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Both histamine and polyamines are important for maintaining basophilic cell function and viability. The synthesis of these biogenic amines is regulated by histidine decarboxylase and ornithine decarboxylase, respectively. In other mammalian tissues, an interplay between histamine and polyamine metabolisms has been suspected. In this report, the interplay between histamine and ornithine-derived polyamines was studied in a non-transformed mouse mast cell line (C57.1) treated with phorbol ester and dexamethasone, a treatment previously used to increase histidine decarboxylase expression in mastocytoma and basophilic leukemia. Treatment with phorbol ester and dexamethasone increased histidine decarboxylase expression and intracellular histamine levels in C57.1 mast cells to a greater extent than those found for other transformed basophilic models. The treatment also induced a reduction in ornithine decarboxylase expression, intracellular polyamine contents, and cell proliferation. These results indicate that the treatment induces a co-ordinate response of polyamine metabolism and proliferation in mast cells and other immune-related cells. The decrease in the proliferative capacity of mast cells caused by phorbol ester and dexamethasone was simultaneous to an increase in histamine production. Our results, together with those reported by other groups working with polyamine-treated mast cells, indicate an antagonism between histamine and polyamines in basophilic cells.

This study set out to examine the possible role of liver macrophages in histamine synthesis in the injured liver. The effects of the hepatotoxins Escherichia coli lipopolysaccharide (LPS) and CCl 4 on histamine synthesis in the liver of mice were evaluated. C3H/HeJ mice were resistant to LPS in including histidine decarboxylase (HDC) in the liver compared with C3H/HeN mice and mast cell-deficient W/W V mice. However, C3H/HeJ mice did respond strongly to another hepatotoxin, CCl 4 , leading to a significant increase in HDC activity. CCl 4 also caused a marked increase in HDC activity and histamine levels in the liver of W/W V mice. In addition, injection of CCl 4 produced a large increase in the activity of HDC in the spleen and lung of W/W V mice. HDC activity was confined to the nonparenchymal cells, with parenchymal cells expressing essentially no HDC activity. The CCl 4 -induced increase in HDC activity was confined, at least in part, to the liver macrophages. These results indicate that the macrophages are responsible for the increase in HDC-dependent histamine production in the liver caused by the injection of hepatotoxins. The possible role of histamine in liver regeneration after injury is discussed.

The human leukemic cell line KU-812-F is known to differentiate into mature basophil-like cells under serum-free culture conditions. In the present study, the activity of histidine decarboxylase (HDC), a histamine-forming enzyme, in KU-812-F cells was found to be high, ranging from 10 to 57 pmol/min/mg protein. The great variation in HDC activity appeared to be due to different percentages and degrees of maturity of basophil-like cells during differentiation of this cell line. The enzyme was inhibited by α-fluoromethylhistidine but not by carbidopa, was unable to form dopamine from l -3, 4-dihydroxyphenylalanine, and had a K m value for histidine of 0.27 mM, indicating that it was HDC and not aromatic amino acid decarboxylase. The HDC activity increased 1.8-fold when the cells were stimulated by phorbol myristate acetate, which is known to activate protein kinase C, and this increase was blocked by staurosporine, a potent inhibitor of protein kinase C.

Since the histidine-containing dipeptide carnosine (beta-alanyl-L-histidine) is believed to have many physiological functions in the brain, we investigated the neuroprotective effects of carnosine and its mechanisms of action in an in vitro model of neurotoxicity induced by N-methyl-d-aspartate (NMDA) in differentiated PC12 cells. Pretreatment with carnosine increased the viability and decreased the number of apoptotic and necrotic cells measured by MTT and Hoechst 33342 and propidium iodide (PI) double staining assays. Carnosine also can inhibit the glutamate release and increase HDC activity and the intracellular and extracellular contents of carnosine, histidine and histamine detected by high-performance liquid chromatography (HPLC). The protection by carnosine was reversed by alpha- fluoromethylhistidine, a selective and irreversible inhibitor of histidine decarboxylase (HDC). Pyrilamine and thioperamide, selective central histamine H(1) and H(3) antagonists also significantly reversed the protection of carnosine. Further, the inhibition of glutamate release by carnosine was reversed by thioperamide. Therefore, the protective mechanism of carnosine may not only involve the carnosine-histidine-histamine pathway, but also H(1)/H(3) receptors and the effective inhibition of glutamate release. This study indicates that carnosine may be an endogenous protective factor and calls for its further study as a new antiexcitotoxic agent.

Using insulin-secreting cells, we previously demonstrated that specific proteins associated with the cytosolic, secretory granule, and mitochondrial fractions undergo a novel type of phosphorylation on their histidine residues. Subsequently, we identified these proteins as the nucleoside diphosphate kinase (NDPK) [Kowluru and Metz, Biochemistry 1994;33:12495-503], the beta subunit of trimeric GTP-binding proteins [Kowluru et al., Biochem J 1996;313:97-107], and the alpha subunit of succinyl-CoA synthetase [Kowluru, Diabetologia 2001;44:89-94], respectively. Since several other enzymes of intermediary metabolism (e.g. ATP-citrate lyase and glucose-6-phosphatase) also undergo histidine phosphorylation, these initial findings may have a more generalized significance to beta cells. Herein, we characterized a novel protein histidine kinase in pancreatic beta cells, and determined it to be acid- and heat-labile as well as alkali-resistant in its phosphorylation of histone 4. Such an activity was detected in normal rat islets, human islets, and clonal beta (HIT-T15 and INS-1) cells, and could utilize either ATP or GTP as a phosphoryl donor (with K(m) values in the range of 60-100 microM). On a size-exclusion column, its molecular mass was estimated to be in the range of 60-70 kDa. It was stimulated by divalent cations (Mg(2+)>Mn(2+)>control=Ca(2+)=Zn(2+)=Co(2+)), but was resistant to polyamines. It was inactivated by known in vitro inhibitors of protein histidine phosphorylation (e.g. UDP or cromoglycate). Mastoparan, a global activator of G-proteins and insulin secretion from isolated beta cells, but not mastoparan-17, its inactive analog, stimulated histidine kinase activity and histidine phosphorylation of G(beta) subunit and insulin secretion from isolated rat islets. These studies identify, for the first time, a protein kinase activity in the pancreatic beta cell that does not act on traditional -Ser, -Tyr, or -Thr residues. They also establish a possible link between histidine kinase activity and G(beta) phosphorylation in isolated beta cells.

