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.     

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.     

Biexponential Kinetics of (R)-α-[3H]Methylhistamine Binding to the Rat Brain H3 Histamine Receptor

The H3 histamine receptor is a high-affinity receptor reported to mediate inhibition of CNS histidine decarboxylase activity and depolarization-induced histamine release. We have used (R)-alpha-[3H]methylhistamine, a specific, high-affinity agonist, to characterize ligand binding to this receptor. Saturation binding studies with rat brain membranes disclosed a single class of sites (KD = 0.68 nM; Bmax = 78 fmol/mg of protein). Competition binding assays also yielded an apparently single class of sites with a rank order of potency for ligands characteristic of an H3 histamine receptor: N alpha-methylhistamine, (R)-alpha-methylhistamine greater than histamine, thioperamide greater than impromidine greater than burimamide greater than dimaprit. In contrast, kinetic studies disclosed two classes of sites, one with fast, the other with slow on-and-off rates. Density of (R)-alpha-[3H]methylhistamine binding followed the order: caudate, midbrain (thalamus and hippocampus), cortex greater than hypothalamus greater than brainstem greater than cerebellum. These data are consistent with an H3 histamine receptor, distinct from H1 and H2 receptors, that occurs in two conformations with respect to agonist association and dissociation or with multiple H3 receptor subtypes that are at present pharmacologically undifferentiated.     

Pyridoxal phosphate and glutamate decarboxylase in subcellular particles of mouse brain and their relationship to convulsions

The subcellular distribution of pyridoxal phosphate (PLP) was studied in mouse brain, as well as the effect of pyridoxal phosphate-γ-glutamyl hydrazone (PLPGH—a convulsant drug which decreases both PLP levels and glutamate decarboxylase activity [GAD] in whole brain) upon both the PLP concentration and the GAD activity in subcellular fractions. An electron microscopic evaluation of the subcellular particles of control and PLPGH-treated animals was also carried out. The main findings were the following: (1) PLP was localized mainly in the supernatant and crude mitochondrial fractions; two-thirds of the amount present in the latter were located in the subfraction containing pure mitochondria, and the remainder was in the synaptosomal fraction. After osmotic disruption of synaptosomes, PLP was found in both the intrasynaptosomal mitochondria and the synaptoplasm. (2) Treatment of mice with PLPGH decreased levels of PLP in several brain fractions, this effect being much more notable in the soluble fractions than in the particulate fractions. After osmotic disruption of the synaptosomes, a specific decrease of PLP in the synaptoplasm was observed. (3) Treatment with PLPGH produced also an inhibition of GAD activity in most of the fractions studied, when this enzyme was assayed in the absence of PLP. In general, the inhibition was greater in those fractions in which levels of PLP were also affected. In synaptosomes, this correlation between the decreased levels of PLP and decreased activity of GAD occurred only in the synaptoplasm. (4) The activation of GAD by PLP added to incubation mixtures was much greater in those fractions from PLPGH-treated animals which displayed extensive inhibition of GAD, in comparison to the corresponding fractions from control animals. (5) No ultrastructural changes were detected in the subcellular fractions from treated animals. Our results show that the decreases of both the levels of PLP and the activity of GAD (as previously found in whole brain) actually occur in the synaptosomes, a finding that supports the hypothesis that the role of PLP in the mechanisms controlling excitability can be explained, at least in part, by its regulatory action on GAD activity, which in turn determines the rate of GABA synthesis at the nerve endings.     

