Isaac Blanco

Constitutive activity of H3 autoreceptors modulates histamine synthesis in rat brain through the cAMP/PKA pathway

We previously described that agonist-activated histamine H3 autoreceptors inhibit the stimulation of histamine synthesis mediated by calcium/calmodulin- and cAMP-dependent protein kinases (CaMKII and PKA respectively) in histaminergic nerve endings. In the absence of an agonist H3 receptors show partial constitutive activity, so we hypothesized that suppression of constitutive activity by an inverse agonist could stimulate these transduction pathways. We show here that the H3 inverse agonist thioperamide increases histamine synthesis in rat brain cortical slices independently from the amounts of extracellular histamine. Thioperamide effects were mimicked by the inverse agonists clobenpropit and A-331440, but not by the neutral antagonist VUF-5681. In contrast, coincubation with VUF-5681 suppressed thioperamide effects. The effects of thioperamide were completely blocked by the PKA inhibitor peptide myristoyl-PKI14-22, a peptide that did not block depolarization stimulation of histamine synthesis. In addition, thioperamide effects required depolarization and were impaired by blockade of N-type calcium channels (mediating depolarization), but not by CaMKII inhibition. These results indicate that constitutive activity of H3 receptors in rat brain cortex inhibits the adenylate cyclase/PKA pathway, and perhaps also the opening of N-type voltage sensitive calcium channels, but apparently not CaMKII.

The presence of two cellular pools of rat brain histamine

HISTAMINE (HA) is now believed to be a neurotransmitter in the central nervous system of mammals (for a review see SNYDER & TAYLOR, 1972; SCHWARTZ, 1975). In adult rat brain, although HA has been found to be concentrated in fractions enriched in synaptosomes (CARLINI & GREEN, 1963; MICHAELSON & COFFMAN, 1967; KATAOKA & DE ROBERTIS, 1967; KUHAR et al., 1971b; SNYDER et al., 1974), around 30% of the amine has been reported to be present in the crude nuclear fraction (SCHWARTZ, 1975) in contrast to the low amounts of other neurotransmitters found in that fraction (DE ROBERTIS et al., 1962; KUHAR et al., 1971a; COYLE & KUHAR, 1974). If brain HA is only considered a neurotransmitter, its ontogenic development in rat brain would seem anomalous since HA levels are found to be 5-6 times higher in the 5-day-old rat brain than in the adult brain (PEARCE & SCHANBERG, 1969; RONNBERG & SCHWARTZ, ~ ~ ~ ~ ; T I L L E M E N T et al., 1971). This pattern differs markedly from that described for other neurotransmitters (ABDEL-LATIF et al., 1970; COYLE & AXELROD, 1971; TISARI & RAUNU, 1975) whose development with age parallels synaptogenesis (AGHAJANIAN & BLOOM, 1967). In contrast, the development of L-histidine decarboxylase activity (HD), the HA synthesizing enzyme, follows a pattern (SCHWARTZ et al., 1971) similar to that described for other neurotransmitter synthesizing enzymes (SCHMIDT & SANDERS-BUSH, 1971 ; COYLE & AXELROD, 1972). During the first week of postnatal life, rat brain H D activity is very low increasing later to reach adult values during the fourth week. The discrepancies between the developmental patterns of HA and HD activity suggested the presence of a pool of brain HA other than the synaptosomal one. In the neonatal rat brain, HA has been found to be concentrated in the crude nuclear fraction (YOUNG et al., 1971). These findings could be related to the recently reported presence of mast cells, known to be rich in HA and poor in H D activity (SCHAYER, 1966), in rat brain (IBRAHIM, 1974; KRUGER, 1974; DROPP, 1976). In the present work we compare the subcellular distribution of HA in neonatal and adult rat brain with that of peritoneal mast cells homogenized together with brain tissue.

Histamine and mast cells in developing rat brain.

The number and distribution of mast cells in rat brain were determined at different postnatal ages. The number of brain mast cells was found to change during ontogenic development following the same pattern as brain histamine (HA) levels. The calculated HA content of brain mast cells was close to the HA content of the crude nuclear fraction at every age studied. Since most of the brain HA in the newborn sediments with the crude nuclear fraction, these results suggest that the developmental pattern of brain HA reflects changes in the number of brain mast cells, that is, in the size of the mast cell HA pool. The HA content of the supernatant of the crude nuclear fraction corrected for mast cell HA contamination, on the other hand, follows a developmental pattern similar to that of other known neurotransmitters.

Effect of Histamine on the Development of Astroglial Cells in Culture

The effect of histamine on different aspects of the growth of astrocytes was studied using primary cultures derived either from forebrain or from cerebellum of the rat. The influence on general growth and differentiation was monitored in terms of the activities of ornithine decarboxylase and glutamine synthetase enzymes, whereas [3H]thymidine incorporation into DNA was used as a specific index of cell proliferation. Treatment with 500 nM histamine of cells grown for 6 days in vitro, caused a time-dependent significant increase in ornithine decarboxylase activity of astrocytes from both sources. The maximum increase was observed at 4 h after histamine treatment, at that time the elevation in ornithine decarboxylase activity being about 80% and 300% over control values in the forebrain and the cerebellar astrocytes, respectively. Under similar experimental conditions, addition of histamine (500 nM) to medium resulted in a significant increase in [3H]thymidine incorporation into DNA in both types of cultures: in comparison with control, the elevation was about 45% at 48 h in forebrain astrocytes and at 24 h in cerebellar astrocytes. On the other hand, the specific activity of glutamine synthetase in cerebellar astrocytes was markedly enhanced (about 100%) by treatment with histamine (500 nM) for 4 days, but forebrain astrocytes were little affected. Addition of histamine to the culture medium produced no significant alteration in the activity of lactate dehydrogenase and protein content of either type of astroglial cells. The present findings, which support our earlier proposal that the biochemical properties of astrocytes differ between various brain regions, provide direct evidence for the involvement of histamine in the regulation of growth and development of astrocytes.