We have reported previously that the histamine H(2) receptor (H(2)R) can stimulate the phospholipase C (PLC) signaling pathway in mouse keratinocytes. In the present work, we examined the physiological mechanisms involved in this activation by studying histamine metabolism and H(2)R expression and coupling during mouse keratinocyte differentiation. Ca(2+)-induced differentiation decreased histidine decarboxylase (HDC) mRNA, the enzyme responsible for histamine synthesis, by 68.9+/-5.0%. Concomitantly, intracellular histamine content and its release into the extracellular medium were reduced significantly by 68.2+/-2.0 and 74.1+/-1.7%, respectively. Binding of [3H]tiotidine to H(2)Rs present on the surface of whole cells was also decreased by cellular differentiation [(18.17+/-2.1)x10(4) vs. (6.27+/-0.87)x10(4) sites/cell, undifferentiated and differentiated cells, respectively], without affecting H(2)R affinity. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the H(2)R mRNA showed that the expression was also down-regulated at the transcriptional level. Moreover, the inhibition of H(2)R expression strongly affected the ability of the receptor to induce PLC activation. Our findings suggest that H(2)R signaling through the PLC second messenger system is inhibited during keratinocyte differentiation by an autocrine loop involving down-regulation of H(2)R expression and inhibition of histamine metabolism.

Mammalian ornithine decarboxylase and histidine decarboxylase present common structural and functional features, and their products also share pharmacological and physiological properties. Although accumulated evidence pointed for years to a direct involvement of polyamines and histamine in tumour growth, it has been only in the last few years that new molecular data have contributed to the clarification of this topic. The aim of this commentary is to review the molecular grounds of the role of histamine and polyamines in cancer and to point to possible directions for future research in emerging areas of interest.

The actions of S-alpha-fluoromethylhistidine (FMH), an irreversible inhibitor of the histamine biosynthetic enzyme histidine decarboxylase (HD), were studied on rat brain HD, as measured by a recently developed CO2-trapping enzymatic method. As expected, FMH induced a virtually complete inhibition of HD in the hypothalamus both in vivo and in vitro. In the frontal cortex, however, maximal doses of FMH did not maximally inhibit HD, suggesting the existence of an FMH-resistant form of HD. Careful studies of the conditions under which the assays were performed (homogenate dilution, preincubation times, incubation times, temperatures), as well as experiments with inhibitors of other decarboxylases, were unable to provide an explanation for this. When comparable studies of the effects of FMH in these brain regions were performed by alternative methods for measuring HD activity, no evidence for the existence of an FMH-resistant form of HD could be found. Thus, even though the CO2-trapping method appears to be accurate for measuring HD activity in rat hypothalamic homogenates, the present results show that this method may not be specific when studying brain regions other than the hypothalamus.

L-Histidine decarboxylase was purified to electrophoretic homogeneity from mouse stomach according to a procedure described previously [Ohmori E, Fukui T, Imanishi N, Yatsunami K and Ichikawa A, J Biochem (Tokyo) 107: 834-839, 1990]. The purified enzyme exhibited a specific activity of 750 nmol histamine formed per min per mg protein, which constituted a 37,500-fold purification compared to the crude extract, with a 1.6% yield. The molecular mass of the enzyme was estimated to be 54 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 100 kDa by gel filtration. The isoelectric point of the enzyme was determined to be pH 5.4. The Km value for L-histidine was estimated to be 0.29 mM. The single mRNA encoding the amino acid sequence of the mouse stomach enzyme was examined and its size was found to be 2.7 kb. These molecular and catalytic property values of the L-histidine decarboxylase of mouse stomach are quite similar to those of the enzyme from mouse mastocytoma P-815 cells.

A method for measuring histidine decarboxylase (HDC) in crude rat brain homogenates was developed by modification of existing 14 CO 2 -trapping methods. The addition of EDTA to tissue homogenates and assay buffer reduced non-enzymatic decarboxylation, and improved assay sensitivity and reliability. Addition of polyethylene glycol (molecular weight 300, PEG300) to the homogenizing buffer increased enzyme stability, permitting storage of crude homogenates. Studies of time course, tissue dilution and blanks showed that up to 8 mg of tissue could be assayed successfully with a 3.5-hr incubation. S - α -Fluoromethylhistidine (FMH) and α-hydrazinohistidine, specific inhibitors of HDC, induced concentration-dependent reductions of enzyme activity by up to 90%, whereas inhibitors of other decarboxylases had little or no effect. Kinetic studies of the enzyme in crude homogenates yielded K m and V max values similar to those found previously with other HDC methods, although a poor fit was found to a single enzyme model. When determined by the new method, the distribution of HDC in seven regions of the rat brain agreed well with previous results. The method is rapid, simple to perform, and requires no specialized equipment other than a scintillation counter.

