Studies on a Capillary-Rich Fraction Isolated from Brain: Histaminic Components and Characterization of the Histamine Receptors Linked to Adenylate Cyclase

Abstract: A fraction enriched in capillaries has been prepared from the guinea pig cerebral cortex. The purity of this fraction was checked by light- and electron-microscopic examination and by its high enrichment in alkaline phosphatase and γ-glutamyl transpeptidase. In the capillary-rich fraction, the endogenous level of histamine was 1.9%'of that measured in the initial hornogenate. The histamine-synthesizing enzyme, I-histidine decarboxylase, and the metabolizing enzyme, histamine-N-methyltransferase, were barely detectable. In addition, histamine elicits a twofold stimulation in the accumulation of cyclic AMP in this capillary fraction with an EC₅₀ of 5 γM. Agonists and antagonists of the two types of histamine receptors (H₁ and H₂) were used for the characterization of the receptors mediating this action: H₂-receptor agonists were able to activate the adenylate cyclase with "relative potencies" similar to that found on typical H₂-receptors, and cimetidine, a specific H₂-receptor antagonist, competitively inhibited the response to histamine with a K₁ value reflecting its interaction with a single population of H₂-receptors. On the contrary, data obtained with H₁-receptor agonists and antagonists reflect their interaction with H₂-receptors rather than H₁-receptors. Thus H₂-receptors are involved in the activation of adenylate cyclase of the capillary fraction.     

Ca2+/Calmodulin-Mediated Regulation of Agonist-Induced Sequestration of Gq Protein-Coupled Histamine H1 Receptors in Human U373 MG Astrocytoma Cells

Abstract: We investigated the regulation by intracellular Ca²⁺ of agonist-induced sequestration of G_q protein-coupled histamine H₁ receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H₁ receptors from the cell surface membrane was detected as the loss of [³H]mepyramine binding sites on intact cells accessible to the hydrophilic H₁-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [³H]mepyramine were mirrored by changes in the subcellular distribution of H₁ receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H₁ receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca²⁺ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca²⁺/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H₁-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of G_q protein-coupled receptors is transiently inhibited by Ca²⁺/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.     

Histamine Evokes Greater Increases in Phosphatidylinositol Metabolism and Catecholamine Secretion in Epinephrine-Containing than in Norepinephrine-Containing Chromaffin Cells

Abstract: Chromaffin cells have H₁ histamine receptors. Histamine, acting at these receptors, increases the metabolism of inositol-containing phospholipids and stimulates catecholamine secretion from Chromaffin cells. We have investigated the effects of histamine and other agents on the accumulation of inositol monophosphate (InsP₁) and catecholamine secretion in purified cultures of norepinephrine-containing and epinephrine-containing bovine Chromaffin cells. Histamine-stimulated InsP, accumulation in epinephrine cells was three times greater than that in norepinephrine cells. In contrast, bradykinin caused roughly equivalent increases in InsP¹ accumulation in the two Chromaffin cell subtypes. Histamine-stimulated catecholamine secretion was also greater in epinephrine cells than in norepinephrine cells, whereas high K⁺, bradykinin, phorbol 12, 13-dibutyrate, and angiotensin II all caused greater secretion from norepinephrine cells than from epinephrine cells. The density of H₁ receptors in epinephrine cells was approximately three times greater than that in norepinephrine cells. The greater density of H₁ receptors on epinephrine cells may account for the greater effects of histamine on InsP₁ accumulation and catecholamine secretion in these cells.     

Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [³²P]P_i-prelabeled rat brain cortical and hippocampal slices. The accumulation of [³²P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC₅₀ 18 µ M ) was inhibited by the H₁ receptor antagonist mepyramine with a K _i constant of 0.7 n M and was resistant to H₂ and H₃ receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC₅₀ 44 n M ) was not blocked by BQ-123, a specific antagonist of the ET_A receptor. Endothelin-3 and the specific ET_B receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC₅₀ values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H₁ and ET_B receptors, respectively.     

