Zsuzsanna Huszti

Glial cells participate in histamine inactivation in vivo

The ability of glial cells to take up histamine in vitro suggests that these cells may be involved in histamine inactivation. This prompted us to study the possible interactions between neuronal and glial processes which determine the histamine concentration in the synaptic cleft. In vitro experiments showed that the glial metabolic toxin, fluoroacetate (20 and 40mmol/l) depressed histamine uptake into cultured astroglial cells and dissociated hypothalamic cells of rats. For in vivo experiments, the push-pull superfusion technique was used. In anaesthetized rat, the anterior hypothalamic area was superfused through the push-pull cannula with artificial cerebrospinal fluid (aCSF) or with aCSF which contained fluoroacetate and the release of endogenous histamine was determined in the superfusate.Hypothalamic superfusion with fluoroacetate (20mmol/l) led to a pronounced increase in extracellular histamine. The effect of fluoroacetate was inhibited by 5μmol/l tetrodotoxin. Superfusion with Ca++-free, Mg++-rich (12mmol/l) aCSF inhibited the basal release rate of histamine. Under these conditions, 20mmol/l fluoroacetate did not modify the level of the amine in the superfusate.These data demonstrate that depression of glial function enhances the concentration of histamine in the extracellular space by slowing down the uptake of the amine into the glial cells. Thus, under in vivo conditions, glial cells are directly involved in the continuous removal of neuronal histamine from the synaptic cleft.

Carrier-mediated uptake and release of histamine by cultured rat cerebral endothelial cells.

The present study demonstrates that histamine could be taken up by and released from endothelial cells of brain capillaries. Incubation of cultured endothelial cells, with low (0.01-0.50 microM) concentrations of [3H]histamine, resulted in a rapid uptake of the amine. The uptake was saturable, Na(+)-dependent and yielded an apparent Km 0.3 +/- 0.02 microM and a Vmax 4.6 +/- 0.04 pmol/mg protein per min. After a 10-min incubation in a histamine-free medium, about 65% of [3H]histamine was released from the cells. Na(+)-deprivation and high K+, as well as the treatment of the cells with ouabain affected the release, resulting in significantly higher rates of the efflux. The ability of cerebral endothelial cells to take up histamine from both luminal and abluminal sides but to release it mainly luminally, may function as an important mechanism to protect the neural tissue from the harmful effects of this endogenous mediator of inflammation.

[3H]histamine uptake and release by astrocytes from rat brain: effects of sodium deprivation, high potassium, and potassium channel blockers.

Histamine transport has been characterized in cultured astroglial cells of rat brain. The kinetics of [3H]-histamine uptake yielded a Km of 0.19±0.03 μM and a Vmax of 3.12±0.75 pmol×mg protein−1×min−1. Transport system revealed high affinity for histamine and an approximately ten times higher capacity than that shown in cultured glial cells of chick embryonic brain. Ouabain which interferes with utilization of ATP to generate ion gradients, and the replacement of Na+ with choline inhibited the initial rate of uptake showing a strong Na+-dependency and suggesting the presence of a tightly coupled sodium/histamine symporter. Dissipation of K+-gradient (in>out) by high K+ or by K+-channel blockers, BaCl2, (100 μM), quinine (100 μM) or Sparteine (20 μM) produced also remarkable inhibitions in the uptake of [3H]-histamine. Impromidine, a structural histamine-analogue could inhibit the uptake non-competitively in a range of concentrations of 1 to 10 μM with a Ki value of 2.8 μM, indicating the specificity of the uptake. [3H]histamine uptake measurements carried out by using a suspension of dissociated hypothalamic cells, of rat brain showed a strong gliotoxin-sensitivity and yielded a Km of 0.33±0.08 μM; and a Vmax of 2.65±0.35 pmoles×mg protein−1×min−1. The uptake could be reversed by incubating the cells in histamine-free Krebs medium. The [3H]histamine efflux was sensitive to Na+ omission, ouabain treatment and high K+ or K+ channel blockers, resulting in marked elevations in the efflux. Data indicate that glial uptake of histamine is a high affinity, Na+-dependent and electrogenic, driven by an inward-oriented sodium ion gradient and an outward-oriented potassium ion gradient and functions as part of histamine inactivation, at least in a shunt mechanism.

Potassium-induced histamine release from mast cells and its inhibition by ketotifen

Potassium chloride induced a dose-dependent release of histamine from rat peritoneal mast cells at concentrations from 5 to 150 mM in the absence of extracellular Ca2+. Potassium concentrations greater than 150 mM produced less histamine release. The release was energy-dependent and was complete within one minute. The histamine liberating effect of KCl could be inhibited by NaCl and by preincubation with ketotifen. The monovalent cations, Rb+ and Cs+ also evoked histamine release, whereas Na+ and Li+ were ineffective.

Regulation of histidine decarboxylase activity in rat hypothalamus in vitro by ATP and cyclic AMP: Enzyme inactivation under phosphorylating conditions

In vitro, hypothalamic HD1 from rat, could strikingly be inhibited by ATP and cyclic AMP. The enzyme inhibition is partially dependent upon Mg2+ and the circumstances favourable for a cAMP-dependent phosphorylation. An almost complete inhibition could be achieved by incubating the homogenate of the hypothalamus under phosphorylating conditions (ATP, cAMP, Mg2+ and IBMX) in the presence of a cAMP-dependent protein kinase (obtained from bovine thymus). Cyclic nucleotides and ATP alone elicit only moderate inhibitions on the hypothalamic HD activity. Neither ATP, nor cAMP, added alone or in combinations, alter the total brain or the hypothalamic HNMT from guinea-pigs or rats in concentrations up to 10−3M.Results suggest that hypothalamic HD is regulated through a cAMP-dependent process, probable a direct phosphorylation, via a cAMP-dependent protein kinase.

