Blockade of histamine receptor H1 augments immune checkpoint therapy by enhancing MHC-I expression in pancreatic cancer cells.

BACKGROUND: Although immune checkpoint blockade (ICB) therapy has proven to be extremely effective at managing certain cancers, its efficacy in treating pancreatic ductal adenocarcinoma (PDAC) has been limited. Therefore, enhancing the effect of ICB could improve the prognosis of PDAC. In this study, we focused on the histamine receptor H1 (HRH1) and investigated its impact on ICB therapy for PDAC. METHODS: We assessed HRH1 expression in pancreatic cancer cell (PCC) specimens from PDAC patients through public data analysis and immunohistochemical (IHC) staining. The impact of HRH1 in PCCs was evaluated using HRH1 antagonists and small hairpin RNA (shRNA). Techniques including Western blot, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-PCR), and microarray analyses were performed to identify the relationships between HRH1 and major histocompatibility complex class I (MHC-I) expression in cancer cells. We combined HRH1 antagonism or knockdown with anti-programmed death receptor 1 (αPD-1) therapy in orthotopic models, employing IHC, immunofluorescence, and hematoxylin and eosin staining for assessment. RESULTS: HRH1 expression in cancer cells was negatively correlated with HLA-ABC expression, CD8+ T cells, and cytotoxic CD8+ T cells. Our findings indicate that HRH1 blockade upregulates MHC-I expression in PCCs via cholesterol biosynthesis signaling. In the orthotopic model, the combined inhibition of HRH1 and αPD-1 blockade enhanced cytotoxic CD8+ T cell penetration and efficacy, overcoming resistance to ICB therapy. CONCLUSIONS: HRH1 plays an immunosuppressive role in cancer cells. Consequently, HRH1 intervention may be a promising method to amplify the responsiveness of PDAC to immunotherapy.

Histamine H1 Receptor-Mediated JNK Phosphorylation Is Regulated by Gq Protein-Dependent but Arrestin-Independent Pathways.

Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.

Histamine H1- and H4-receptor expression in human colon-derived cell lines.

In previous studies, we demonstrated the involvement of H4R in inflammatory bowel disease (IBD) and IBD-associated colon cancer in mice and could ascribe H4R-mediated histamine function to colon epithelial cells. The transferability of obtained data to humans is however lacking. Functional expression of H4R on colon epithelial cells is a prerequisite to pursue the hypothesis of involvement of H4R in carcinogenesis. Thus, we here compared the expression of histamine receptor subtypes in a series of cell lines. Out of these, three colon-derived cell lines displaying different combinations of H1R and H4R expression were submitted to functional analyses. Human hematopoietic HMC-1, HL-60, and U937, lung-derived A549 and Calu-3, and colorectal LoVo, SW 480, Caco-2, HT-29, and HCT116 cells were included in the study. mRNA expression was quantified by RT-qPCR. For functional analyses, Caco-2, HT-29, and HCT116 cells were treated by incubation with 1 - 10 µM histamine in the presence or absence of selective histamine receptor antagonists. Calcium mobilization, cAMP accumulation, and cell proliferation were measured by fluorimetry, mass spectrometry, and real-time bioimpedance measurements, respectively. Histamine receptor expression was heterogeneous in the cell lines tested. In most cell lines, we detected H1R mRNA while H4R mRNAs were found only occasionally. The colon-derived epithelial cell lines LoVo, SW480, and HT-29 expressed H1R mRNA exclusively, while in HCT116 cells H1R and H4R mRNAs and in CaCo-2 H2R mRNA were detectable. Subsequent functional analyses in HT29, Caco-2, and HCT116 cells, however, indicated that only HT-29 responded to histamine stimulation, by means of H1R. For a detailed analysis of histamine receptor function, esp. that of H1R and H4R, in human colon-derived cell lines, the cell lines tested here are not fully convenient unless genetically modified.

Histamine promotes angiogenesis through a histamine H1 receptor-PKC-VEGF-mediated pathway in human endothelial cells.

