THE SUBCELLULAR LOCALIZATION OF HISTIDINE DECARBOXYLASE IN VARIOUS REGIONS OF RAT BRAIN

Abstract— The subcellular distribution of histidine decarboxylase (assayed by two different isotopic methods) and several biochemical markers (lactate dehydrogenase, DOPA decarboxylase and protein) was determined in rat cerebral cortex. After differential centrifugation, the enzyme activity was found mainly in the crude mitochondrial and soluble fractions. Further separation of the former on discontinuous sucrose gradients showed that the particulate histidine decarboxylase (HD) was found in the synaptosomal fraction. After osmotic shock, HD activity appeared in the supernatant fraction suggesting that a major portion of the enzyme is localized in the cytoplasm of cortical nerve endings. By analogy with other brain amines, this finding, together with the presence of histamine in synaptic vesicles (K ataoka and de R obertis , 1967), can be taken as further support for the hypothesis of a role as neurotransmitter for histamine.
Various brain regions were homogenized under conditions leading to synaptosome formation. The distribution of HD between 'particulate' and soluble fractions differed from one region to the other, but did not give any clear-cut indication of regions rich in cell bodies or nerve terminals.     

The subcellular localization of histamine and histamine methyltransferase in rat brain

Abstract— We have examined the subcellular localization of histamine and histamine methyl-transferase (S-adenosylmethionine: histamine 7V-methyltransferase; EC 2.1.1.8) in rat brain. The highest levels of histamine and histamine methyltransferase activity were found in the hypothalamus. A large proportion of hypothalamic histamine and histamine methyltransferase activity was found in particles with sedimentation properties in sucrose gradients similar to synaptosomes storing norepinephrine and serotonin. Histamine displayed a bimodal distribution in sucrose gradients. A substantial amount of a tracer dose of [³H]histamine added to hypothalamic homogenates at 4°C was bound to particulate fractions, suggesting that endogenous histamine may redistribute and bind to subcellular fractions during homogenization. The second, lighter peak of histamine in sucrose gradients was thought to be due to histamine that redistributed during homogenization.     

Dynamics of the regulation of histamine levels in mouse brain

Abstract— The intraperitoneal administration of L-histidine in a dose of 1000 mg/kg increased threefold the whole brain levels of histamine in the mouse. This increase was evident in all brain regions except the medulla oblongata-pons. The subcellular localization of histamine and histidine was the same in mice administered bhistidine as in salinetreated animals. Cold exposure and restraint further augmented the elevation of histamine elicited by histidine treatment. a-Hydrazino-histidine and 4-bromo-3-hydroxybenzyloxyamine (NSD-1055) but not a-methyl-DOPA inhibited histidine decarboxylase [EC 4.1.1.221 activity in mouse brain homogenates and prevented the increase in brain histamine after histidine administration. NSD-1055 and a-hydrazino-histidine also lowered brain levels of histamine by 50 per cent. NSD-1055 lowered whole brain levels of histamine rapidly, with a half-life for the depletable histamine pool of about 5 min. Assuming that inhibition of histidine decarboxylase accounted for the reduction in histamine, then the rate of histamine decline reflects the rate of histamine turnover, and our results suggest that a portion of mouse brain histamine turns over quite rapidly. Reserpine lowered brain levels of histamine by about 50 per cent, whereas the antihistaminic agent, dexbrompheniramine, and sodium pentobarbital elevated histamine levels.     

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.     

Isotopic microassay of histamine, histidine, histidine decarboxylase and histamine methyltransferase in brain tissue

