Histamine H3 Receptor Activation Research Articles

Involvement of Protein Kinase Cδ/Extracellular Signal-regulated Kinase/Poly(ADP-ribose) Polymerase-1 (PARP-1) Signaling Pathway in Histamine-induced Up-regulation of Histamine H1 Receptor Gene Expression in HeLa Cells

The histamine H(1) receptor (H1R) gene is up-regulated in patients with allergic rhinitis. However, the mechanism and reason underlying this up-regulation are still unknown. Recently, we reported that the H1R expression level is strongly correlated with the severity of allergic symptoms. Therefore, understanding the mechanism of this up-regulation will help to develop new anti-allergic drugs targeted for H1R gene expression. Here we studied the molecular mechanism of H1R up-regulation in HeLa cells that express H1R endogenously in response to histamine and phorbol 12-myristate 13-acetate (PMA). In HeLa cells, histamine stimulation caused up-regulation of H1R gene expression. Rottlerin, a PKCδ-selective inhibitor, inhibited up-regulation of H1R gene expression, but Go6976, an inhibitor of Ca(2+)-dependent PKCs, did not. Histamine or PMA stimulation resulted in PKCδ phosphorylation at Tyr(311) and Thr(505). Activation of PKCδ by H(2)O(2) resulted in H1R mRNA up-regulation. Overexpression of PKCδ enhanced up-regulation of H1R gene expression, and knockdown of the PKCδ gene suppressed this up-regulation. Histamine or PMA caused translocation PKCδ from the cytosol to the Golgi. U0126, an MEK inhibitor, and DPQ, a poly(ADP-ribose) polymerase-1 inhibitor, suppressed PMA-induced up-regulation of H1R gene expression. These results were confirmed by a luciferase assay using the H1R promoter. Phosphorylation of ERK and Raf-1 in response to PMA was also observed. However, real-time PCR analysis showed no inhibition of H1R mRNA up-regulation by a Raf-1 inhibitor. These results suggest the involvement of the PKCδ/ERK/poly(ADP-ribose) polymerase-1 signaling pathway in histamine- or PMA-induced up-regulation of H1R gene expression in HeLa cells.

Proadrenergic effects of cardiac natriuretic peptides: inhibition by histamine H3‐receptor (H3R) activation

Natriuretic peptides (NP) are generally viewed as cardioprotective. Yet, NP were recently found to increase myocardial infarct size in mice, while their deletion decreased it. Also, mice lacking NP receptor-A are resistant to endotoxic shock. Further, a recombinant NP (nesiritide) was shown to increase death risk in heart failure patients. Excessive norepinephrine (NE) release may have been pivotal in these unexpected events. Langendorff guinea-pig hearts were perfused with NP; this resulted in a ≈ 73% increase in NE overflow. NP treatment of guinea-pig cardiac synaptosomes and NGF-differentiated PC12 cells also increased NE and dopamine release (≈ 34% and ≈ 48% increase, respectively). Thus, we investigated the mechanisms of the pro-adrenergic effect of NP. We found that NP markedly increased PKG activity, cAMP levels and PKA activity in differentiated PC12 cells; these effects derived from a cGMP/PKG-mediated prevention of cAMP hydrolysis by PDE3. Because of the antiexocytotic properties of H3R, we next questioned whether H3R activation might inhibit the proadrenergic effects of NP. Activation of H3R, constitutively expressed in heart synaptosomes and permanently transfected in PC12 cells, inhibited NP-induced catecholamine release and prevented the increases in cAMP level and PKA activity. Since the clinical use of NP in congestive heart failure (CHF) remains controversial, our findings may foster the design of selective H3R agonists to enable a safe and effective treatment of CHF with NP. This work was supported by NIH Grant HL 034215 and by a PhRMA Pre-doctoral fellowship.