Sulfur Amino Acid Metabolism in the Developing Rhesus Monkey Brain: Subcellular Studies of Taurine, Cysteinesulfinic Acid Decarboxylase, γ-Aminobutyric Acid, and Glutamic Acid Decarboxylase

Abstract: Taurine, cysteinesulfinic acid decarboxylase (CSAD), glutamate, γ-aminobutyric acid (GABA), and glutamic acid decarboxylase (GAD) were measured in subcellular fractions prepared from occipital lobe of fetal and neonatal rhesus monkeys. In addition, the distribution of [³⁵S]taurine in subcellular fractions was determined after administration to the fetus via the mother, to the neonate via administration to the mother prior to birth, and directly to the neonate at various times after birth. CSAD, glutamate, GABA, and GAD all were found to be low or unmeasurable in early fetal life and to increase during late fetal and early neonatal life to reach values found in the mother. Taurine was present in large amounts in early fetal life and decreased slowly during neonatal life, arriving at amounts found in the mother not until after 150 days of age. Significant amounts of taurine, CSAD, GABA, and GAD were associated with nerve ending components with some indication that the proportion of brain taurine found in these organelles increases during development. All subcellular pools of taurine were rapidly labeled by exogenously administered [³⁵S]taurine. The subcellular distribution of all the components measured was compatible with the neurotransmitter or putative neuro-transmitter functions of glutamate, GABA, and taurine. The large amount of these three amino acids exceeds that required for such function. The excess of glutamate and GABA may be used as a source of energy. The function of the excess of taurine is still not clear, although circumstantial evidence favors an important role in the development and maturation of the CNS.     

The Significance of Mast Cells as a Source of Histamine in the Mouse Brain

Abstract: Knowledge of the relative contributions of mast cells and neurons to the overall pool of histamine in the brain is a prerequisite to determining the significance and role of this amine in brain function. Consequently, we analyzed the levels of brain histamine in four genotypes (+/+, W/+, W^v/+ , and WIW^v ) of WBB6F₁ mice, whose numbers of brain-associated mast cells vary in a genotypically specific manner. Although mast cell numbers ranged from a total absence of mast cells (W/ W^v ) to an average of about 500 mast cells/brain ( W/+ ), no significant differences between genotypes were found in the quantities of histamine in whole brains, brain regions, or crude subcellular fractions. Thus, in this strain of mice, mast cells are not a significant source of histamine in the brain. This suggests that most of the histamine is of neuronal origin. Since neuronal histamine levels are maintained only by continued histidine decarboxylase activity, complete inhibition of this enzyme by α-fluoromethylhistidine, a "suicide" inhibitor of histidine decarboxylase, would totally deplete W/W^v mice of brain histamine. This was not found to occur in the W/W^v mice, suggesting that neuronal stores of histamine can be maintained in the absence of histidine decarboxylase, or that an additional nonneuronal, non-mast cell source of histamine exists in the W/W^v mouse brain.     

Rapid and simple determination of histamine-N-methyl transferase activity by high-performance liquid chromatography with UV detection.

A rapid, simple and low-cost assay method of histamine-N-methyltransferase activity was developed. Methylhistamine, which was separated from the enzymatic reaction system on reversed-phase high-performance liquid chromatography using an ion-paired chromatographic technique, was detected spectrophotometrically at 226 nm. The mobile phase used for the separation of methylhistamine was 0.05M NH4H2PO4 (pH 3.0) containing 2 mM of sodium octanesulfonate. The new assay technique could detect methylhistamine as an enzyme activity product of histamine-N-methyltransferase in the brain and kidney of rats. Chloropheniramine maleate, an antihistamine, activated the histamine-N-methyltransferase. Whether neurotransmitter or neuromodulator, the role of histamine in the brain has not yet been made clear. Therefore, the present method could be applicable for the enzymatic investigation of histamine metabolism in central nervous system or inflammatory reactions.     

Metabolism of intracerebrally administered histidine, histamine and imidazoleacetic acid in mice and frogs