Abstract— Microassays are described for histamine, histidine, and the activities of the enzymes histidine decarboxylase (EC 4.1.1.22) and histamine niethyltransferase (EC 2.1.1.8) in brain tissue. The enzymic-isotopic microassay for histamine is based on the methylation of tissue histamine by added histamine methyl-transferase and [¹⁴C]- or [³H]-labelled S-adenosyl-l -methionine. In a double-isotopic form of the assay, a tracer of [³H]histamine is employed along with [¹⁴C]S-adenosyl-l -methionine, and the ratio [¹⁴C]:[³H] reflects the amount of histamine in the sample. Because the methylation of histamine is uniform in brain samples studied, a single isotopic assay with [³H]S-adenosyl-l -methionine as the methyl donor is possible and increases sensitivity, so that 10 pg of tissue histamine can be estimated reliably. The assay for histidine involves decarboxylation of histidine by a bacterial histidine decarboxylase and measurement of the histamine formed by the enzymicisotopic procedure. In the histidine decarboxylase assay, histamine synthesized from added histidine is measured. The assay for histamine methyltransferase involves measuring the formation of [¹⁴C]methylhistamine with [¹⁴C]S-adenosyl-l -methionine serving as the methyl donor.     

Adenosine triphosphate:creatine phosphotransferase of rat cerebral cortex: evidence for a bimodal subcellular localization

—(1) ATP: creatine phosphotransferase of rat cerebral cortex is soluble to the extent of 57 per cent when the tissue is homogenized in 0.25 M-sucrose and 80 per cent when distilled water is used for tissue dispersion. Among particulate fractions, the crude mitochondria] fraction contains the highest percentage of enzyme activity. (2) Discontinuous sucrose gradient fractionation of the crude mitochondrial fraction yields about 55 per cent of the particulate activity in the nerve ending fractions and 24 per cent in the mitochondrial pellet. (3) Rupturing of the nerve-ending particles by a moderate osmotic shock designed to spare the mitochondria results in about 60 per cent of the ATP:creatine phosphotransferase becoming soluble, the remainder preserving the association with heavy particles, presumably mitochondria. (4) Subfractionation of the microsomal fraction on a discontinuous sucrose gradient reveals that this particulate component of the enzyme is an adsorption artifact. (5) The overall evidence points to at least two distinct subcellular localizations of the enzyme in rat brain cortex, a major soluble component and a particulate component. It has not been unequivocally shown whether the latter, in turn, reflects the presence of a single, mitochondrial component or whether the soluble matrix of the nerve ending particles represents a third locale for the enzyme.     

Quantitative analysis of histidine decarboxylase gene (hdcA) transcription and histamine production by Streptococcus thermophilus PRI60 under conditions relevant to cheese making

This study evaluated the influence of parameters relevant for cheese making on histamine formation by Streptococcus thermophilus. Strains possessing a histidine decarboxylase (hdcA) gene represented 6% of the dairy isolates screened. The most histaminogenic, S. thermophilus PRI60, exhibited in skim milk a high basal level of expression of hdcA, upregulation in the presence of free histidine and salt, and repression after thermization. HdcA activity persisted in cell extracts, indicating that histamine might accumulate after cell lysis in cheese.     

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.     

A cytochemical study on the pancreas of the guinea pig. I. Isolation and enzymatic activities of cell fractions

Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.     

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.     

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.     

Increased synthesis of phosphatidylserine decarboxylase in a strain of Escherichia coli bearing a hybrid plasmid. Altered association of enzyme with the membrane.

A strain of Escherichia coli bearing a hybrid plasmid containing the psd gene, starved for isoleucine by the addition of valine, produces amounts of phosphatidyl-serine decarboxylase, a membrane-bound enzyme, about 40-fold higher than wild type. At least 98% of the enzyme from cells with high levels of decarboxylase is isolated in the inner, cytoplasmic membrane fraction if the cells are broken by osmotic lysis of spheroplasts following treatment with lysozyme/EDTA. In contrast, if cells containing these large amounts of enzyme are disrupted by sonication, 40 to 45% of the activity is recovered in the 100,000 times g supernatant fraction, whereas with wild type cells, only 5 to 10% is recovered in this fraction. About half of the decarboxylase in membranes saturated with the enzyme is thus only loosely bound, and readily removed by sonication, but not by osmotic lysis. This apparent saturation of the membrane with decarboxylase seems specific, since two other membrane-bound enzymes, phosphatidyl-glycerophosphate synthetase, and CDP-diglyceride synthetase, are not displaced into the supernatant fraction upon sonication. Fractionation on columns of agarose and by centrifugation through gradients of sucrose revealed that the decarboxylase in the supernatant is associated with lipid, in a complex with an apparent molecular weight of at least 5 times 10(6).     