A280V Polymorphism in the Histamine H3 Receptor as a Risk Factor for Migraine

Activation of histamine H3 receptors blocks the release of peptides responsible for headache. Our objective was to investigate the association between the genotypes of A280V polymorphism in the H3 receptor and migraine risk. We evaluated the frequency of the genotypes of A280V, polymorphism A280V and allelic variants of H3 receptor in 147 migraine patients and 186 healthy controls using a PCR-RLFP method. V allele frequency was 6.46% and 2.68% for the cases and controls, respectively (p = 0.02) (OR 2.67; 95% CI 1.20-5.93). The frequency of V/V + V/A genotypes was 12.92% in migraine patients, significantly higher when compared to the 3.22% frequency in the control group (p = 0.001) (OR 4.45; 95% CI 1.7-11.46). The results of this study suggest that V-allele genotypes in the H3 receptor gene are related to migraine risk in the Mexican population. We propose the hypothesis that the V-allele genotypes in the H3 receptor gene increase the population of inactive receptors, enhancing the inhibition of the negative feedback mechanism on the H3 receptor and increasing histamine release, which correlates with migraine attacks in susceptible patients. The case-control study reinforces the role of histamine in migraine pathogenesis.

Pre-synaptic histamine H3 receptors modulate glutamatergic transmission in rat globus pallidus

The globus pallidus, a neuronal nucleus involved in the control of motor behavior, expresses high levels of histamine H3 receptors (H3Rs) most likely located on the synaptic afferents to the nucleus. In this work we studied the effect of the activation of rat pallidal H3Rs on depolarization-evoked neurotransmitter release from slices, neuronal firing rate in vivo and turning behavior. Perfusion of globus pallidus slices with the selective H3R agonist immepip had no effect on the release of [3H]-GABA ([3H]-γ-aminobutyric acid) or [3H]-dopamine evoked by depolarization with high (20 mM) K+, but significantly reduced [3H]-d-aspartate release (−44.8±2.6% and −63.7±6.2% at 30 and 100 nM, respectively). The effect of 30 nM immepip was blocked by 10 μM of the selective H3R antagonist A-331440 (4′-[3-[(3(R)-dimethylamino-1-pyrrolidinyl]propoxy]-[1,1-biphenyl]-4′-carbonitrile). Intra-pallidal injection of immepip (0.1 μl, 100 μM) decreased spontaneous neuronal firing rate in anaesthetized rats (peak inhibition 68.8±10.3%), and this effect was reversed in a partial and transitory manner by A-331440 (0.1 μl, 1 mM). In free-moving rats the infusion of immepip (0.5 μl; 10, 50 and 100 μM) into the globus pallidus induced dose-related ipsilateral turning following systemic apomorphine (0.5 mg/kg, s.c.). Turning behavior induced by immepip (0.5 μl, 50 μM) and apomorphine was partially prevented by the local injection of A-331440 (0.5 μl, 1 mM) and was not additive to the turning evoked by the intra-pallidal injection of antagonists at ionotropic glutamate receptors (0.5 μl, 1 mM each of AP-5, dl-2-amino-5-phosphonovaleric acid, and CNQX, 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione). These results indicate that pre-synaptic H3Rs modulate glutamatergic transmission in rat globus pallidus and thus participate in the control of movement by basal ganglia.

A novel proadrenergic effect of cardiac natriuretic peptides: inhibition by histamine H3‐receptor activation

Natriuretic peptides (NP) are generally viewed as cardioprotective. Yet, NP were recently found to increase myocardial infarct size in mice, while their deletion decreased it. Further, a recombinant NP (nesiritide) was shown to increase death risk in heart failure patients. Excessive norepinephrine release may have been pivotal in these unexpected events. Thus, we questioned by which mechanisms NP could exert a pro‐adrenergic effect and whether this could be inhibited by histamine H3‐receptor (H3R) activation. NP treatment of sympathetic nerve endings isolated from guinea‐pig hearts (cardiac synaptosomes) and NGF‐differentiated PC12 cells markedly increased cAMP levels and PKA activity, thus promoting catecholamine exocytosis. These pro‐adrenergic effects derived from a cGMP/PKG‐mediated prevention of cAMP hydrolysis by PDE3. Activation of H3R, constitutively expressed in synaptosomes and permanently transfected in PC12 cells, inhibited NP‐induced catecholamine release and prevented the increases in cAMP levels and PKA activity. These effects most likely resulted from a H3R‐mediated enhancement of PDE3 activity and/or PKG inhibition. These findings elucidate new means to control catecholamine exocytosis in the heart, and may foster the design of new agents (e.g., selective H3R agonists) to enable a safe and effective treatment of congestive heart failure with NP.This work was supported by NIH Grant HL 034215 and by a PhRMA Pre‐doctoral fellowship.

Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways

It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G 11-coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changes in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP β2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or β2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [ 3H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq–PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.

Differential role of β‐catenin in VEGF and histamine‐induced MMP‐2 production in microvascular endothelial cells

Increases in endothelial cell permeability and production of matrix-degrading enzymes are two early steps in the angiogenic process. Factors such as vascular endothelial growth factor (VEGF) and histamine induce the angiogenic process through alterations in both permeability and proteolysis. We hypothesized that beta-catenin acts as a positive regulator of MMP-2 and MT1-MMP transcription following VEGF or histamine stimulation. Rat microvascular endothelial cells were exposed to VEGF or histamine overnight and MMP-2 protein production was assessed by gelatin zymography. Latent MMP-2 protein levels were increased following VEGF and histamine treatment as were MMP-2 mRNA transcript levels. Endothelial cells exposed to VEGF and histamine had an increased level of nuclear beta-catenin, which was sensitive to inhibition of the PI3-kinase signaling pathway. Promoter assays indicated increased transcriptional activity of both MMP-2 and MT1-MMP in endothelial cells co-transfected with luciferase reporter constructs and beta-catenin. Inhibition of beta-catenin signaling with inhibitor of catenin and T cell factor (ICAT) revealed that the VEGF-induced increase in MMP-2 mRNA is beta-catenin dependent. Interestingly, while MMP-2 mRNA levels increased in response to histamine H1 or H2 receptor activation, significantly larger increases were observed in cells co-treated with ICAT and histamine or the histamine receptor agonists, HTMT and dimaprit. While both VEGF and histamine increase nuclear beta-catenin and MMP-2 production, the role of beta-catenin in MMP-2 regulation differs between the two stimuli.

Marked changes in signal transduction upon heteromerization of dopamine D1 and histamine H3 receptors

Functional interactions between the G protein-coupled dopamine D1 and histamine H3 receptors have been described in the brain. In the present study we investigated the existence of D1-H3 receptor heteromers and their biochemical characteristics. D1-H3 receptor heteromerization was studied in mammalian transfected cells with Bioluminescence Resonance Energy Transfer and binding assays. Furthermore, signalling through mitogen-activated protein kinase (MAPK) and adenylyl cyclase pathways was studied in co-transfected cells and compared with cells transfected with either D1 or H3 receptors. Bioluminescence Resonance Energy Transfer and binding assays confirmed that D1 and H3 receptors can heteromerize. Activation of histamine H3 receptors did not lead to signalling towards the MAPK pathway unless dopamine D1 receptors were co-expressed. Also, dopamine D1 receptors, usually coupled to G(s) proteins and leading to increases in cAMP, did not couple to G(s) but to G(i) in co-transfected cells. Furthermore, signalling via each receptor was blocked not only by a selective antagonist but also by an antagonist of the partner receptor. D1-H3 receptor heteromers constitute unique devices that can direct dopaminergic and histaminergic signalling towards the MAPK pathway in a G(s)-independent and G(i)-dependent manner. An antagonist of one of the receptor units in the D1-H3 receptor heteromer can induce conformational changes in the other receptor unit and block specific signals originating in the heteromer. This gives rise to unsuspected therapeutic potentials for G protein-coupled receptor antagonists.

Effects of Histamine H3 Receptor Activation on the Behavioral-Stimulant Effects of Methamphetamine and Cocaine in Mice and Squirrel Monkeys

Background: Cocaine and methamphetamine (METH) are two commonly abused drugs that have behavioral-stimulant properties. These stimulant effects are partially mediated by the dopaminergic system. Recent evidence has suggested that the histamine H₃ receptor (H₃R) may modulate the release of dopamine induced by METH. The aim of the present study was to examine the role of H₃R in the behavioral-stimulant effects of cocaine and METH in mice and monkeys. Methods: Nonhabituated, experimentally naïve mice (n = 5–6) were pretreated with the H₃R agonist imetit 30 min before METH or cocaine, and activity was measured for 90 min. The behavioral-stimulant effects of METH and cocaine were also studied in squirrel monkeys (n = 3) under a fixed-interval schedule of stimulus termination. Monkeys were pretreated with imetit 30 min before the peak behavioral-stimulant doses of METH or cocaine derived from individual subjects. Results: Pretreatment with imetit did not affect basal activity in mice. Imetit significantly attenuated the behavioral-stimulant effects of METH, but not cocaine. In monkeys, no dose of imetit tested significantly altered the behavioral-stimulant effects of METH or cocaine. Conclusion: These results suggest a role of H₃R in the behavioral-stimulant effects of METH, but not cocaine, in mice and no role in monkeys.