Abstract— After intracerebral administration of [¹⁴C]histidine to mice the major labelled substance found in the brain extracts was histidine itself; small amounts of labelled carnosine and homocarnosine were detected. No other labelled substances were detected on radio- autographs of two-dimensional TLC's of the extracts. In the case of the frog, radioactive histidine, N-acetylhistidine, carnosine and homocarnosine were found in the brain extracts at various times after intracerebral injection of the labelled histidine. With time, approximately 90 per cent of the radioactivity in the extracts was found in the N-acetylhistidine. In neither the mouse nor frog could we find unequivocal evidence for the formation either of histamine or imidazoleacetic acid from intracerebrally administered histidine, but our analytical procedures may have lacked sufficient sensitivity to pick up extremely low activities of histamine and imidazoleacetic acid. Experiments with [¹⁴C]histamine administered intracerebrally into mice demonstrated the major pathway of metabolism in brain to be histamine → methylhistamine → methylimidazoleacetic acid. No detectable label appeared in inlidazoleacetic acid. In the frog intracerebral administration of the labelled histamine led to the formation of methylhistamine and imidazoleacetic acid, but at most only traces of methylimidazoleacetic acid were found. The injection of [¹⁴C]imidazoleacetic acid intra- cerebrally into mice and frogs resulted in virtually no loss of the label in the form administered in the frog brain over a period of 4 h and in a slow rate of decrease in the mouse brain. No radioactive metabolites of imidazoleacetic acid were found in either species. The limitations of trying to determine natural functions of substances in brain by following the fate of exogenously administered materials is discussed.     

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.     

Presence and distribution of histaminergic components in rat and bovine retina

The presence of histamine and its related enzymes, histidine decarboxylase and histamine N-methyltransferase and the subcellular distribution of the amine and of H₁-receptors were studied in the retina of two mammalian species. Histamine is present in rat and bovine retinas in concentrations (113 ± 10 and 72 ± 9 ng/g wet tissue, respectively) similar to those found in the brain. Histological examination and release experiments with Compound 48/80 performed in rat retina indicate a non mast cell location for the amine. Histidine decarboxylase and histamine N-methyltransferase activities in rat and bovine retinas were also comparable to those found in brain cortex suggesting that histamine can be synthesized and catabolyzed in situ. Subcellular fractionation of bovine retina showed that both the amine and H₁-receptors are concentrated in particulate fractions where small sized synaptosomes sediment, presumably derived from horizontal and amacrine cells. These results are in agreement with a neurotransmitter or neuromodulator role for histamine in cells of the retinal inner nuclear layer.     

Developmental effects of alpha-fluoromethylhistidine, an irreversible inhibitor of histidine decarboxylase, on growth and on levels and turnover of catecholamines

To examine the potential participation of histamine in cellular development, neonatal rats were given daily 50 mg/kg doses of alpha-fluoromethylhistidine (FMH), an irreversible inhibitor of histidine decarboxylase; previous studies have shown this regimen to deplete both neurotransmitter and nonneurotransmitter pools of histamine. No inhibition of growth was observed for either body weight, brain weight, heart weight or kidney weight; indeed, kidney weights tended to become supranormal toward weaning in the FMH-treated pups. Similarly, FMH failed to affect protein synthesis, confirming the lack of systemic toxicity of this amino acid as well as indicating that maintenance of histamine levels is not required for growth to proceed. In contrast, FMH did have a deleterious effect on development of the cardiac-sympathetic axis, with deficits in norepinephrine levels appearing during the third postnatal week. The deficits were not present in other catecholaminergic systems (brain noradrenergic or dopaminergic neurons and renal sympathetic neurons). The subnormal cardiac norepinephrine levels were preceded by a sharp increase in the turnover of norepinephrine at precisely the age at which central control of sympathetic tone first appears. The developmental effects of FMH indicate that, although it is unlikely that histamine participates in a major way in general control of cellular maturation, a more selective role for histamine as a trophic agent or neurotransmitter may exist during defined periods in nervous system development.     