A Cytochemical Study on the Pancreas of the Guinea Pig : I. Isolation and Enzymatic Activities of Cell Fractions

Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.     

Histidine Decarboxylaseless Mutants of Lactobacillus 30a: Isolation and Growth Properties

Mutants of Lactobacillus 30a deficient in their ability to form an inducible histidine decarboxylase (EC 4.1.1.22) were selected by plating nitrosoguanidine-treated cultures on a medium containing histidine and methyl red. Wild-type organisms produce histamine, thus raising the pH and forming yellow colonies; mutant colonies remain red. In the presence of added histidine, decarboxylase-producing cultures grow more heavily than mutant cultures when the initial pH of the growth medium is low or when the lactic acid produced lowers the pH to growth-limiting values. Addition of the decarboxylation products, histamine and carbon dioxide, did not favor growth in crude medium.     

A rapid histidine decarboxylase assay

Histidine decarboxylase (EC 4.1.1.22) catalyzes the conversion of histidine to histamine. Because current assays for enzyme activity are time consuming and require additional enzymes or large amounts of tissue, a rapid radioisotopic assay was devised. Using commercially available radioactive histidine (without additional purification), the enzyme mediates the formation of histamine. The product is resolved from precursor by paper electrophoresis in a formic acid-acetic acid solution for 15 min. After drying and ninhydrin staining, radioactive histamine is measured by liquid seintillation spectrometry. This assay procedure is sensitive enough to measure decarboxylase activity in milligram quantities of rat brain.     

THE REGIONAL AND SUBCELLULAR DISTRIBUTION OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Catechol-O-methyl transferase (COMT) activities determined in different regions of rat brain showed small variations. Highest activities were found in the hypothalamus and corpora quadrigemina, and lowest activities in the hippocampus and corpus striatum. The regional distribution of COMT was thus at variance with the distribution of DOPA decar- boxylase in this study and with the distribution of catecholamines and tyrosine hydroxylase reported in the literature. Determinations of the subcellular distribution of COMT in rat forebrain showed that 50 per cent of the activity was recovered in the high speed supernatant fluid and about 33 per cent in the crude mitochondrial fraction. Further separation of the latter by discontinuous sucrose gradients showed that the particulate COMT was found in the synaptosomal fraction in an occluded form. Full enzyme activity was only obtained after treatment with a detergent or after resuspension in water. After hypo-osmotic rupture of the crude mitochondrial fraction, COMT was recovered in the cytoplasmic fraction. The subcellular distribution of COMT was very similar to the ones of lactate dehydrogenase and DOPA decarboxylase. The proportions of soluble COMT obtained from homogenates of various regions of the brain differed from that of choline acetyl transferase and DOPA decarboxylase but were similar to that of lactate dehydrogenase. In conclusion, COMT is a cytoplasmic enzyme almost evenly distributed in the CNS. Its distribution does not resemble the distributions of the catecholamines or of the enzymes participating in the synthesis of catecholamines.     

Histamine formation in rat brain in vivo: effects of histidine loads

Abstract— Administration of l -histidine at the rate of 500 mg/kg induced an increase of nearly 50 per cent in the level of histamine in rat brain which lasted several hours. The augmentation of histamine level was not significant 3 h after lower doses or after d -histidine α-methyl DOPA and Ro 4-4602 neither affected the cerebral level of histamine nor its elevation induced by l -histidine. Brocresine, a known histidine decarboxylase inhibitor not only prevented the effect of histidine load but also induced a prompt fall in the amine level. These results confirm those from earlier experiments in vitro indicating that histamine synthesis in rat brain depends on a specific decarboxylase (EC 4 , 1.1.22) which is not normally saturated by the endogenous level of its substrate. When histamine levels were enhanced by histidine treatment, histidine decarboxylase activity, as evaluated on hypothalamus homogenates, was significantly reduced; intracisternal administration of cycloheximide, an inhibitor of protein synthesis, had similar effects. On the other hand, enzyme activity was not altered by the addition of histamine to hypothalamus homogenates. These results are compatible with the existence of a regulation mechanism of histidine decarboxylase involving repression by its end-product.     

Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri.

Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histidine uptake, histamine efflux, and histidine/histamine exchange are electrogenic processes. Histidine/histamine exchange is much faster than the unidirectional fluxes of these substrates, is inhibited by an inside-negative delta psi and is stimulated by an inside positive delta psi. These data suggest that the generation of metabolic energy from histidine decarboxylation results from an electrogenic histidine/histamine exchange and indirect proton extrusion due to the combined action of the decarboxylase and carrier-mediated exchange. The abundance of amino acid decarboxylation reactions among bacteria suggests that this mechanism of metabolic energy generation and/or pH regulation is widespread.     

In vitro demonstration of histamine biosynthesis from carnosine by kidneys of pregnant mice

Kidneys of pregnant mice synthesize histamine when incubated in the presence of carnosine, manganese, and pyridoxal phosphate. Intensity of biosynthesis increases linearly with the amount of enzyme and the incubation time. The reaction can only be catalysed by two enzymes that are located in kidneys and act in succession: carnosinase, which hydrolyzes carnosine into its two moieties, and histidine decarboxylase, which transforms histidine, a product of carnosine degradation, into histamine. The biosynthesis of histamine from carnosine seems to increase with the progress of pregnancy. In nonpregnant mice, kidneys do not effect this biosynthesis. The above results directly demonstrate that carnosine may be used for histamine synthesis when the activity of histidine decarboxylase is high, as in pregnant mouse kidney. Vertebrate carnosine, its role still enigmatic, might thus be mainly a potential histidine reservoir that would be mobilized any time there is a significant requirement for histidine, such as for histamine biosynthesis.     

Identification and measurement of acid (specific) histidine decarboxylase activity in rabbit gastric mucosa: ending an old controversy?

One of the main obstacles in assigning any distinct function to histamine in health and disease was the longlasting controversy on the existence of any physiological, endogenous histamine formation in man and most of the other mammals except the rat. Using a modification of Schayer's isotope dilution method, a renewed attempt was made to identify the very low activities of an acid (specific) histidine decarboxylase in rabbit gastric mucosa capable of producing endogenous histamine in physiological conditions, to develop tests for its identification in crude enzyme extracts and to demonstrate the specificity of the enzymatic assay by excluding any relevant Dopa decarboxylase activity and also nonenzymatic decarboxylation interfering with the determination of acid (specific) histidine decarboxylase. To achieve this aim five tests were developed: In the pH profile (test 1), a pH optimum was found at 7.0 in the presence of a low substrate concentration (1.6 X 10(-6)M L-[ring-2-14C]-histidine). The apparent Michaelis concentration at the pH optimum (test 2) was 1.8 X 10(-4)M, the maximum rate 12.5pmol [14C]histamine formed X min-1. To increase the specificity of inhibition experiments with alpha-methylhistidine and alpha-methyl-L-Dopa a pH profile was determined in the presence of these two enzymatic inhibitors (test 3 and 4). alpha-Methylhistidine was used for a reliable diagnostic confirmation test, alpha-methyl-L-Dopa for a reliable exclusion test. Benzene showed no influence on either blanks or recovery rates, but inhibited the enzymic activity at pH 7.0, not however that of unspecific histidine decarboxylase and hence was very valuable as an additional diagnostic exclusion test (test 5). Although these new tests identifying acid (specific) histidine decarboxylase and demonstrating the specificity of its determination were tedious, despite the use of the modified isotope dilution method, they excluded the presence of any Dopa decarboxylase activity in mixtures with crude enzyme preparations as well as of any kind of nonspecific and nonenzymatic histidine decarboxylation. An endogenous histidine decarboxylase in rabbit gastric mucosa is postulated, capable of forming histamine in vivo.