Our studies suggest that the fluctuation of HDC activity in fetal liver in late gestation is regulated by the plasma glucocorticoid level through the pituitary-adrenal system. Taken together, these results support the conclusion that glucocorticoid promotes a rapid increase in HDC synthesis in fetal liver histamine-forming cells, as well as in mouse mastocytoma P-815 cells and rat glandular stomachs.

The human leukemic cell line KU-812-F is known to differentiate into mature basophil-like cells under serum-free culture conditions. In the present study, the activity of histidine decarboxylase (HDC), a histamine-forming enzyme, in KU-812-F cells was found to be high, ranging from 10 to 57 pmol/min/mg protein. The great variation in HDC activity appeared to be due to different percentages and degrees of maturity of basophil-like cells during differentiation of this cell line. The enzyme was inhibited by alpha-fluoromethylhistidine but not by carbidopa, was unable to form dopamine from L-3,4-dihydroxyphenylalanine, and had a Km value for histidine of 0.27 mM, indicating that it was HDC and not aromatic amino acid decarboxylase. The HDC activity increased 1.8-fold when the cells were stimulated by phorbol myristate acetate, which is known to activate protein kinase C, and this increase was blocked by staurosporine, a potent inhibitor of protein kinase C.

The effects of α-fluoromethylhistidine (α-FMH), a new suicide inhibitor [Kollonitsch et al., Nature, Lond. 274 , 906 (1978)], on histidine decarboxylase (HDC) activities and histamine contents of the skin, fundic stomach and brain of mice were investigated. Four hours after i.p. administration of α-FMH to ddy mice, HDC activities in the brain, stomach and skin had decreased in a dose-dependent way (1–25 mg/kg), by a maximum of 90–95%. The histamine levels in the brain and stomach decreased to 50% of the control levels, whereas the level in the skin did not change at all. The time courses of changes in HDC activities and histamine levels were examined. After i.p. administration of 25 mg/kg of α-FMH, HDC activities in these tissues dropped rapidly within 1 hr. Recovery of HDC activities in the stomach and skin began within 12 hr, but the activity in the brain remained low for 24 hr, confirming the result of Garbarg et al. [ J. Neurochem. 35 , 1045 (1980)]. The histamine content of the stomach decreased to 40% of the original level in 8 hr and recovered within 12 hr, whereas that in the brain decreased to 50% and remained low for more than 24 hr. The histamine content of the skin did not change. These results suggest that the histamine level that was not reduced by α-FMH was derived from mast cells. During the above experiments, no behavioral changes of the animals were detected. α-FMH prevented the increase in HDC activity in mouse kidney on day 18 of gestation when administered i.p. every 12 hr from day 13. No abnormalities were seen in fetuses and neonates after this treatment. It is concluded that α-FMH causes depletion of newly synthesized histamine in situ and, thus, is useful for studies on histamine.

The injection of recombinant interleukin-1 (IL-1) into mice induced histidine decarboxylase (HDC) activity in the bone marrow, spleen, lung and liver and ornithine decarboxylase (ODC) activity in the spleen and liver. The ability of IL-1 to induce these responses was the most potent of the various cytokines tested. The induction of these responses by IL-1 seemed to be more rapid than that produced by a lipopolysaccharide. The potency of IL-1 alpha to induce both HDC and ODC activities was similar to that of IL-1 beta, and their combination did not potentiate the induction of these responses. In contrast, although the ability of recombinant tumor necrosis factor-alpha (TNF alpha) to induce these responses was less potent than that of IL-1 alpha or IL-1 beta, the combination of TNF alpha and IL-1 beta produced higher HDC and ODC activities in some tissues tested than those induced by the combination of IL-1 alpha and IL-1 beta. These results suggest that the syntheses of histamine and putrescine are regulated by IL-1 and/or TNF alpha in inflammatory or immune responses. Through these experiments, it was noticed that, in spite of a marked induction of HDC activity in the bone marrow, there was no detectable induction of ODC activity in this tissue. The meaning of HDC induction in the bone marrow is discussed.

The injection of Escherichia coli lipopolysaccharide (LPS) into mice produced simultaneous induction of histidine and ornithine decarboxylases in the liver, lung, spleen and kidney. The time courses of the changes in activities of the two enzymes were similar in all the tissues. After the injection, both activities increased within 1.5 hr, peaked at 4.5 hr and returned to the basal levels within 15 hr. The induction of these enzymes was very sensitive to this agent, i.e. as little as 1 μg/kg of the E. coli lipopolysaccharide produced significant increases in these enzyme activities. An increase in the product amines, histamine and putrescine, followed the rise of enzyme activities. The levels of histamine changed more rapidly than those of putrescine. In spite of the increase in putrescine, there was no increase in spermidine and spermine. In the brain and thymus the LPS induced ornithine decarboxylase, but not histidine decarboxylase. In the blood, the histamine level increased without an increase in the activity of histidine decarboxylase. These results are discussed in relation to the actions of lipopolysaccharide. A simple method for the simultaneous assay of the activities of histidine and ornithine decarboxylases without using radioisotope substrates was used in this study.

