Fiona C. Shenton

The histamine H4 receptor is functionally expressed on neurons in the mammalian CNS

Background and purpose:  The histamine H4 receptor is the most recently identified of the G protein‐coupled histamine receptor family and binds several neuroactive drugs, including amitriptyline and clozapine. So far, H4 receptors have been found only on haematopoietic cells, highlighting its importance in inflammatory conditions. Here we investigated the possibility that H4 receptors may be expressed in both the human and mouse CNS.

Molecular and biochemical pharmacology of the histamine H4 receptor

The elucidation of the human genome has had a major impact on histamine receptor research. The identification of the human H4 receptor by several groups has been instrumental for a new appreciation of the role of histamine in the modulation of immune function. In this review, we summarize the historical developments and the molecular and biochemical pharmacology of the H4 receptor.

Human Inflammatory Dendritic Epidermal Cells Express a Functional Histamine H4 Receptor

Expression of histamine H(4) receptor (H(4)R) on leukocytes suggests an immunomodulatory role of this receptor. Here we investigated the expression and function of H(4)R on human inflammatory dendritic epidermal cells (IDECs). H(4)R is expressed by IDEC of the skin. On monocyte-derived IDECs (Mo-IDECs), H(4)R is also expressed and upregulated by IFN-gamma. Functionally, histamine and H(4)R agonists clobenpropit and 4-methylhistamine downregulated the production of the Th2-linked chemokine CCL2 and the Th1 cytokine IL-12 on Mo-IDEC, whereas agonists for the other histamine receptors did not. An H(4)R-selective antagonist (JNJ7777120) blocked the effect of H(4)R agonists. Downregulation of CCL2 also led to a decreased migration of monocytes. Thus, IDEC express a functionally active H(4)R, which upon stimulation leads to downregulation of CCL2 and IL-12. This might have implications for the treatment of atopic dermatitis, since H(4)R agonists may have beneficial effects in downregulating inflammation.

Immunohistochemical localization of histamine H3 receptors in rodent skin, dorsal root ganglia, superior cervical ganglia, and spinal cord: Potential antinociceptive targets

Abstract Activation of histamine H3 receptors (H3Rs) reduces inflammation and nociception, but the existence of H3Rs on peripheral innervation has never been demonstrated. Here we use antibodies to locate H3Rs in whisker pads, hairy and glabrous hind paw skin, dorsal root ganglia (DRGs), and spinal cords of rats, wild type mice, and H3R knockout (H3KO) mice. Although H3Rs have been hypothesized to be on C and sympathetic fibers, H3R‐like immunoreactivity (H3R‐LI) was only detected on presumptive periarterial Aδ fibers and on Aβ fibers that terminated in Meissner’s corpuscles and as lanceolate endings around hair follicles. The H3R‐positive periarterial fibers were thin‐caliber and coexpressed immunoreactivity for calcitonin gene‐related peptide (CGRP), substance P, acid sensing ion channel 3, and 200 kDa neurofilament protein (NF). H3R‐LI was also detected on epidermal keratinocytes and Merkel cells, but not on Merkel endings, C fibers, any other Aδ fibers, or sympathetic fibers. In DRGs, H3R‐LI was preponderantly on medium to large neurons coexpressing NF‐LI and mostly CGRP‐LI. In dorsal horn, CGRP‐positive fibers with and without H3R‐LI ramified extensively in lamina II; many of the former formed a plexus in lamina V. Low levels of H3R‐LI were also present on Aβ fibers penetrating superficial and into deeper laminae. The distribution of H3R‐LI was similar in rats and wild type mice, but was eliminated or strongly reduced in Aδ fibers and Aβ fibers, respectively, in H3KO mice. Taken with recently published behavioral results, the present findings suggest that periarterial, peptidergic, H3R‐containing Aδ fibers may be sources of high threshold mechanical nociception.

Oligomerization of recombinant and endogenously expressed human histamine H4 receptors

In this study, we report the homo- and hetero-oligomerization of the human histamine H4R by both biochemical (Western blot and immobilized metal affinity chromatography) and biophysical [bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer (tr-FRET)] techniques. The H4R receptor is the most recently discovered member of the histamine family of G-protein-coupled receptors. Using specific polyclonal antibodies raised against the C-terminal tail of the H4R, we demonstrate the presence of H4R oligomers in human embryonic kidney 293 and COS-7 cells heterologously overexpressing H4Rs and putative native H4R oligomers in human phytohaemagglutinin blasts endogenously expressing H4Rs. Moreover, we show that H4R homo-oligomers are formed constitutively, are formed at low receptor densities (300 fmol/mg of protein), and are present at the cell surface, as detected by tr-FRET. The formation of these oligomers is independent of N-glycosylation and is not modulated by H4R ligands, covering the full spectrum of agonists, neutral antagonists, and inverse agonists. Although we show H4R homo-oligomer formation at physiological expression levels, the detection of H1R-H4R hetero-oligomers was achieved only at higher H1R expression levels and are most likely not physiologically relevant.