Characterization of catecholamine release from deer adrenal medullary chromaffin cells

Isolated adrenal medullary chromaffin cells maintained in culture have been widely used to study neurosecretory events. Many of these studies have been conducted using cells obtained from the bovine adrenal. In this study we have cultured chromaffin cells from an alternative large animal model, the deer, and have conducted the first characterization of secretion from this preparation. Cervine chromaffin cells, preloaded with [ 3H]noradrenalin, displayed a strong secretory response to the cholinergic agonist carbachol, with a maximal secretion of approximately 28% cell content over 15 min. This response was reproduced by nicotinic but not muscarinic agonists and was similarly inhibited by nicotinic but not muscarinic antagonists. Nicotine-evoked secretion measured over a 15 min time period was inhibited approximately 50% by the L-type Ca 2+-channel antagonist nifedipine and approximately 20% by N-type (ω-conotoxin GVIA) or N, P/Q-type (ω-conotoxin MVIIC) antagonists. In contrast the response was unaffected by ω-agatoxin IVA, a P/Q-type antagonist. In addition to nicotinic receptor stimulation, activation of PACAP or histamine H1 receptors resulted in a concentration-dependent increase in secretion. PACAP was approximately two-fold more effective than histamine although both were weaker secretagogues than nicotine. In contrast, cervine chromaffin cells did not respond to angiotensin II or bradykinin, two agents known to stimulate secretion from bovine chromaffin cells. These data provide an initial characterization of the secretory response from cervine adrenal medullary chromaffin cells indicating that there are marked similarities but also potentially significant differences between them and their far more extensively described bovine counterparts.

4-Benzyl-1H-imidazoles with Oxazoline Termini as Histamine H3 Receptor Agonists

Research on the therapeutic applications of the histamine H3 receptor (H3R) has traditionally focused on antagonists/inverse agonists. In contrast, H3R agonists have received less attention despite their potential use in several disease areas. The lower availability of H3R agonists not only hampers their full therapeutic exploration, it also prevents an unequivocal understanding of the structural requirements for H3R activation. In the light of these important issues, we present our findings on 4-benzyl-1H-imidazole-based H3R agonists. Starting from two high throughput screen hits (10 and 11), the benzyl side chain was altered with lipophilic groups using combinatorial and classical chemical approaches (compounds 12-31). Alkyne- or oxazolino-substituents gave excellent affinities and agonist activities up to the single digit nM range. Our findings further substantiate the growing notion that basic ligand sidechains are not necessary for H 3R activation and reveal the oxazolino group as a hitherto unexplored functional group in H3R research.

Histamine Suppresses Fibulin-5 and Insulin-like Growth Factor-II Receptor Expression in Melanoma

We previously showed that transgenic enhancement of histamine production in B16-F10 melanomas strongly supports tumor growth in C57BL/6 mice. In the present study, gene expression profiles of transgenic mouse melanomas, secreting different amounts of histamine, were compared by whole genome microarrays. Array results were validated by real-time PCR, and genes showing histamine-affected behavior were further analyzed by immunohistochemistry. Regulation of histamine-coupled genes was investigated by checking the presence and functional integrity of all four known histamine receptors in experimental melanomas and by administering histamine H1 receptor (H1R) and H2 receptor (H2R) antagonists to tumor-bearing mice. Finally, an attempt was made to integrate histamine-affected genes in known gene regulatory circuits by in silico pathway analysis. Our results show that histamine enhances melanoma growth via H1R rather than through H2R. We show that H1R activation suppresses RNA-level expression of the tumor suppressor insulin-like growth factor II receptor (IGF-IIR) and the antiangiogenic matrix protein fibulin-5 (FBLN5), decreases their intracellular protein levels, and also reduces their availability in the plasma membrane and extracellular matrix, respectively. Pathway analysis suggests that because plasma membrane-bound IGF-IIR is required to activate matrix-bound, latent transforming growth factor-beta1, a factor suggested to sustain FBLN5 expression, the data can be integrated in a known antineoplastic regulatory pathway that is suppressed by H1R. On the other hand, we show that engagement of H2R also reduces intracellular protein pools of IGF-IIR and FBLN5, but being a downstream acting posttranslational effect with minimal consequences on exported IGF-IIR and FBLN5 protein levels, H2R is rather irrelevant compared with H1R in melanoma.