A variety of histamine agonists, antagonists and their analogs were tested for their ability to inhibit histamine-N-methyltransferase. In earlier studies, we had shown that the enzyme is inhibited noncompetitively by the histamine H2-receptor agonist, Dimaprit, and we had suggested that the enzyme possessed an inhibitory site with an affinity for both Dimaprit and histamine. The most potent inhibitors had, in addition to a side chain ammonium group, a thiourea or imidazole group which, like the imidazole group of histamine, is capable of prototropic tautomerism. The H1-receptor agonists, 2-pyridyl-β-ethylamine and 2-thiazolyl-β-ethylamine, which do not undergo prototropic tautomerism, were only weakly inhibitory. There was no correlation, however, between pharmacological and inhibitory activity. The most potent inhibitor (Ki∼ 10−6 M) was a Dimaprit analog, SKF 91488, which has no histamine agonist activity. SKF 91488, therefore, may be especially useful in studies of histamine metabolism.

alpha-Fluoromethyl histidine is an irreversible inhibitor of histidine decarboxylase. The injection of a single dose to pyloric-ligated rats inhibits gastric mucosal histidine decarboxylase in a dose-dependent manner but does not modify histamine content and gastric acid secretion even at the highest dose used. Administration of cimetidine increases histidine decarboxylase activity, decreases histamine level in gastric mucosa and inhibits gastric acid secretion. The co-administration of alpha-fluoromethyl histidine blocks the augmentation in enzyme activity, maintains lowered histamine level and prolongs the antisecretory action of cimetidine.

Histidine decarboxylase activity in the gastric mucosa of the rat stomach is markedly increased by the H 2 receptor antagonists, burimamide and metiamide. The increase in enzyme activity is reduced by cycloheximide but not by actinomycin D. An inhibitor of histidine decarboxylase, 4-imidazolyl-3-amino-2-butanone (McN-A-1293), is also effective in reducing the enzyme activity stimulated by the H 2 receptor antagonists. The time course and the magnitude of the response of the enzyme with maximum doses of burimamide are similar to those obtained with pentagastrin. The histamine content of the mucosa is also reduced by burimamide and the data are discussed in relation to the hypothesis that H 2 receptor antagonists increase histidine decarboxylase activity through the release of endogenous gastrin.

Rat glandular stomach histamine concentration increases from 15.5 μg/g at 25 days of age to 78 μg/g at 179 days of age, whereas histamine levels in the forestomach do not change with aging. This increase in histamine occurs primarily in the muscular layer of the glandular stomach but not the mucosal and serosal layers of this organ. There is little change in the histamine-forming capacity of the stomach in this age group, nor does Compound 48/80 lower histamine levels in vivo in glandular stomach ; it does lower levels in the forestomach and diaphragm. These data suggest that changes in rat stomach histamine storage capacity but not storage properties occur with aging.

Preincubation with (S)-alpha-fluoromethylhistidine, an irreversible inhibitor of histidine decarboxylase, was found to markedly reduce, but not eliminate, the uptake of [3H]histidine by rat peritoneal mast cells. The Vmax for histidine transport for cells in which decarboxylation of histidine had been completely inhibited was 11.9 pmoles per min per 10(6) cells, compared to a Vmax of 18.9 pmoles per min per 10(6) cells in the presence of active mast cell histidine decarboxylase. The Km of uptake was 139 microM in the presence of alpha-fluoromethylhistidine, several times higher than the Km of 44.0 microM in the uninhibited cell. alpha-Fluoromethylhistidine did not inhibit mast cell uptake of phenylalanine, a competitive inhibitor of histidine uptake but not a substrate for histidine decarboxylase; nor did it inhibit the uptake of histidine by non-mast cells, which lack histidine decarboxylase. Levels of intracellular [3H]histidine in mast cells were similar in the presence and absence of the decarboxylase inhibitor. Based on these observations, we propose that intracellular decarboxylation of histidine in the mast cell serves to specifically enhance the uptake of histidine by the relatively non-specific amino acid transporter present in the plasma membrane of the cell.

(S)-alpha-Fluoromethylhistidine (alpha-FMH) is a Kcat or "suicide-substrate" inhibitor of partially purified mammalian histidine decarboxylase; i.e. the agent is converted enzymatically to a more active form which effects a time-dependent, irreversible inhibition. Incubation of a alpha-FMH[4-3H] with enzyme and pyridoxal phosphate resulted in an apparently irreversible labeling of protein, with no demonstratable formation of free-amine product, suggesting a very low to non-existent turnover ratio. alpha-FMH was accumulated in isolated mastocytoma cells and effected a time-dependent inhibition of the conversion histidine[3H]----histamine[3H], the latter product having a markedly different distribution between cells and medium than the pre-existing histamine pool. Inhibition of whole-body histidine decarboxylase activity, as specifically measured by alpha-methylhistidine-14COOH----14CO2, was also time dependent. Concomitant reduction in histamine levels was seen only in the rapidly turning-over pools of stomach and brain. However, over the course of 13 weeks of chronic treatment, depletion of the relatively inert mast-cell histamine pool(s) was seen as well.

In freely fed rats, an intraperitoneal injection of 0.688 m-mole per kg of 4-bromo-3-hydroxybenzyloxyamine (NSD 1055) produces a transient drop in both the histidine decarboxylase activity of the glandular stomach and the diamine oxidase activity of the intestine. Both enzymes are inhibited by approximately 50 per cent within 30 min, but recovery to normal levels occurs within a few hours. The results obtained in vivo are in sharp contrast to the data in vitro , which show that the inhibitor is much more active against the decarboxylase. The transient effect of NSD 1055 against the two enzymes in vivo may be due primarily to the rapid clearance or metabolism of the drug, while the change in potency ratio from a situation in vitro to one in vivo may reflect the greater rate of turnover of histidine decarboxylase. The drug, however, is very effective in preventing the rise in histidine decarboxylase produced by insulin, pentagastrin or refeeding of starved animals. Presumably under these circumstances there is a greater opportunity for the drug to complex with the cofactor, pyridoxal phosphate.