IN VIVO FORMATION AND CATABOLISM OF [14C]HISTAMINE IN MOUSE BRAIN

Abstract— The formation of histamine in brain was studied in mice injected with l -[¹⁴C]-histidine (ring 2-¹⁴C) intravenously (i.v.) or intracerebrally; [¹⁴C]histamine appeared rapidly and exhibited a rapid rate of turnover. Drugs known to block various pathways of histamine catabolism were tested for effects on brain–[¹⁴C]histamine and [¹⁴C]-methyl-histamine in mice given (1) [¹⁴C]histamine i.v., (2) [¹⁴C]histamine intracerebrally, and (3) l -[¹⁴C]histidine i.v. Blood-borne histamine did not enter brain; brain histamine was formed locally by decarboxylation of histidine Methylhistamine did cross the blood-brain barrier. Methylation was the major route of histamine catabolism in mouse brain and some of the methylhistamine formed was destroyed by monoamine oxidase. No evidence for catabolism by the action of diamine oxidase was found.     

Effects of α-fluoromethylhistidine (FMH), an irreversible inhibitor of histidine decarboxylase,on development of brain histamine and catecholamine systems in the neonatal rat

Daily administration of FMH to neonatal rats produced long-lasting inhibition of histidine decarboxylase in hypothalamus and cerebral cortex and led to depletion of histamine in both brain regions. The onset of depletion was more rapid in cerebral cortex, a region in which non-neurotransmitter pools of histamine predominate in early postnatal life, appearing as early as postnatal day 3; depletion in the hypothalamus, a region rich in histaminergic neuronal projections, appeared later. No effects were seen on body or brain growth, nor was development of other biogenic amine systems affected. FMH thus provides a selective probe for examining the role of histamine in brain development.     

METHYLHISTAMINE: EVIDENCE FOR SELECTIVE DEAMINATION BY MAO B IN THE RAT BRAIN IN VIVO

Abstract— A new method has been developed for the separation of histamine and its metabolites after intracisternal injection of [³H]histamine into the rat brain, involving solvent extraction and subsequent thin-layer chromatography. The effect of graded doses of the MAO inhibitors deprenil and pargyline, which at relatively low doses inhibit preferentially the B form (phenethylamine deaminating) of the enzyme, and clorgyline, which mainly inhibits the A form (serotonin, noradrenaline and dopamine deaminating) on the brain levels of intracisternally injected [³H]histamine and its labelled metabolites was studied and compared to MAO A and B activity as determined with the substrates serotonin and phenethylamine, respectively. In addition, the time-course of the effects of a single dose of pargyline (50mg/kg subcutaneously) was investigated. No [³H]imidazoleacetic acid could be detected in any of the control or treated animals. [³H]Histamine accounted for 9–12% of the total extracted radioactivity and this was not altered significantly by pretreatment with any of the MAO inhibitors up to high doses, at which both MAO A and B activities were completely inhibited. In the controls, 40–43% of the total extracted radioactivity was [³H]methylhistamine and 28–30% was [³H]methylimidazoleacetic acid. Deprenil and pargyline caused [³H]methylhistamine levels to increase in a dose-dependent manner up to about 150% of control levels and those of [³H]methylimida-zoleacetic acid to decrease concomitantly to about 10% of control levels. Clorgyline in doses up to 10 mg/kg subcutaneously (s.c.) had no effect on the levels of these two metabolites. The dose-response curves of the effects of deprenil and pargyline on [³H]methylimidazoleacetic acid levels were congruent with those of the MAOI effects on MAO B activity and not with those on MAO A activity. Pargyline (50 mg/kg s.c.) had a long lasting effect on the accumulation of [³H]methylhistamine and [³H]methylimidazoleacetic acid. Recovery occurred within 21 days, and the half-lives observed were 5.3 and 5.6 days, respectively. This compares well to the half-life for the recovery of MAO B activity reported earlier after the same dose of pargyline (5.5 days). These results suggest that methylhistamine is metabolized selectively by MAO B in rat brain. Moreover, the fact that clorgyline, at doses where phenethylamine deamination is already considerably inhibited, did not affect the deamination of methylhistamine, suggests that the latter is an even more selective substrate for MAO B than phenethylamine itself. Therefore, small doses of deprenil (0.3–3 mg/kg s.c.) or pargyline (1–3 mg/kg) can be used to influence histamine catabolism without interfering with catecholamine or serotonin deamination.     