Effect of Mast Cell Degranulation on Chicken Ileal Ion Transport In Vitro

Histamine is a primary mediator of the inflammatory response in mammals. Degranulation of intestinal mast cells results in the release of mast cell mediators such as histamine. Histamine stimulates epithelial ion transport in a range of mammalian tissues via specific histamine receptors. The aim of this study was to assess a potential role of tissue mast cells and of exogenous histamine in the regulation of ion transport in avian mucosa. Broiler chicken ileal histamine release and secretory responses to mast cell degranulation were determined in vitro with the use of ELISA and Ussing chamber techniques. Pharmacological degranulation of mucosal mast cells using compound 48/80 (15 μg/mL) resulted in histamine release and an immediate-onset transient increase in transmural short-circuit current. The response to compound 48/80 was subject to tachyphylaxis and was significantly reduced in the presence of the histamine H1 antagonist mepyramine, but was unaffected by the cyclooxygenase inhibitor piroxicam. Prior incubation with the mast cell stabilizer ketotifen prevented compound 48/80-induced increase in transmural short-circuit current. In conclusion, degranulation of avian intestinal mast cells would appear to result in histamine release that stimulates epithelial ion transport via histamine H1 receptor activation. Although prostaglandin E2 is a potent secretagogue in the avian small intestine epithelium, prostanoid production appears to have little role to play in mast cell-mediated epithelial ion transport.

Central Histamine H3 Receptor Signaling Negatively Regulates Autoimmune Inflammation (129.31)

Abstract Histamine is a ubiquitous regulator of diverse physiologic processes including inflammation, immune modulation and neurotransmission. Four subtypes of histamine receptors are currently recognized and genetic and pharmacological studies have shown that the H1 and H2 receptors play a role in susceptibility to experimental allergic encephalomyelitis (EAE), the primary autoimmune model of multiple sclerosis. Histamine H3 receptor (H3R), which is not expressed in hematopoietic cells, is a presynaptic auto- and hetero-receptor. Here we show that H3RKO mice develop significantly more severe acute phase clinical disease and neuropathology compared to wild-type controls. In H3RKO mice this is preceded by disruption of the blood brain barrier and increased chemokine/chemokine receptor expression in peripheral T-cells. These data are consistent with inhibition of H3R-mediated neurogenic control of cerebrovascular tone and T-cell function. Additionally, genetic studies indicate that an H3R polymorphism leading to differential expression of H3R isoforms underlies eae8, a locus controlling disease associated weight loss, a phenotype known to be regulated by central H3R activity.

DIFFERENTIAL DEVELOPMENT OF CARBACHOL‐INDUCED DESENSITIZATION IN RECEPTOR‐MEDIATED Ca2+ INFLUX AND Ca2+ RELEASE PATHWAYS IN SMOOTH MUSCLE OF GUINEA‐PIG TAENIA CAECI