HISTAMINE-SENSITIVE ADENYLATE CYCLASE IN HYPOTHALAMUS OF RAT BRAIN: H1 AND H2 RECEPTORS

Abstract— In in vitro experiments on rat hypothalamic homogenates the effects of biogenic amines such as histamine (HA), noradrenaline (NA), dopamine (DA), serotonin (5-HT) and drugs such as isoprenaline (ISP), 2-(2-pyridyl)ethylamine (H₂ stimulant—H_ls), 4-methyl-histamine (H₂ stimulant H_2s), mepyramine (H₁ antagonistp H_la), cimetidine (H₂ antagonist—H_2a) were tested on adenylate cyclase activity. HA possessed a powerful stimulating effect on hypothalamic adenylate cyclase activity, higher than that shown by the other substances.
The stimulating effect of HA was greatest in hypothalamic tissue from male rats, while tissue from females showed only a modest stimulation. H_2s, induced a greater stimulation of adenylate cyclase than H_ls. On the other hand, the H_2a inhibited HA stimulation to a greater extent than the H_la, H_la and H_2a, when used together, completely inhibited the HA stimulation. HA may have a neurotrans-mitter role in the hypothalamus, and in this area there appears to be a mixed population of H₁ and H₂ receptors, with a majority of H₂ receptors.     

Involvement of Presynaptic Histamine H3 Receptors in the Modulation of Somatostatin Binding and Its Effects on Adenylyl Cyclase Activity in the Rat Frontoparietal Cortex

Abstract: Thioperamide (2 mg/kg, i.p.), a histamine H₃-receptor antagonist, increased the number of somatostatin (SS) receptors, with no change in the affinity constant, in the rat frontoparietal cortex. This effect was prevented by treatment with ( R )-α-methylhistamine (3.2 mg/kg, i.p.), a histamine H₃-receptor agonist. Thioperamide also induced an increase in SS binding in rats pretreated with mepyramine, a histamine H₁-receptor antagonist, or cimetidine, a histamine H₂-receptor antagonist. Pretreatment with mepyramine plus cimetidine administered simultaneously antagonized the thioperamide effect on SS binding. The increase in the number of SS receptors was accompanied by a greater SS-mediated inhibition of basal and forskolin-stimulated adenylyl cyclase (AC) activity in frontoparietal cortical membranes in the thioperamide group. Furthermore, the functional activity of the guanine nucleotide-binding inhibitory protein (G_i protein) was not altered by thioperamide or ( R )-α-methylhistamine administration in frontoparietal cortical membranes. In rats treated with mepyramine plus thioperamide or cimetidine plus thioperamide, the increase in the number of SS receptors was also accompanied by an increased SS inhibition of AC activity. Thioperamide induced a significant increase in SS-like immunoreactivity content in the frontoparietal cortex. Altogether, these results suggest that frontoparietal cortical histamine may play, at least in part, a role in the regulation of the somatostatinergic system.     

Histamine induces neural stem cell proliferation and neuronal differentiation by activation of distinct histamine receptors

Histamine has neurotransmitter/neuromodulator functions in the adult brain, but its role during CNS development has been elusive. We studied histamine effects on proliferation, cell death and differentiation of neuroepithelial stem cells from rat cerebral cortex in vitro . RT-PCR and Western blot experiments showed that proliferating and differentiated cells express histamine H₁, H₂ and H₃ receptors. Treatments with histamine concentrations (100 nM–1 mM) caused significant increases in cell numbers without affecting Nestin expression. Cell proliferation was evaluated by BrdU incorporation; histamine caused a significant increase dependent on H₂ receptor activation. Apoptotic cell death during proliferation was significantly decreased at all histamine concentrations, and cell death was promoted in a concentration-dependent manner by histamine in differentiated cells. Immunocytochemistry studies showed that histamine increased 3-fold the number of neurons after differentiation, mainly by activation of H₁ receptor, and also significantly decreased the glial (astrocytic) cell proportion, when compared to control conditions. In summary, histamine increases cell number during proliferative conditions, and has a neuronal-differentiating action on neural stem cells, suggesting that the elevated histamine concentration reported during development might play a role in cerebrocortical neurogenesis, by activation of H₂ receptors to promote proliferation of neural precursors, and favoring neuronal fate by H₁-mediated stimulation.     

