catecholamines

This modification of the catechol-O-methyltransferase (COMT) based radioenzymatic assay for norepinephrine (NE) and epinephrine (E) improves sensitivity, selectivity and eliminates many inhibitors of COMT. Prior to assay, samples are extracted into heptane with diphenylborate, then into dilute acetic acid. This extraction procedure has an efficiency of 78% for NE but less than 2% for S-adenosylmethionine (SAM). The extraction procedure also excludes calcium and other COMT inhibitors present in urine, plasma and every tissue tested. This eliminates the requirement for individual standardization of tissue and urine samples. Sensitivity of the assay for NE and E is 10 and 6 pg/ml respectively in 1 ml of plasma. The intraassay coefficients of variation for NE and E are 4 and 13% and the interassay coefficients of variation for NE and E are 10 and 16% in a human plasma sample containing low catecholamine levels. The assay permits quantitation of plasma E levels that were undetectable in prior assays.     

Theoretical 3D model of histamine N-methyltransferase: insights into the effects of a genetic polymorphism on enzymatic activity and thermal stability

Histamine N-methyltransferase (HNMT) catalyzes the N-methylation of histamine in mammals. The experimentally determined HNMT three-dimensional (3D) structure is not available. However, there is a common genetic polymorphism for human HNMT (Thr105Ile) that reduces enzymatic activity and is a risk factor for asthma. To obtain insights into mechanisms responsible for the effects of that polymorphism on enzymatic activity and thermal stability, we predicted the 3D structure of HNMT using the threading method and molecular dynamics simulations in water. Herein, we report a theoretical 3D model of human HNMT which reveals that polymorphic residue Thr105Ile is located in the turn between a beta strand and an alpha helix on the protein surface away from the active site of HNMT. Ile105 energetically destabilizes folded HNMT because of its low Chou-Fasman score for forming a turn conformation and the exposure of its hydrophobic side chain to aqueous solution. It thus promotes the formation of misfolded proteins that are prone to the clearance by proteasomes. This information explains, for the first time, how genetic polymorphisms can cause enhanced protein degradation and why the thermal stability of allozyme Ile105 is lower than that of Thr105. It also supports the hypothesis that the experimental observation of a significantly lower level of HNMT enzymatic activity for allozyme Ile105 than that with Thr105 is due to a decreased concentration of allozyme Ile105, but not an alternation of the active-site topology of HNMT caused by the difference at residue 105.     

An improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine N-methyltransferase

Radioenzymatic assays have been developed for norepinephrine (NE) using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for NE but have been considered less sensitive than the more complex assay procedures employing COMT. An improved purification procedure for bovine PNMT has permitted development of a NE assay with substantially improved sensitivity (less than 0.5 pg), reproducibility, and decreased manipulative effort. PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sephacryl S-200 and Phenyl Boronate-agarose. Recovery of PNMT activity through the purification scheme was 50% while blank recovery was less than 0.001%. Norepinephrine can be directly quantified in 25 microliters of human plasma and a seventy-tube assay can be routinely completed within 4 h. The capillary to venous plasma NE gradient was examined in eight normotensive male subjects. Capillary plasma NE (211 +/- 21.7 pg/ml) was significantly lower than venous plasma NE (367 +/- 32.7 pg/ml) in all subjects (p less than 0.005). This difference suggests the concentration of NE in capillary blood may be a unique indicator of sympathetic nervous system activity in vivo.