Tyrosine hydroxylase and dopamine β-hydroxylase inhibiting properties of a new series of pyridazinyl hydrazones☆

New pyridazinyl hydrazones are described as a novel class of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) inhibitors. A great number of these substances showed potent TH- or DBH-inhibiting capacities. The structure-activity analysis suggested an important role for the N2 substitution of hydrazine in the inhibiting characteristics of the compounds. β-Substituted pyridazinyl hydrazones showed favourable TH- or DBH-inhibiting activities, whereas the free hydrazines were only slightly effective on tyrosine hydroxylation and noneffective on dopamine hydroxylation. The β-ketoester derivatives of 6-chloro-3-pyridazinyl hydrazones were characterized as potent DBH-inhibiting substances while the cyclohexylidene or the bicycloheptylidenepyridazinyl hydrazones were found to be marked TH- and DBH-blocking agents. Among the above substances, GYKI 11679 {chemically 1-(6-morpholino-3-pyridazinyl)-2-[(1-tertiary-butoxycarbonyl)-2-propylidene] hydrazine} has been found to be the most potent inhibitor of tyrosine hydroxylation both in vitro and in vivo. Its in vitro as well as its in vivo TH-blocking activity was comparable with that of α-methyltyrosine. The most effective representative of this new series on dopamine and tyramine hydroxylations in vitro and in vivo, compound GYKI 11473 {chemically 1-(6-chloro-3-pyridazinyl)-2-[(1-ethoxycarbonyl)-2-propylidene] hydrazine}, has been characterised as a reversible and competitive inhibitor of DBH with dopamine and a noncompetitive inhibitor of DBH with tyramine as substrate at concns of 10−5–10−6 M. The compound has also been found to inhibit the neuronal and the vesicular uptake of dopamine in hypothalamic slices. GYKI 11473 showed a greater effect in the heart than in the brain and its in vivo efficiency has been established as being more potent than those of fusaric acid in both tissues. The noradrenaline (NA) determinations suggested similar, but not equal, and exclusive contributions of TH and DBH in the maintenance of NA pools in these tissues.

2-Hydroxy-5-carbomethoxybenzyloxyamine: A new potent inhibitor of histidine decarboxylase

Abstract Histidine decarboxylase inhibiting and histamine lowering effects of 2-hydroxy-5-carbomethoxybenzyloxyamine have been studied in detail. The compound proved to be a potent inhibitor of both rat stomach pyloric histidine decarboxylase and guinea-pig liver aromatic l -amino acid decarboxylase, showing a more pronounced effect on the former enzyme. The type of inhibitions were ascribed as reversible and competitive with respect to substrate and coenzyme in both cases. The K i values for histidine and pyridoxal phosphate were 1 × 10 −7 and 2.5 × 10 −7 M; for5-hydroxytryptamine and pyridoxal phosphate, 1 × 10 −6 and 5 × 10 −6 M. Studies with the pyridoxal phosphate oxim of this substance showed no remarkable inhibition of the nonspecific enzyme, and substantially less effect on specific decarboxylase than that obtained with 2-hydroxy-5-carbomethoxybenzyloxyamine itself. These results suggested that the inhibiting effect of the compound is partly directly on the coenzyme, forming a pyridoxal phosphate inactivator complex, but mainly on the apoenzyme, displacing pyridoxal phosphate from the apoenzyme. The fact that the inhibitory activity of the compound mainly depended on the relative strength of binding of pyridoxal phosphate to apoenzyme, showing substantially less effect on aromatic l -amino acid decarboxylase and on diamine oxidase than that on histidine decarboxylase, confirmed this assumption. Administration of the inhibitor to male rats resulted in lower levels of histamine in stomach, lungs, heart and skin. The minimum effective dose of the compound was 15 mg/kg maximal effects were obtained with doses of 45 mg/kg in lungs, heart and skin and 135 mg/kg in stomach. The maximal effects were observed 1–2 hr after the treatment and were significantly less at 5 hr. Repeated administration resulted in lower histamine levels than after single treatments. No toxic effects were apparent during repeated and prolonged administrations of the inhibitor.

Decarboxylase inhibition and structure-activity relationship studies with some newly synthetized benzyloxyamine and pyridylmethoxyamine derivatives.

Abstract 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.

Mercury-stimulated histamine uptake and binding in cultured astroglial and cerebral endothelial cells.

The effects of mercuric compounds on histamine uptake and binding to uptake carrier in cultured rat astroglial and cerebral endothelial cells were investigated. Experimental results showed that mercuric compounds produced strong stimulation of glial and cerebroendothelial histamine uptake over a concentration range of 25–500 μM. The stimulated histamine uptake showed characteristics similar to those described for basal uptake in terms of sensitivity to inhibitory agents (e.g., impromidine) and the requirement of external Na+. Mercury‐induced stimulation of histamine uptake could be abolished by sulfhydryl agents, dithiotreitol and cysteamine, indicating a complete reversal of, and not simply a protection from, the action of mercury.

Regulation of histamine synthesis: Altered synthesis and level of histamine in the hypothalamus of rats by repeated administration of histamine H1 and H2 receptor antagonists

Repeated administration of histamine H1 and H2 receptor antagonists resulted in significant alterations in the hypothalamic histamine level of rats and similar changes in the HD activity of the hypothalami were observed after these treatments. The changes in the level run parallel with the modifications in the HD activity.The effects of classical antihistaminergic agents were however opposite to that of the specific H2 antagonists, namely chloropyramine and tripelennamine markedly enhanced while metiamide and cimetidine diminished the hypothalamic level of histamine.