R. Rodriguez

Properties and Ontogenic Development of Membrane‐Bound Histidine Decarboxylase from Rat Brain

Abstract: Histidine decarboxylase (HD) activity was determined in high‐speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates. Twenty‐eight percent of the HD activity was associated with membranes, and the remaining was soluble. Several properties of the soluble and membrane‐bound HD were compared. No significant differences in the values of Km for histidine and pyridoxal 5′‐phosphate were observed. The solubilization of membrane‐bound HD with Triton X‐100 resulted in an increase of 60% over the nonsolubilized activity with no changes in the Km for substrate and cofactor. The proportion of free pyridoxal 5′‐phosphate‐independent activity was identical in both fractions. The soluble and membrane‐bound forms of the enzyme differ slightly in their pH‐activity profiles, although both enzymes showed an optimum pH near 6.5. The HD activities present in soluble and membrane fractions were determined at different postnatal ages. The soluble activity increased until day 90, whereas the membrane‐bound activity became stabilized from day 20.

Histamine H2-receptor mediated activation of neonatal rat brain ornithine decarboxylase in vivo

The effect of histamine (HA) administered via intracerebroventricular injection on ornithine decarboxylase (ODC) activity was studied in neonatal rat brain. The HA effect was dose and time dependent. Maximal increase in ODC activity was achieved 2 hr after administration of 10 micrograms HA (38% over control levels). Impromidine (HA H2-agonist) mimicked the effect of HA on ODC and ranitidine (HA H2-antagonist) inhibited the response to HA. Neither 2-thiazolylethylamine (HA H1-agonist) nor mepyramine (HA H1-antagonist) modified control ODC activity. The HA-releasers, compound 48/80 and polymixin B sulfate, elicited an increase in brain ODC activity of 35% and 32%, respectively, over the control value.

Histamine stimulated synaptosomal Ca2+ uptake through activation of calcium channels.

Histamine stimulated Ca2+ uptake in synaptosomes was completely inhibited by the slow Ca2+ channel antagonists verapamil, cinnarizine and flunarizine, and slightly inhibited by nifedipine and diltiazem. Ca2+ uptake in synaptosomes depolarized or predepolarized with varying K+ concentrations was increased by histamine, in both conditions, until 30mM K+. At higher K+ concentrations histamine was not able to alter K+ effects in either conditions. 30mM K+ stimulated uptake of Ca2+ in the absence or presence of histamine was not inhibited by verapamil and diltiazem. However nifedipine slightly inhibited K+ and K+ +histamine effects. 3-Isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP potentiated (10%) the uptake of Ca2+ in synaptosomes induced by histamine. Dibutyryl cyclicAMP alone however decreased the basal Ca2+ uptake in a concentration-dependent manner. Verapamil, but not diltiazem, antagonized the effects elicited by 3-isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP in the presence of histamine. The data suggest that the increase in synaptosomal Ca2+ uptake induced by histamine is mediated by the activation of the voltage sensitive calcium channels, and possibly a cyclicAMP-dependent protein kinase phosphorylation can modulate the opening of Ca2+ channels.

Subcellular localization of brain mast cell histamine in developing rat

The intracerebroventricular administration of compound 48/80 or polymixin B to rats 0 to 60 days old, produced a decrease both in the histamine which sediments in the crude nuclear fraction, as well as in the number of mast cells in the brain. In contrast, the histamine-releasers did not affect histamine levels in subcellular fractions where neuronal histamine is found. Once released, histamine disappeared rapidly (t 1/2 = 3.8 min). In untreated animals and in those treated with histamine releasers, the number of mast cells/g in the whole brains of developing rats and in the cerebral regions of adult rats showed a close correlation with the histamine levels in the crude nuclear fraction. The content of histamine per mast cell in adult rat brain was estimated to be about 13 pg/cell. Histologic examination of the subcellular fractions revealed the presence of intact mast cells in the crude nuclear fraction obtained from untreated animals, and of degranulated mast cells in the same fraction obtained from animals treated with histamine releasers. The mast cell contribution to adult rat brain histamine levels was about 22%. Our results strongly support that most of the histamine which sediments in the crude nuclear fraction of the rat brain is located in mast cells. Determination of histamine in the crude nuclear fraction and in the supernatant of this fraction is proposed as an easy way for identifying the cellular pool altered by any treatment affecting brain histamine levels.