Glucocorticoids Enhance Histamine-Evoked Catecholamine Secretion from Bovine Chromaffin Cells

Abstract: The synthetic glucocorticoid dexamethasone enhanced histamine-evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine-rich) and noradrenergic (norepinephrine-rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine-evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC₅₀ for dexamethasone was ∼1 n M . The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine-evoked catecholamine secretion in both dexamethasone-treated and untreated cells was blocked by the H₁ histamine receptor antagonist mepyramine but was not affected by the H₂ antagonist cimetidine; thus, dexamethasone appeared to enhance an H₁ receptor-mediated process. In the absence of glucocorticoids, H₁ receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H₁ receptor mRNA levels in both cell types. The increased expression of H₁ receptors presumably contributes to the enhancement of histamine-evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.     

Regulation of the Release of Interleukin-6 from Human Astrocytoma Cells

Abstract: Recent evidence suggests that the level of interleukin-6 (IL-6) is elevated in Alzheimer's disease (AD) brains. IL-6 is produced by reactive glial cells and could potentially affect neuronal survival. Understanding the biochemical mechanism that regulates the production and release of IL-6 by astrocytic cells may help to identify potential targets for therapeutic intervention in AD. In the present study, glial fibrillary acidic protein-positive human U373MG astrocytoma cells were used as a model of reactive astrocytes. Production of IL-6 in response to drug treatment was monitored with an ELISA assay. Histamine (1–100 µ M ), substance P (SP; 1–100 n M ), and human interleukin-1β (IL-1β; 1–30 p M ) stimulated the release of IL-6 in a time- and concentration-dependent manner, with EC₅₀ values of 4.5 µ M , 8 n M , and 4.5 p M , respectively. The respective effects of histamine, SP, and IL-1β were effectively blocked by the histamine H₁, SP, and IL-1 receptor antagonists, supporting a receptor-mediated event for these agents. Both histamine and SP enhanced the formation of inositol phosphates and increase intracellular calcium levels, suggesting that the phosphatidyl-inositol bisphosphate/protein kinase C pathway may be involved in the IL-6 release process. Indeed, phorbol 12-myristate 13-acetate, a protein kinase C activator, also evoked IL-6 release from the U373MG cells. On the other hand, IL-1β, which produces a much more robust release of IL-6 than histamine or SP, has no effect on inositol phosphate formation or intracellular calcium levels. The biochemical mechanism of the release of IL-6 in response to IL-1β remains to be elucidated.     

Histamine H1-Receptor Binding Sites in Guinea Pig Brain Membranes: Regulation of Agonist Interactions by Guanine Nucleotides and Cations

Abstract: Agonist, but not antagonist, interactions with histamine H₂-receptors labeled by [³H]mepyramine are regulated selectively by sodium, divalent cations, and guanine nucleotides. Sodium decreases the affinity of histamine and the agonist 2-amino-ethylpyridine for [³H]mepyramine sites in guinea pig brain membranes up to 10-fold. The effect of sodium is exerted to a lesser extent by lithium, while potassium and rubidium are much weaker. Guanine nucleotides also decrease the affinity of histamine for H₁ binding sites about twofold. GTP and its nonmetabolized analogue GMP-PNP as well as GDP exert similar effects, while GMP, ATP, ADP, and AMP are inactive. The effects of GTP and sodium on histamine interactions with H₁-receptors are additive. By contrast, certain divalent cations enhance the potency of histamine at H₁-receptors. Manganese is most potent, while magnesium is almost as active as manganese and calcium is essentially inactive. Sodium, divalent cations, and guanine nucleotides have negligible effects on the interactions of antihistamines with H₁-receptors.     