1. We have found that development of carbachol (CCh)-induced desensitization to receptor agonists, but not to receptor by-passed stimulation, is transiently interrupted by a Ca2+-dependent resensitization during the early stage in the smooth muscle of guinea-pig taenia caeci. To further characterize the receptor-mediated signal transduction pathways involved in this peculiar desensitization process, we examined the desensitization processes during Ca2+ influx- and Ca2+ release-mediated contractions in response to activation of muscarinic receptors or histamine H1 receptors. 2. Desensitization treatment with 10(-4) mol/L CCh for 30 min in the presence of extracellular Ca2+ resulted in desensitization to the muscarinic agonists McN-A-343 or AHR-602, which are known to induce contraction only in the presence of extracellular Ca2+ in taenia caeci. The development of desensitization to these agonists was interrupted by a transient resensitization at 1 min. In contrast, the transient resensitization phase was lost following removal of extracellular Ca2+ during the desensitization treatment with CCh; under these conditions, the desensitization developed gradually without an apparent resensitization phase. 3. Contractions to 10(-4) mol/L CCh and 10(-4) mol/L histamine in the absence of extracellular Ca2+ were gradually desensitized without a resensitization phase following the CCh desensitization treatment, irrespective of the presence or absence of extracellular Ca2+ during CCh treatment, although the onset of the desensitization was delayed under Ca2+-free conditions. 4. These results suggest that the receptor-mediated Ca2+ influx and Ca2+ release pathways are differentially desensitized to CCh and that the transient resensitization appears to regulate the desensitization process in response to Ca2+ influx-mediated contraction. Such differential processes of desensitization in receptor-mediated bifurcated signalling pathways may determine cellular responsiveness to certain types of stimuli, depending on the different Ca2+ sources required for contraction.

Stimulation of Histamine H1 Receptor Up-Regulates Histamine H1 Receptor Itself Through Activation of Receptor Gene Transcription

Histamine is a major mediator in allergy acting mainly through the histamine H1 receptor (H1R). Although H1R up-regulation has been suggested as an important step for induction of allergic symptoms, little is known about the regulation of H1R level. Here we report that the activation of H1R up-regulates H1R through augmentation of H1R mRNA expression in HeLa cells. Histamine stimulation significantly increased both H1R promoter activity and mRNA level without alteration in mRNA stability. H1R protein was also up-regulated by histamine. An H1R antagonist but not histamine H2 receptor antagonist blocked histamine-induced up-regulation of both promoter activity and mRNA expression. A protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate, increased H1R mRNA expression, whereas an activator of PKA or PKG (8-Br-cAMP or 8-Br-cGMP, respectively) did not. Furthermore, histamine-induced upregulation of both promoter activity and mRNA level were completely suppressed by the PKC inhibitor Ro-31-8220. H1R antagonists have long been thought to block H1R and inhibit immediate allergy symptoms. In addition to this short-term effect, our data propose their long-term inhibitory effect against allergic diseases by suppressing PKC-mediated H1R gene transcription. This finding provides new insights into the therapeutic target of H1R antagonist in allergic diseases.

Fluorescent styryl dyes FM1‐43 and FM2‐10 are muscarinic receptor antagonists: intravital visualization of receptor occupancy

The fluorescent styryl dyes FM1-43 and FM2-10 have been used to visualize the endocytic and exocytic processes involved in neurotransmission in a variety of central and peripheral nerve preparations. Their utility is limited to some extent by a poorly understood vesicular-independent labelling of cells and tissues. We show here that one likely cause of this troublesome background labelling is that FM1-43 and FM2-10 are selective and competitive antagonists at both cloned and endogenously expressed muscarinic acetylcholine receptors. In radioligand binding studies, FM1-43 and FM2-10 bound with moderate affinity (23-220 nM) to membranes of Chinese hamster ovary (CHO) cells expressing cloned human muscarinic receptors (M1-M5). In functional studies in vitro, FM1-43 and FM2-10 inhibited electrical field stimulation (EFS) and acetylcholine-induced cholinergic contractions of guinea-pig tracheal strips (IC50: FM1-43, 0.4 +/- 0.1; FM2-10, 1.6 +/- 0.1 microM; concentration of antagonist producing a 2-fold leftward shift in the acetylcholine concentration-response curve (Kb): FM1-43, 0.3 +/- 0.1; FM2-10, 15.8 +/- 10.1 microM). Neither compound inhibited EFS-evoked, non-adrenergic non-cholinergic nerve-mediated relaxations or contractions of the airways, or contractions mediated by histamine H1 receptor or tachykinin NK2 receptor activation. Incubating freshly excised tracheal whole-mount preparations with 5 microM FM1-43 resulted in intense fluorescence labelling of the smooth muscle that was reduced by up to 90% in the presence of selective M2 and M3 receptor antagonists. The potency of the FM dyes as muscarinic receptor antagonists is within the concentration range used to study vesicular cycling at nerve terminals. Given that muscarinic receptors play a key role in the regulation of neurotransmitter release from a variety of neurones, the anticholinergic properties of FM dyes may have important implications when studying vesicular events in the nervous system. In addition, these dyes may provide a novel tool for visualizing muscarinic receptor occupancy in living tissue or cell preparations.