Calcium effects on the solubilization of membrane-bound histidine decarboxylase in the rat brain.

Abstract: In a previous work we have shown that histidine decarboxylase (HD) activity is found in a soluble and a membrane‐bound form. A major part (82%) of the membrane‐bound HD activity in the crude mitochondrial fraction (P2) was present in the synaptic plasma membrane‐containing subfraction. Physiological concentrations of Ca2+ had no direct effect on HD activity but caused a solubilization of ∼50% of membrane‐bound HD in the P2 fraction. Mg2+ had similar but lower effects (20% solubilization) than Ca2+. Incubation with depolarizing concentrations of K+ in the presence of 1 mM CaCl2 caused a significant (30%) solubilization of HD.

A comparative study of histamine and K+ effects on (Ca2+Mg2+)-ATPase activity in synaptosomes

Histamine (10(-4) M) and 60 mM K+, but not 60 mM Na+ or 60 mM choline+, increased the maximal synaptosomal (Ca(2+)-Mg2+)-ATPase activity by 15 and 36% respectively and decreased the extrasynaptosomal Ca2+ concentration necessary to reach it. Histamine and K+ enhanced the synaptosomal (Ca(2+)-Mg2+)-ATPase activity in a concentration-dependent manner. In synaptic plasma membranes histamine (10(-4) M) and 60 mM choline+ were not able to alter the enzymatic activity, however 60 mM K+ and 60 mM Na+ elevated (Ca(2+)-Mg2+)-ATPase activity by 20 and 15%, respectively, without altering the affinity for Ca2+. Histamine effects in synaptosomes were mediated by H2 receptor stimulation. 3-Isobutyl-1-methyl-xanthine (10(-4) M) potentiated (15%) the maximal histamine effect. The slow Ca2+ channel antagonists verapamil and diltiazem, both at 10(-6) M, completely inhibited K+ effects in synaptosomes, however histamine effects were only blocked by verapamil. The data suggest that K+ and histamine effects on synaptosomal (Ca(2+)-Mg2+)-ATPase activity are mediated by increases of intrasynaptosomal Ca2+ levels. Moreover, histamine effects on synaptosomal enzyme activity were mediated by cAMP.

Synaptosomal (Ca2+-Mg2+)-ATPase activity modulation by cyclic AMP

Dibutyryl cyclic AMP, in a concentration-dependent manner, increased synaptosomal (Ca2+-Mg2+)-ATPase activity, but in synaptic plasma membranes lacked any effect. The maximal enzyme activity in synaptosomes was increased by 38%, leaving unaltered the extrasynaptosomal Ca2+ concentration necessary to reach it. In the presence of 5 microM cyclic AMP, cyclic AMP-dependent protein kinase increased (30%) maximal (Ca2+-Mg2+)-ATPase activity in synaptic plasma membranes, but the apparent affinity for Ca2+ was not modified. This effect was partially inhibited (60%) by a cyclic AMP-dependent protein kinase inhibitor. The data suggest that synaptosomal (Ca2+-Mg2+)-ATPase activity is modulated by a cyclic AMP-dependent phosphorylation reaction.

Histamine increases ornithine decarboxylase activity in different neonatal rat brain subcellular fractions.

The subcellular localization of ornithine decarboxylase activity was investigated in control or histamine-treated 6-day-old rat brains. Ornithine decarboxylase activity was located mainly in the cytosolic fraction (75%), whereas significant lower activity was observed in the crude nuclear (7%), crude mitochondrial (15%) and crude microsomal (3%) fractions. Cytosolic and nuclear ornithine decarboxylase activity were increased after treatment with histamine (35 and 400%, respectively). Histamine did not affect ODC activity in crude mitochondrial and crude microsomal fractions. The present findings suggest that the major part of ODC activity in the neonatal rat brain is located in the cytosolic fraction and are the first showing an induction of nuclear ODC activity by a neuromodulator.