Endothelin-1 Increases Arachidonic Acid Release in C6 Glioma Cells Through a Potassium-Modulated Influx of Calcium

Abstract: Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca²⁺, and the demonstration of an increase in phospholipase A₂ (PLA₂) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA₂ in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca²⁺]_i triggered by Et-1, whereas valinomycin, which causes K⁺ efflux, not only caused a rapid rise in [Ca²⁺]_i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA₂ in this cell type requires calcium influx dependent on K⁺ efflux.     

Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

The role of protein kinase C (PKC) and calcium in the stimulation of prostaglandin D₂ (PGD₂) synthesis was investigated in primary rat astroglial cultures using the phorbol esters phorbol 12-myristate, 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and the calcium ionophore A₂₃₁₈₇. Both phorbol esters and the ionophore were able to stimulate PGD₂ synthesis in a concentration dependent manner. The inactive stereoisomers of PMA and PDB had no significant effect. Combinations of subthreshold concentrations of phorbol esters (10 nM PMA or 10 nM PBD) potentiated PG formation induced by 100 nM A₂₃₁₈₇. An even more pronounced effect was observed when phorbol ester concentrations were increased to 100nM. The contribution of extra- and intracellular calcium in phorbol ester or A₂₃₁₈₇ stimulated PGD₂ synthesis was evaluated by carrying out experiments with calcium-free media plus EGTA or with the intracellular calcium-chelating agent TMB-8. Ionophore stimulated PGD₂ release was shut down to basal values upon removal of extracellular calcium, whereas phorbol ester stimulated PGD₂ formation persisted at a reduced level. It was unabated also upon further addition of EGTA. In the presence of TMB-8, however, phorbol ester stimulated PGD₂ synthesis was completely suppressed. These data strongly suggest that PKC has an additional effect on the activation of phospholipase A₂ and subsequent prostanoid synthesis, which is independent from extracellular calcium and, thus, support the concept of more than one metabolic pathway in astrocytes that synergistically regulate phospholipase A₂ activity.     

Opposing Actions of D1 and D2-Dopamine Receptors on Arachidonic Acid Release and Cyclic AMP Production in Striatal Neurons

Abstract: D₁-and D₂-dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D₁ or D₂ receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [³H]AA, D₂-receptor stimulation enhanced release of [³H]AA produced by application of the Ca²⁺ ionophore A23187 or of the purinergic agonist ATP. By contrast, D₁-receptor stimulation inhibited [³H]AA release. This inhibitory effect of D₁ receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D₂ and D₁ receptors in striatum, potentiation and inhibition, respectively, of Ca²⁺-evoked AA release.     

The C Terminal Tail of the Histamine H2 Receptor Contains Positive and Negative Signals Important for Signal Transduction and Receptor Down-Regulation

Abstract: To examine the role of the C terminal tail in H₂ receptor regulation, three cDNAs, encoding truncated histamine H₂ receptor mutants (H₂T295, H₂T307, and H₂T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H₂ receptor binding of the H₂T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H₂T307) did not affect the binding properties of H₂ antagonists and histamine or histamine-induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H₂T341), which was able to form a ternary complex but was unable to fully activate the G_s protein on histamine exposure. Agonist-induced but not the cyclic AMP-dependent H₂ receptor down-regulation was more profound for the H₂T307 receptor, indicating that different structural elements of the H₂ receptor protein are involved in the cyclic AMP-dependent and independent pathways of H₂ receptor down-regulation. Taken together, in this study we identified regions in the C terminal tail of the H₂ receptor that act as positive and/or negative signals in H₂ receptor signaling and down-regulation.     