Profile of phospholipase A2 activity in subcellular fractions and lamellar bodies of developing, neonatal and adult rabbit lung. Correlation with intracellular levels of disaturated phosphatidylcholine

Phospholipase A2 activity was determined in subcellular fractions and lamellar bodies of fetal, neonatal and adult rabbit lungs. Specific activity in most fractions decreased from the 24th to the 28th day of gestation. All fractions except the mitochondrial and the nuclear fractions exhibited a sharp increase in activity in the newborn lung. Specific activity in the adult lung generally declined in comparison to neonatal values. During gestation total enzyme activity per gram of lung was concentrated in the cytosolic fraction. With the exception of the lamellar body fraction, the total content of phospholipase A2 activity increased dramatically in all fractions from the neonatal lung. The lamellar body fractions displayed both low specific activity and low total enzyme activity during gestation. Specific activity increased dramatically in the neonatal and adult lung but still accounted for only a small fraction of the activity in comparison to the other subcellular fractions. The subcellular content of disaturated phosphatidylcholine (PC) appeared to correlate well with the activity of phospholipase A2 in the neonatal mitochondrial, microsomal and cytosolic fractions. Since decreasing prenatal enzyme levels are associated with increasing disaturated PC content, the alkaline and calcium-dependent phospholipase A2 may not be directly involved in disaturated PC synthesis in the fetus. However, postnatally, the correlation between the pattern of production of disaturated PC and the activity of the phospholipase A2 indicates a role for this enzyme in surfactant-related disaturated PC synthesis.     

TAURINE IN DEVELOPING RAT BRAIN: SUBCELLULAR DISTRIBUTION AND ASSOCIATION WITH SYNAPTIC VESICLES OF [35S]TAURINE IN MATERNAL, FETAL AND NEONATAL RAT BRAIN

The concentration of taurine in the brain of the fetus in several species is higher than that found in the mature animal. In order to explore the functional significance of this, we have studied the subcellular distribution of taurine and [³⁵S]taurine in the brain of the mother, the fetus and the neonate after [³⁵S]taurine was administered to pregnant rats. In maternal brain, the distribution of taurine and of radioactivity (all of which was recovered from brain as taurine) in the subcellular fractions of maternal brain were essentially identical and were recovered primarily in two fractions (72% taurine, 71% [³⁵S]taurine was soluble, S₃; 16% and 17%, respectively, was in the crude mitochondrial and synaptosomal fraction, P₂). After further fractionation of P₂, most of the taurine and [³⁵S]taurine were in the cytoplasmic, O, and the synaptosomal, B, fractions. In the neonatal brain, shortly after birth there was a decrease in taurine and [³⁵S]taurine recovered in the supernatant fraction, S₃, accompanied by an increase in the percentage of taurine and [³⁵S]taurine recovered in the crude mitochondrial fraction. A small percentage of taurine and [³⁵S]taurine was consistently recovered in the synaptic vesicle fraction. Fractionation of the synaptic vesicles on a gel column separated the vesicle bound taurine completely from the free taurine: approx 1% of the taurine in the synaptic vesicle fraction was eluted with vesicles and could not be released by hypo-osmotic shock. The pattern of development in subcellular fractions of neonatal rat brain labelled with [³⁵S]taurine via intraperitoneal injections of the pregnant mother may be an indication of maturation or protection of putative taurinergic nerve endings.     

Subcellular alterations in cyclic AMP-binding and protein kinase activities during ontogeny in the rat cerebellum