4-Imidazolyl-3-amino-2-butanone (McN-A-1293) was shown to be an effective inhibitor of fetal rat histidine decarboxylase, but not guinea pig kidney aromatic l -amino acid decarboxylase in vitro . McN-A-1293 was a more potent inhibitor than α-methylhistidine, but less potent than brocresine (NSD-1055) and other aminooxyamines. Kinetic studies using fetal rat histidine decarboxylase indicated the inhibition to be reversible and competitive with histidine. McN-A-1293 in vitro was a less potent inhibitor of diamine oxidase than brocresine and was only a weak inhibitor of imidazole- N -methyltransferase. Administration of McN-A-1293 (200 mg/kg, i.p.) to fed rats resulted in 55 per cent inhibition of gastric histidine decarboxylase, while gastric aromatic l -amino acid decarboxylase was only slightly inhibited (17 per cent). Brocresine (200 mg/kg, i.p.) inhibited both gastric decarboxylases > 60 per cent. Doses of McN-A-1293 as low as 50 mg/kg, i.p., inhibited gastric histidine decarboxylase activity. In fasted rats, McN-A-1293 (200 mg/kg, i.p.) was found to inhibit the elevation in gastric histidine decarboxylase induced by gastrin or insulin. 4-Imidazolyl-3-amino-2-butanone (McN-A-1293) is an effective and specific inhibitor of histidine decarboxylase both in vitro and in vivo and should be a useful agent for studies on the role of histidine decarboxylase in histamine physiology.

Some of the characteristics of the histidine decarboxylase derived from fetal rat liver have been determined. The pH optimum is 6.6; pyridoxal 5'-phosphate is required as a coenzyme; the enzyme is not appreciably inhibited by a-methyl DOPA; and benzene neither activates nor inhibits the enzyme. The fetal enzyme activity appears shortly after the appearance of liver cells, reaches a peak at the fourteenth day of gestation and apparently disappears rapidly after birth. At the present time the functional significance of this enzyme remains obscure.

Inhibition of histamine synthesis in vivo was examined in terms of effects on the kinetics of decline of exogenous 3 H-histamine in various tissues of the female rat. Inhibition of tissue histidine decarboxylase activity by dietary pyridoxine deficiency coupled with semicarbazide administration greatly reduced rates of decline in vivo of labeled amine. On the assumption that exogenous 3 H-histamine equilibrates with endogenous amine in nonmast-cell pools, estimated rates of turnover of loosely bound histamine were reduced 30–75 per cent. The histidine decarboxylase inhibitor, NSD-1055 (4-bromo-3-hydroxybenzyloxyamine) was ineffective in reducing endogenous histamine levels and did not affect the kinetics of decline of 3 H-histamine. It is suggested that the kinetics of 3 H-histamine in vivo decline and the tissue histidine decarboxylase activity in vitro may reflect the same biochemical phenomenon—presumably the synthesis and catabolism of loosely bound nonmast-cell histamine in dynamic endogenous pools.

The specific histidine decarboxylase of the thyroid gland of pigs and of the stomach of rats and the nonspecific histidine decarboxylase of the kidney of guinea pigs are inhibited by inhibitors of intrathyroid hormone synthesis in vitro. m-Fluortyrosine, an inhibitor of iodide ion trapping, and 5-fluoruracil, a cytostatic drug, are ineffective. 2-mercaptobenzimidazole-1,3-dimethylol is a relatively strong inhibitor of the specific and unspecific histidine decarboxylase, 2-carbethoxythio-1-methylimidazole is effective too, but 6-propyl-2-thiouracile and 6-methyl-2-thiouracile are only weak inhibitors. The Km of the unspecific histidine decarboxylase and histidine is 1.7 × 10−3M, if the velocity of reaction is calculated as initial velocity and not after an incubation time of 3 hr, when values between 10−2 and 10−1 M have been reported. Newer results do not agree with the suggestion, that the nonspecific histidine decarboxylase does not play a role in the decarboxylation of histidine in vivo. 2-Mercaptobenzimidazole-1,3-dimethylol inhibits the nonspecific histidine decarboxylase competitively with respect to histidine and pyridoxal phosphate. The Ki-values for histidine and pyridoxal phosphate are 1.7 × 10−4 and 2.7 × 10−4 M, the Km for pyridoxal phosphate being 6 × 10−5 M. From this 2-mercaptobenzimidazole-1,3-dimethylol shows a 10-fold higher affinity to the active center of the nonspecific histidine decarboxylase than histidine. During the incubation and complete inhibition of histidine decarboxylase the histamine content in the incubation mixture is diminished though aminoguanidine and chlorpromazine have been added. In the presence of antithyroid drugs, such as 2-carbethoxythio-1-methylimidazole, in concentrations greater than 1 × 10−3 M, the fluorescence of the condensation product of histamine and o-phthaldialdehyde is diminished. Histamine therefore has been estimated on the isolated guinea pig's ileum, because the substances can be separated from histamine by the extraction procedure only to 50 per cent. The diminishing effect is not caused by quenching, but by reaction of phthaldialdehyde with the inhibitors and partial absorption of the exciting light. This effect can be used for estimation of the antithyroid drugs. An influence of histamine on the intrathyroid synthesis of thyroid hormones is suggested. The importance of the unspecific histidine decarboxylase for the metabolism of histamine in mammals is discussed.

The stomach of the male rat contains both a soluble and an insoluble histidine decarboxylase. The soluble enzyme in the glandular stomach is the one which is generally assumed to be associated with gastric secretion. The insoluble enzyme is located in the debris fraction of the forestomach and the activity varies widely in different animals. In female rats the activity of the insoluble enzyme is very low, whereas the activity of the soluble enzyme in the glandular stomach is similar to that found in male rats. In male rats starvation causes a more rapid depletion of the insoluble enzyme than of the soluble histidine decarboxylase. Refeeding or an injection of insulin causes a sharp rise in histidine decarboxylase activity in the glandular stomach, but does not affect the insoluble enzyme in the forestomach. At the present time there is little evidence to suggest that the insoluble enzyme is of major importance in studying the role of histamine in gastric secretion.