Activation of D1 and D2 Dopamine Receptors Inhibits Protein Kinase C Activity in Striatal Synaptoneurosomes

Abstract: The effects of D₁ and D₂ dopamine ligands on protein kinase C (PKC) activity were examined in synaptoneurosomes. Incubation with D₁ agonists (SKF 38393, fenodopam), in the presence of calcium, decreased the soluble and increased the particulate PKC activity. These effects were reversed by SCH 23390, which by itself had the opposite effect of increasing the soluble and decreasing the particulate PKC activity. In contrast, incubation with the D₂ agonists [LY 171555, (+)-3-(3-hydroxyphenyl)- N - n -propylpiperidine, RU 24213] increased the soluble and decreased the particulate PKC activity. These effects were reversed by sulpiride. (−)-3-(3-Hydroxyphenyl)- N - n -propylpiperidine had a D₂ antagonist profile. Apomorphine showed a biphasic dose-response change; i.e., it decreased particulate PKC activity at the D₂ receptor at low concentrations (0.1 µ M ) and increased it at the D₁ receptor at higher concentrations (10 µ M ). Pretreatment with tetrodotoxin or omission of calcium in the incubation medium did not alter the responses of the D₂ agonists, but it reversed the changes in PKC activity induced by the D₁ agonists and converted the biphasic response of apomorphine to a monophasic inhibition. These results indicate that (1) D₁ and D₂ dopamine receptors are negatively coupled to PKC and (2) the increase in particulate PKC activity seen with the D₁ drugs in the presence of calcium is mediated indirectly via a transneuronal effect.     

Photoaffinity Labeling and Electrophoretic Identification of the H1-Receptor: Comparison of Several Brain Regions and Animal Species

Abstract: The photoaffinity probe [^l25I]iodoazidophen-pyramine was used to label irreversibly the H₁-receptor in membranes of several guinea pig brain regions and of the cerebral cortex of the rat, mouse, and pig. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, two main bands were specifically labeled in all tissues: a 56-kilodalton (kDa) peptide and a 41-47-kDa peptide whose relative importance diminished in the presence of protease inhibitors. This indicates that, in all tissues examined, in spite of evidence for pharmacological heterogeneity, the ligand recognition domain of the H₁-receptor resides in a 56-kDa peptide.     

Tyrosine Hydroxylase Phosphorylation in Bovine Adrenal Chromaffin Cells: The Role of Intracellular Ca2+ in the Histamine H1 Receptor-Stimulated Phosphorylation of Ser8, Ser19, Ser31, and Ser40

Abstract: Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H₁ receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca²⁺. In contrast, the H₁-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 µ M ) and significantly reduced by prior exposure to caffeine (10 m M for 10 min) to deplete intracellular Ca²⁺. Trypticphosphopeptide analysis by HPLC revealed that the H₁ response in the presence or absence of extracellular Ca²⁺ resulted in a major increase in the phosphorylation of Ser¹⁹ with smaller increases in that of Ser⁴⁰ and Ser³¹. In contrast, although a brief stimulation with nicotine (30 µ M for 60 s) also resulted in a major increase in Ser¹⁹ phosphorylation, this response was abolished in the absence of extracellular Ca²⁺. These data indicate that the mobilization of intracellular Ca²⁺ plays a crucial role in supporting H₁-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.     

Ontogenetic Development of Histamine H1- Receptor Binding in Rat Brain

Abstract: Histamine H_1- receptors labeled with [³H]mepyramine in rat brain show an age-dependent development. [³H]Mepyramine receptor density and histidine decarboxylase activity in whole rat brain reach adult levels at 25–30 days after birth and they attain 50% of adult level at day 10 and 17, respectively. The apparently later development of histidine decarboxylase activity in whole rat brain is partly accounted for by a biphasic developmental increase of this enzymatic activity in cerebral cortex. For all other brain regions examined, the development of histamine H_1- receptors parallels that of histidine decarboxylase. The increase in [³H]mepyramine binding can be accounted for by an absolute increase in the numbers of the receptor sites, with no change in affinity. Subcellular fractionation studies indicate that histamine H_1- receptors are predominantly associated with synaptosomal fractions derived from both newborn and adult rat.