CCK- and leptin-induced vagal afferent activation: a model for organ-specific endocrine modulation of visceral sensory information

the vagus nerve supplies sensory innervation to nearly all of the viscera, including the heart, respiratory passages, great vessels of the thoracic cavity, esophagus, stomach, small intestine, cecum, colon, pancreas, and liver ([29][1]). Although the vagus supplies the majority of parasympathetic

Opposite Functions of Histamine H1 and H2 Receptors and H3 Receptor in Substantia Nigra Pars Reticulata

The substantia nigra pars reticulata (SNr) is a key basal ganglia output nucleus. Inhibitory outputs from SNr are encoded in spike frequency and pattern of the inhibitory SNr projection neurons. SNr output intensity and pattern are often abnormal in movement disorders of basal ganglia origin. In Parkinson's disease, histamine innervation and histamine H3 receptor expression in SNr may be increased. However, the functional consequences of these alterations are not known. In this study, whole cell patch-clamp recordings were used to elucidate the function of different histamine receptors in SNr. Histamine increased SNr inhibitory projection neuron firing frequency and thus inhibitory output. This effect was mediated by activation of histamine H1 and H2 receptors that induced inward currents and depolarization. In contrast, histamine H3 receptor activation hyperpolarized and inhibited SNr inhibitory projection neurons, thus decreasing the intensity of basal ganglia output. By the hyperpolarization, H3 receptor activation also increased the irregularity of the interspike intervals or changed the pattern of SNr inhibitory neuron firing. H3 receptor-mediated effects were normally dominated by those mediated by H1 and H2 receptors. Furthermore, endogenously released histamine provided a tonic, H1 and H2 receptor-mediated excitation that helped keep SNr inhibitory projection neurons sufficiently depolarized and spiking regularly. These results suggest that H1 and H2 receptors and H3 receptor exert opposite effects on SNr inhibitory projection neurons. Functional balance of these different histamine receptors may contribute to the proper intensity and pattern of basal ganglia output and, as a consequence, exert important effects on motor control.

Evidence for histamine as a neurotransmitter in the cardiac sympathetic nervous system

The colocalization of histamine (HA) and norepinephrine (NE) immunoreactivities was identified within the superior cervical ganglia neurons of the guinea pig. HA and NE immunoreactivity levels were significantly attenuated after chemical sympathectomy with 6-hydroxydopamine (6-OHDA). Coexistence of NE and HA was also visualized in the cardiac sympathetic axon and varicosities labeled with anterograde tracer biotinylated dextran amine. Depolarization of cardiac sympathetic nerve endings (synaptosomes) with 50 mM potassium stimulated endogenous HA release, which was significantly attenuated by 6-OHDA or a vesicular monoamine transporter 2 (VMAT2) inhibitor reserpine pretreatments. Compound 48/80, a mast cell releaser, did not affect cardiac synaptosome HA exocytosis. Furthermore, K+ -evoked HA release was abolished by the N-type Ca2+ -channel blocker omega-conotoxin but was not affected by the L-type Ca2+ -channel blocker lacidipine. Cardiac synaptosome HA exocytosis was augmented by the enhanced synthesis of HA or the inhibition of HA metabolism. HA H3-receptor activation by (R)-alpha-methylhistamine inhibited high K+ -evoked histamine release. The HA H3 receptor antagonist thioperamide enhanced K+ -evoked HA release and blocked the (R)-alpha-methylhistamine effect. The K+ -evoked endogenous NE release was attenuated by preloading the cardiac synaptosomes with L-histidine or quinacrine. These inhibitory effects were reversed by thioperamide or antagonized by alpha-fluoromethylhistidine. Our findings indicate that high K+ -evoked corelease of NE and HA may be inhibited by endogenous HA via activation of presynaptic HA H3-receptors. The H3-receptor may function as an autoreceptor, rather than a heteroreceptor, in the regulation of sympathetic neurotransmission and HA may be a novel sympathetic neurotransmitter.