Histidine decarboxylase activity was identified in the gastric mucosa of monkeys and man and in a human gastric carcinoid tumor. Crude homogenates of gastric mucosal tissue but not of the carcinoid tumor also demonstrated 5-hydroxytryptophan decarboxylase activity; this activity disappeared completely during purification procedures that resulted in increased specific activity of histidine decarboxylase. In all respects studied these histidine decarboxylases had properties that were very similar to or identical with histidine decarboxylases prepared from other mammalian sources: in particular, the apparent K m varied inversely with pH; the pH optimum varied inversely with substrate concentration; activity of the enzyme was enhanced by pyridoxal 5′-phosphate but not by benzene and inhibited by both brocresine and hydrazino-analog of histidine but not by α-methyl-dopa. In both monkeys and men histidine decarboxylase activity was greater in the fundus than it was in the antrum of the stomach. These findings indicate that the enzyme necessary for histamine biosynthesis is present in gastric tissues of primates and thus remove one theoretical objection to the hypothesis that histamine may mediate gastric acid secretion in primates.

Upon administration of brocresine to man, rat or dog, the blood plasma becomes inhibitory to a preparation of fetal histidine decarboxylase in vitro. The inhibitory activity of the plasma quickly disappears 2 hr after treatment. Brocresine when added to normal plasma and assayed immediately inhibits histidine decarboxylase to the same extent as brocresine assayed in the absence of plasma. This inhibitory activity quickly disappears when the brocresine is incubated in plasma prior to assay. It is concluded that brocresine is rapidly cleared from the plasma and/or metabolized.

Relatively high activities of the specific histidine decarboxylases were found in the gastric mucosa of men, monkeys, pigs, cows, dogs, cats, guinea-pigs, rabbits and rats. Some improvements of the assay of specific histidine decarboxylase were necessary, before these enzymes could be demonstrated in all stomachs investigated. The specific histidine decarboxylase could also be shown in the human gastric carcinomas. By pH-optima, substrate optima, K m , inhibition by α-methylhistidine but not by α-methyldopa, inhibition by benzene and activation by pyridoxal-5′-phosphate the histidine decarboxylases in the gastric mucosa could be characterized as specific histidine decarboxylases. The enzyme in the stomach of guinea-pigs has been purified 22-fold by ultracentrifugation and gelfiltration on Sephadex G 100. The demonstration of the specific histidine decarboxylases (isoenzymes) in the gastric mucosa of numerous mammals has some importance for the hypothesis of a physiological function of histamine as a chemostimulator of gastric secretion. Problems of the nomenclature of histidine decarboxylases are discussed. The terms “acid” and “alkaline” histidine decarboxylases are proposed.

Kinetic studies have shown that a number of acidic anti-inflammatory drugs (salicylate, “Ibufenac”, phenylbutazone, Indomethacin and Flufenamic acid) inhibit histamine formation by competing with pyridoxal phosphate for the coenzyme binding site, believed to be a lysyl ϵ-amino group on mammalian histidine decarboxylase. Apparent inhibitor constants (Ki's) for these drugs were measured. These drugs also inhibited the binding of pyridoxal phosphate and 2,4,6-trinitro-benzaldehyde (TNBAL) to ϵ-amino groups of bovine plasma albumin at pH 7.5. A number of chemical analogues of these drugs were screened for their ability to (i) inhibit histamine formation in vitro and to (ii) retard the formation of the red coloration which forms when TNBAL reacts with albumin at pH 7.5 (and provides a novel colorimetric method for testing potential drug activity). Other compounds with notable drug activity in these two assays [(i) and (ii) above] include Sanochrysin, “Biogastrone”, pentachlorophenol and phenylindanedione—all of which, like the anti-inflammatory drugs named above, also uncouple oxidative phosphorylation.

Decarboxylase inhibiting properties and the structure-activity relationship have been studied with a number of newly synthetized benzyloxyamines and pyridylmethoxyamines. In the case of benzyloxyamines, the 3-hydroxyl group, and in that of pyridylmethoxyamines, the position of methoxyamine group in the molecule, appear to be of special importance for inhibition of aromatic l -amino acid decarboxylase. We did not find any close relationship between specific histidine decarboxylase inhibiting effect and chemical structure. 3-Hydroxy-4-nitrobenzyloxyamine and pyridyl-3-methoxyamine were the most active compounds in these series. The former compound affected markedly both aromatic l -amino acid decarboxylase and histidine decarboxylase in vitro and in vivo and produced a significant decrease in the levels of histamine in the stomach and in the lungs. The latter compound proved to be a specific inhibitor of histidine decarboxylase and had a more pronounced effect on the tissue histamine levels than most of new and old benzyloxyamine derivatives.

A technique has been devised for assay of plasma levels of histidine decarboxylase inhibitors in man. It is based upon determination of the power of plasma to inhibit the activity of a preparation of histidine decarboxylase in vitro after administration of a drug. This method, which should be generally applicable to assay of all histidine decarboxylase inhibitors, was used to study plasma levels of brocresine and d -2 hydrazino-3-4 (5) -imidazole propionic acid (MK - 785) after oral administration of various single doses. Levels obtained with MK-785 were much higher than with equivalent doses of brocresine. Both the rate at which peak plasma levels were attained and the rate of disappearance from plasma were more rapid with brocresine than with MK-785.