Ontogenic relationships between levels of cyclic AMP-binding activity and protein kinase activity were examined in subcellular fractions of the cerebellum during the first 3 weeks of neonatal life. A progressive increase in cyclic AMP levels was paralleled by an increase in cyclic AMP bindign by the nuclear and cytosol fractions, but not by the mitochondrial or microsomal fractions. Utilization of heat-stable protein kinase inhibitor permtited distinction of the cyclic AMP-dependent from the cyclic AMP-independent form of the protein kinase population. Cyclic AMP-dependent protein kinase increased between days 4 and 20 to represent a progressively greater proportion of the protein kinase population. In all subcellular fractions alterations of cyclic AMP-dependent protein kinase during neonatal development paralleled changes in binding of cyclic AMP to protein in these fractions. In both the nuclear and cytosol fractions cyclic AMP-dependent protein kinase activity increased progressively between days 4 and 20, i.e. 64 ± 6 to 176 ± 16 and 79 ± 12 to 340 ± 12 pmol/min per mg protein, respectively. Cyclic AMP-dependent protein kinase activity in the mitochondrial fraction declined during the postnatal period studied, and in the microsomal fraction it rose to a non-sustained peak at 14 days and fell thereafter. Unlike the cyclic AMP-dependent form, cyclic AMP-independent protein kinase activity did not follow the ontogenetic pattern of cyclic AMP-binding activity. The specific activity of nuclear cyclic AMP-independent protein kinase did not change during days 4–20, and a non-sustained rise of cyclic AMP-independent protein kinase activity in both cytosol and microsomal fractions during the 7th–12th day tended to parallel more closely known patterns of postnatal proliferative growth. The findings reported herein indicate that the ontogenic pattern of cyclic AMP-dependent protein kinase varies between different subcellular fractions of the neonatal cerebellum, that these patterns parallel the changes in cyclic AMP-bidign activity, and suggest that the component parts of the cyclic AMP system may develop as a functional unit.     

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.     

ACTIVITIES OF 3,4-DIHYDROXY-l-PHENYLALANINE AND 5-HYDROXY-l-TRYPTOPHAN DECARBOXYLASES IN RAT BRAIN: ASSAY CHARACTERISTICS AND DISTRIBUTION

Abstract— Optimal assay conditions for decarboxylation of 3,4-dihydroxy- l -phenylalanine (DOPA) and 5-hydroxy- l -tryptophan (5-HTP) were determined in homogenates of rat brain by use of a sensitive, precise microradiometric technique. The two activities exhibited widely different optima for pH, temperature and substrate concentrations. The activity of 5-HTP decarboxylase was stimulated 2-fold by added pyridoxal-5-phosphate and was relatively resistant to antagonists of pyridoxal-P. By contrast, the activity of DOPA decarboxylase was stimulated 20-fold by added coenzyme and could be completely inhibited by carboxyl trapping agents. DOPA decarboxylase activity in subcellular fractions of brain was associated predominately with the soluble fractions and its distribution in the various fractions closely paralleled that of lactic acid dehydrogenase. 5-HTP decarboxylase activity in brain was distributed almost equally between soluble and particulate fractions, and its distribution within the particulate fractions differed from that of succinic acid dehydrogenase. The two decarboxylases in brain exhibited a 7-fold divergence in relative specific activity when their respective distributions in subcellular fractions were compared. Similarly, the regional distributions of the two decarboxylases in rat brain did not parallel one another; e.g. there was a 4-fold difference between the ratio of the two activities in cerebellum and that found in the corpus striatum.     

The subsynaptosomal localization of histamine, histidine decarboxylase and histamine methyltransferase in rat hypothalamus

Abstract— We have examined the subcellular localization of histamine, histamine methyltransferase (EC 2.1.1.8) (HMT) and histidine decarboxylase (EC 4.1.1.22) in rat hypothalamus after osmotic lysis of synaptosome-containing primary particulate fractions. When crude mitochondrial fractions are subjected to osmotic lysis, histamine is retained within particulate structures, while HMT is released into the supernatant fluid. The majority of histidine decarboxylase activity is also recovered in the supernatant fluid, although more histidine decarboxylase than HMT is retained in particulate fractions. After sucrose gradient fractionation of osmotically lysed crude mitochondrial or microsomal pellets, histamine is also retained in particulate structures, with the greatest amount occurring in a fraction enriched in synaptic vesicles. In these sucrose gradients histidine decarboxylase activity shows a greater particulate localization than does HMT activity.