Polyamine uptake in cultured astrocytes: characterization and modulation by protein kinases.

Abstract: The properties and regulation of the polyamine transport system in brain are still poorly understood. The present study shows, for the first time, the existence of a polyamine transport system in cerebellar astrocytes and suggests that polyamine uptake is mediated by a single and saturable high‐affinity transport system for putrescine, spermine, and spermidine (Km = 3.2, 1.2, and 1.8 μM, respectively). Although substitution of NaCl by choline chloride produced a decrease in the putrescine, spermine, and spermidine uptake, it seems that polyamine transport in cerebellar astrocytes is not mediated by an Na+ cotransport as in the presence of Na+ and cholinium, polyamine uptake was much lower than when measured in a sucrose‐based medium. On the other hand, ouabain, gramicidin (a Na+ ionophore), and ionomycin (a Ca2+ ionophore) produced a strong inhibition of polyamine uptake, suggesting that membrane potential could have an important role in the functioning of the astroglial polyamine uptake system. Moreover, protein kinase C inhibition produced an enhancement of polyamine uptake, whereas stimulation of protein kinase C with phorbol esters inhibited polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein caused a marked reduction in the uptake. No effects on polyamine uptake were observed with inhibitors and activators of cyclic AMP‐dependent protein kinase or when Ca2+/calmodulin‐dependent protein kinase II was inhibited with KN‐62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities.

PROPERTIES OF RAT BRAIN HISTIDINE DECARBOXYLASE

Abstract– The properties of histidine decarboxylase (l‐histidine carboxylyase EC 4.1,1.22) have been studied in a whole rat brain homogenate. Optimum pH depended upon substrate concentration; the variations of Km and Vmax were determined as a function of pH. pH values lower than 6.0 caused a loss of enzymic activity; activity was stable at pH values higher than 6.0. Enzyme activity was proportional to temperature in the range 30‐45°C; temperature characteristic (μ) and Q10 were determined and thermal inactivation was studied. Addition of pyridoxal 5′‐phosphate increased enzyme activity. Dialysis of homogenates against phosphate buffer caused a partial loss of enzyme activity which could be restored by addition of the coenzyme to the incubation mixture. Enzyme activity was inhibited by α‐methylhistidine and benzene and was unaffected by α‐methyl DOPA. The properties correspond to those of a ‘specific’ histidine decarboxylase. However, the brain enzyme differs from the corresponding enzyme in peripheral tissues in the inability to achieve a total inhibition of activity by dialysis.

DIFFERENT ROUTES OF CA2+ INFLUX IN NMDA-MEDIATED GENERATION OF NITRIC OXIDE AND ARACHIDONIC ACID

Nitric oxide and arachidonic acid act as inter‐ and intracellular messengers in the central nervous system. It is well known that the NMDA‐mediated generation of nitric oxide and arachidonic acid is dependent on extracellular Ca2+. However, the role of voltage‐dependent calcium channels (VDCCs) in this regard is poorly understood. We report here that NMDA‐mediated nitric oxide production in striatal neuron cultures is blocked (80%) by the L‐type VDCC antagonist nifedipine, but not by ω‐conotoxin or ω‐agatoxin IVA, antagonists of the N‐and P‐type VDCCs respectively. By contrast, none of the VDCC antagonists inhibited the NMDA‐mediated release of arachidonic acid. These data indicate that permeation through different Ca2+ channels is responsible for the production of arachidonic acid and nitric oxide in striatal neurons.

Histamine modulation of glutamate release from hippocampal synaptosomes

Histamine is widely believed to act as a neuromodulator in the central nervous system. Histaminergic fibers arriving at the hippocampus could be involved in the modulation of glutamatergic neurotransmission. Therefore, we have investigated the effect of histamine on [3H]glutamate release from hippocampal synaptosomes by using a superfusion system. Calcium-dependent [3H]glutamate release was stimulated by KCI or 4-aminopyridine. When submaximal concentrations of the depolarizing agents were used (15 mM KCI or 50 microM 4-aminopyridine), histamine, acting via histamine H1 and H2 receptors, produced a concentration-dependent increase in the evoked release of glutamate. Maximal effect was obtained with 500 microM histamine. Histamine (up to 1 mM) did not modify basal release. These data suggest that histaminergic afferents may modulate activity-dependent glutamatergic presynaptic terminals in the hippocampus.

Pyridoxal-5'-phosphate as a cofactor for rat brain histidine decarboxylase.

The cofactor requirements of brain histidine decarboxylase activity have been studied. Preincubation with carbonyl reagents caused inhibition of the activity ranging from 90% for 10−2m‐semicarbazide, 10−3m‐phenylhydrazide and 10−3m‐hydroxylamine to 50% for isonicotinic acid hydrazide. Sodium borohydride, a reducing agent, also caused complete inhibition of activity. The histidine decarboxylase activity was maximal at 10−4m‐pyridoxal‐P concentration and was inhibited at higher concentrations of the cofactor.

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.