The regional distribution of DOPA decarboxylase and histidine decarboxylase activities in the gastric mucosa of the rat and the rabbit was studied. In the rat, these enzymes occurred exclusively in the pyloric region of the stomach. In the rabbit, DOPA decarboxylase was found predominantly in the fundic region; in this species the histidine decarboxylase activity was too low to permit estimation. A partially purified preparation of histidine decarboxylase from rat stomach had properties very similar to those of histidine decarboxylase from fetal rat tissues. A histamine-forming enzyme could also be extracted from rabbit stomach but this enzyme had properties identical with those of DOPA decarboxylase which is unspecific and attacks all aromatic amino acids including histidine. DOPA decarboxylase could be demonstrated in the stomach wall of all species studied; so far, the specific histidine decarboxylase has been demonstrated in rat stomach only. After intraperitoneal injection of DOPA or 5-hydroxytryptophan to rats, large amounts of dopamine or 5-HT accumulated in the gastric mucosa, indicating that the DOPA decarboxylase in the stomach wall is active in vivo. The amines were located to an enterochromaffin-like cell system in the pyloric part of the gastric mucosa as revealed by fluorescence microscopy. Preliminary evidence indicate that the amines were produced within these cells. The high DOPA decarboxylase activity in the gastric mucosa of the species studied may indicate an important role for this enzyme in gastric function.

Production of 14 CO 2 from dl -histidine- 14 COOH was observed with both N -(2′-carbomethoxy) cyclopentylidene, cyanoacethydrazide (AY-17,224) and a female rat pyloric stomach preparation. Each exhibited an optimum pH of 7.0. The activities of AY-17,224 and the pyloric enzyme were found to be dependent upon the type and concentration of buffer employed. Protein and pyridoxal phosphate caused a decrease in activity of AY-17,224. AY-17,224 neither inhibited nor potentiated the pyloric enzyme. AY-17,224 was partially inhibited whereas the pyloric enzyme was stimulated maximally by pyridoxamine phosphate. α-Methyl-histidine inhibited AY-17,224 but had no effect on the pyloric enzyme, whereas the reverse was observed with KCN. Phenylhydrazine inhibited whereas phenylethylhydrazine stimulated and NSD-1055 had no effect on the activity of AY-17,224; each of these compounds inhibited the pyloric enzyme. The K m of AY-17,224 with respect to histidine- 14 COOH was 1.35 × 10 −4 M while that of the pyloric enzyme was 1.25 × 10 −3 M.

Histamine synthetic activity which is high in young mast cells decreases as the cells mature [Beaven et al ., J. Pharmac. exp. Ther . 224 , 620 (1983)]. In this study we show that a substantial proportion of newly formed histamine in young mast cells leaked to the extracellular environment. The cells acquired the full ability to sequester newly formed histamine once the numbers of intracellular granules and the supply of sulfated mucopolysaccharide material within them had increased. Rat peritoneal mast cells were separated into successive fractions of increasing size and maturity by counter current elutriation. Loss of histamine from fractions of immature cells was demonstrated by a progressive accumulation of histamine in the medium without any decrease in intracellular histamine content. The estimated turnover time of histamine was less than 10 hr. In fractions of more mature cells, the proportion of cellular histamine released into the medium was substantially lower, giving estimated turnover times of 20 hr or longer. Studies with radiolabeled histidine also indicated that little, if any, newly formed histamine was lost from fractions of mature cells. Both release of endogenous histamine and formation of radiolabeled histamine from labeled histidine were inhibited by the histidine decarboxylase inhibitor α-fluorotnethylhistidine (10 μM). Histamine turnover times were similar in the presence or absence of external histidine, a possible indication that the supply of intracellular histidine was sufficient to maintain normal histamine synthetic activity.

Histamine production by purified intact rat peritoneal mast cells, as measured by formation of [β- 3 H]histamine from [β- 3 H] l -histidine or by release of 14 CO 2 from 14 C-carboxyl-labeled histidine, was ten to thirty times greater than that of disrupted cells or soluble extracts of these cells. Loss of activity was evident whether cells were disrupted by sonification, freezing and thawing, or lysis, both in the absence and presence of inhibitors of proteolytic enzymes and agents known to preserve enzyme activity. Studies with decarboxylase inhibitors indicated that a specific histidine decarboxylase was responsible for histamine formation in both the intact cells and cell extracts. In the presence of subsaturating concentrations of histidine, various histidine analogs and glutamine inhibited histidine uptake and histamine formation in intact mast cells but did not inhibit synthesis in cell extracts. These data indicate that, at physiological concentrations of histidine, blockade of histidine transport (through system N) may limit histamine synthesis in the intact cell and that measurement of histidine decarboxylase activity in tissue homogenates or cell extracts may not reflect actual histidine decarboxylase activity in vivo .

High levels of ornithine decarboxylase are found in the developing rat placenta. Peak activities are observed around day 17 of pregnancy, after which there is a slight decline in activity up to the time of parturition. Most of the ornithine decarboxylase activity is found in the fetal placenta, whereas in the maternal placenta the activity declines steadily throughout the latter part of pregnancy. At term over 90 per cent of the placental ornithine decarboxylase is found in the fetal segment. The putrescine content of the fetal placenta is three to ten times higher than that of the maternal tissue from day 15 onward. In contrast to the pattern of distribution for ornithine decarboxylase, diamine oxidase is found mainly in the maternal placenta, and the activity remains high throughout pregnancy. S -adenosyl methionine decarboxylase is also present in the placenta with significantly higher levels being found in the fetal tissue. The activity of this enzyme, however, was at all times much lower than that of ornithine decarboxylase and diamine oxidase. The alignment of the metabolic pathways favoring the metabolism of putrescine via oxidative deamination rather than through conversion to spermidine suggests that putrescine may have a role to play in the developing rat placenta. The results also indicate that the high levels of diamine oxidase activity found in the placenta are probably associated more with putrescine than with histamine metabolism.