Amiloride-sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles

We investigated whether channels of the epithelial sodium/amiloride‐sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch‐evoked afferent discharge was reduced in a dose‐dependent manner by amiloride and three analogues – benzamil, 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching ≥85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC δ subunit agonists (icilin and capsazepine). HMAs profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P < 0.01 each) at 1 μm, while 10 μm HMA was required for highly significant inhibition (P < 0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low‐affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC α, β and γ subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC α, β or γ relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P < 0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1α) were also highly significantly greater (P < 0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic‐like vesicles which regulate afferent excitability in these mechanosensitive endings.

Heterogeneity of histaminergic neurons in the tuberomammillary nucleus of the rat

Histaminergic neurons of the hypothalamic tuberomammillary nuclei (TMN) send projections to the whole brain. Early anatomical studies described histaminergic neurons as a homogeneous cell group, but recent evidence indicates that histaminergic neurons are heterogeneous and organized into distinct circuits. We addressed this issue using the double‐probe microdialysis in freely moving rats to investigate if two compounds acting directly onto histaminergic neurons to augment cell firing [thioperamide and bicuculline, histamine H3‐ and γ‐aminobutyric acid (GABA)A‐receptor (R) antagonists, respectively] may discriminate groups of histaminergic neurons impinging on different brain regions. Intra‐hypothalamic perfusion of either drug increased histamine release from the TMN and cortex, but not from the striatum. Thioperamide, but not bicuculline, increased histamine release from the nucleus basalis magnocellularis (NBM), bicuculline but not thioperamide increased histamine release from the nucleus accumbens (NAcc). Intra‐hypothalamic perfusion with thioperamide increased the time spent in wakefulness. To explore the local effects of H3‐R blockade in the histaminergic projection areas, each rat was implanted with a single probe to simultaneously administer thioperamide and monitor local changes in histamine release. Thioperamide increased histamine release from the NBM and cortex significantly, but not from the NAcc or striatum. The presence of H3‐Rs on histaminergic neurons was assessed using double‐immunofluorescence with anti‐histidine decarboxylase antibodies to identify histaminergic cells and anti‐H3‐R antibodies. Confocal analysis revealed that all histaminergic somata were immunopositive for the H3‐R. This is the first evidence that histaminergic neurons are organized into functionally distinct circuits that influence different brain regions, and display selective control mechanisms.

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

The H(4)R (histamine H(4) receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H(4)R is primarily expressed in eosinophils and mast cells and has the highest homology with the H(3)R. The occurrence of at least twenty different hH(3)R (human H(3)R) isoforms led us to investigate the possible existence of H(4)R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H(4)R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H(4)R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H(4)R-ligand induced signalling or constitutive activity for these H(4)R splice variants. However, when co-expressed with full-length H(4)R [H(4)R((390)) (H(4)R isoform of 390 amino acids)], the H(4)R splice variants have a dominant negative effect on the surface expression of H(4)R((390)). We detected H(4)R((390))-H(4)R splice variant hetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H(4)R splice variants were detected in various cell types and expressed at similar levels to the full-length H(4)R((390)) mRNA in, for example, pre-monocytes. We conclude that the H(4)R splice variants described here have a dominant negative effect on H(4)R((390)) functionality, as they are able to retain H(4)R((390)) intracellularly and inactivate a population of H(4)R((390)), presumably via hetero-oligomerization.

Histamine H3 and H4 receptors are expressed on distinct endocrine cell types in the rat fundic mucosa.

The histamine H3 receptor (H3R) is expressed in the central nervous system of different mammalian species [1] and is also localized to the endocrine cells of rat gastric mucosa [2]. In this study, the expression and the distribution of histamine H4 receptor (H4R) within the rat gastric fundus and its possible coexistence with H3R have been explored by immunohistochemistry using our unique immunological probes [3, 4].

Evidence for native and cloned H3 Histamine receptor higher oligomers

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