Escherichia coli lipopolysaccharide (LPS) induced a strong secretion of corticosterone in C3H/HeN mice with a concomitant increase in the splenic histidine decarboxylase activity. Treatment of the mice with α-fluoromethyl histidine, a suicide substrate for the enzyme, markedly attenuated both the secretion and the increase. In C3H/HeJ mice, LPS provoked little corticosterone release and induction of the enzyme. However, these mice responded to tetradecanoyl phorbol acetate with a large increase in both this secretion and enzyme activity. Injection of LPS produced a comparable increase in the serum histamine and corticosterone level and activity of histidine decarboxylase in various tissues of genetically mast-cell-deficient W/Wv mice and in closely related +/+ mice. These results suggest that secretion of corticosterone caused by LPS is mediated by histamine produced through induction of histidine decarboxylase in non-mast cells.

Endogenous adenosine 3',5'-monophosphate (cAMP) levels in mastocytoma P-815 cells, synchronized either at the G 1 /S transition by amethopterin- or double thymidine-block or in mitosis by colcemid block, were highest during late S and early G 2 phases and lowest during mitosis. These cell cycle-dependent changes in cAMP levels were largely accounted for by the changes in adenylate cyclase and phosphodiesterase activities. Similar fluctuations occurred simultaneously with specific prostaglandin E 1 (PGE 1 ) binding, histidine decarboxylase activity, histamine content, and [ 35 S]SO 4 2− incorporation into glycosaminoglycans of the cells. In addition, endogenous levels of the E group of prostaglandins (PGEs) and [ 14 C]arachidonic acid incorporations into PGE, phosphatidylcholine and phosphatidylinositol also exhibited fluctuation patterns similar to that of cAMP levels. Since cAMP levels still fluctuated in a serum-depleted medium where DNA synthesis and cell division were inhibited, endogeneous levels of prostaglandin and cAMP appeared not to be regulated solely by serum factor(s). Exposure of cells at G 1 /S transition to 1-methyl-3-isobutylxanthine (MIX) resulted in a 10-fold elevation of cAMP levels throughout the cell cycle without affecting DNA synthesis. On the other hand, PGE 1 and/or MIX added at late S phase elevated cAMP levels, prolonged G 2 phase and retarded the cell division, but these agents added at the beginning of mitosis elevated cAMP levels without affecting the cell division. These results suggest that prostaglandins newly synthesized by the increased metabolism of phospholipids promote the cAMP synthesis via their binding to the receptors and thereby control the division and phenotypic expression of mastocytoma P-815 cells.

Histamine, a major constituent of the amine-storage organelles in pig platelets, is taken up by intact platelets in only trace amounts under conditions where 70% of 14C-serotonin is accumulated. Thrombin caused the release of 70–90% of endogenous histamine but only 5–10% of the newly absorbed 3H-amine; however, after 18 hr 30% of the 3H-amine could be specifically released by thrombin. Isolated storage organelles accumulated histamine in a reserpine-sensitive, ATP-dependent manner but at a rate 80–100-fold less than serotonin uptake. Incubation of intact platelets with 1 mM serotonin until amine uptake was saturated caused no changes in platelet histamine content. Similarly, loading of isolated storage organelles with 1 mM histamine or 1 mM serotonin did not affect the levels of the other amine. These results suggested that the storage of each amine is independent of the other. Histidine decarboxylase was not detected in platelet lysates. Since platelets have a short half-life (1–2 weeks) and pig plasma levels of histamine are higher than in other animals, it is concluded that most of the histamine in the storage organelles is probably accumulated in the platelet precursor, the megakaryocyte, either by slow uptake or by synthesis.

An irreversible inhibitor of histidine decarboxylase, alpha-fluoromethylhistidine (FMH), was used to inhibit histamine formation by mast cells in vivo. Even at doses of FMH sufficient to reduce histamine formation more than 95%, the ability of mast cells to synthesize histamine recovered rapidly. It was possible, however, to sustain levels of histamine-forming activity below 10% of normal with continuous administration of FMH from subcutaneously implanted osmotic pumps. Administration of FMH under these conditions did not deplete significantly mast cell histamine but did prevent the increase in total mast cell histamine that occurs over 14 days and also prevented the reconstitution of mast cell histamine stores after depletion by treatment with polymyxin B.

Histidine decarboxylase (HDC) activity in Ficoll-Hypaque purified human peripheral blood leukocytes (PBL) was determined by measuring the formation of [3H]histamine from L-[3H]histidine. HDC activity was inhibited in vitro to more than 90% by (S)-alpha-fluoromethylhistidine (alpha-FMH) at concentrations of 10(-5) M and above. Both polymorphonuclear and mononuclear cells possessed HDC activity, but on a per cell basis the former had several-fold higher enzyme activity than the latter. In safety and tolerability studies, alpha-FMH was administered orally to healthy human subjects twice daily for 7 days at doses of 2.5, 10, 50 and 100 mg per person. A dose-dependent inhibition of HDC activity was observed in PBL that were isolated both at 12 hr after administration of the first dose of alpha-FMH and after treatment for 1 week. At the 50 and 100 mg doses of alpha-FMH, there was complete inhibition of HDC activity and partial inhibition at the 10 mg dose. Twenty-four hours after the last dose, HDC activity had recovered to 64-100%, 44-46%, and 30-52% of control values in subjects that received 10, 50 and 100 mg alpha-FMH respectively.