Histamine is a well-known inflammatory mediator, but how histamine induces angiogenesis remains poorly understood. In the present study, we demonstrated a dose-dependent dynamic tube formation in the human endothelial cell line EA.hy926 in the presence of histamine that was completely blocked by histamine H1 receptor (H1R) and protein kinase C (PKC) inhibitors. However, histamine H2, H3, and H4 receptor inhibitors did not inhibit tube formation, suggesting that H1R-PKC signaling is involved in histamine-induced tube formation. Moreover, we found an H1-specific induction of vascular endothelial growth factor (VEGF) expression. Inhibition of VEGF receptor 2 (VEGFR2) suppressed the histamine-induced tube formation, indicating that VEGF is downstream of histamine signaling. Additionally, we demonstrated that histamine stimulation induces the expression of critical regulators of angiogenesis such as matrix metalloproteinase (MMP)-9 and MMP-14 metalloproteases, as histamine-induced tube formation is blocked by MMP inhibitors. In summary, our study indicates that histamine can activate the H1R in human endothelial cells and thereby promote tube formation through the PKC, MMP, and VEGF signaling pathways.

Simultaneous activation of CXC chemokine receptor 4 and histamine receptor H1 enhances calcium signaling and cancer cell migration.

C-X-C chemokine receptor 4 (CXCR4) is widely overexpressed in various types of cancer and is involved in several cancer phenotypes including tumor growth, survival, and metastasis. The roles of histamine and histamine receptor H1 (HRH1) in cancer pathogenesis remain controversial. Here, we show that HRH1 is widely expressed in various cancer cell lines and cancer tissues and that coexpression of CXCR4 and HRH1 is associated with poor prognosis in breast cancer. Using bimolecular fluorescence complementation and bioluminescence resonance energy transfer donor saturation assays, we demonstrate that CXCR4 and HRH1 can assemble into a heteromeric complex. Simultaneous activation of CXCR4 and HRH1 synergistically increases calcium flux in MDA-MB-231 cells that endogenously express CXCR4 and HRH1 but not in cells deficient in CXCR4 or HRH1. Costimulation of CXCR4 and HRH1 also significantly enhances CXCL12-induced MDA-MB-231 cell migration, while histamine alone does not induce cell migration. Synergistic effects on calcium flux and cell migration are inhibited by the Gαi inhibitor pertussis toxin and the Gαq inhibitor YM254890, suggesting that the Gαi and Gαq pathways are involved in the synergy. Enhanced calcium signaling and cell migration are also observed in NCI-H23 and HeLa cells, which coexpress CXCR4 and HRH1. Taken together, our findings demonstrate an interplay between CXCR4 and HRH1, and suggest the possibility of the CXCR4-HRH1 heteromer as a potential therapeutic target for anticancer therapy.

LYTIC COCKTAIL ATTENUATES CATECHOLAMINE SURGE AFTER SEVERE BURNS BY BLOCKING HISTAMINE H1 RECEPTOR/PKA/CREB/TYROSINE HYDROXYLASE SIGNALING IN CHROMAFFIN CELLS.

ABSTRACT: Severe burns develop a catecholamine surge, inducing severe damage to the organism, raising the possibility of multisystem organ failure, and even death. The mechanisms of catecholamine surge have not been fully elucidated, and few strategies are generally acceptable to reduce catecholamine surge postburn. Thus, it is valuable to investigate the underlying mechanisms of catecholamine surge postburn to develop targeted interventions to attenuate it. We have found that the lytic cocktail alleviates the surge of catecholamine and organ injury after severe burn; however, the underlying mechanisms were still unclear. Moreover, the lytic cocktail has side effects, such as significant arterial hypotension and breathing depression, limiting its clinical application. This study aims to investigate the therapeutic mechanism of the lytic cocktail in regulating catecholamine levels postburn. We find that promethazine, a classic histamine H1 receptor blocker and a component of the lytic cocktail, can effectively reduce catecholamine surge and organ injury postburn. Our study confirms that blood histamine levels increase after severe burns. We find that histamine can amplify the catecholamine surge by elevating tyrosine hydroxylase expression and catecholamine synthesis in chromaffin cells through the histamine H1 receptor/Protein Kinase A /cAMP-response element binding protein signaling pathway. In summary, for the first time, we find that histamine plays a vital role in catecholamine surge postburn. We also confirm that the lytic cocktail effectively alleviates catecholamine surge and organ injury postburn through promethazine.

Optimization of Peptide Linker-Based Fluorescent Ligands for the Histamine H1 Receptor.

The histamine H1 receptor (H1R) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity H1R-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a H1R fluorescent ligand, bearing a peptide-linker, based on antagonist VUF13816 and sought to further explore structure-activity relationships (SARs) around the linker, orthostere, and fluorescent moieties. Here, we report a series of high-affinity H1R fluorescent ligands varying in peptide linker composition, orthosteric targeting moiety, and fluorophore. Incorporation of a boron-dipyrromethene (BODIPY) 630/650-based fluorophore conferred high binding affinity to our H1R fluorescent ligands, remarkably overriding the linker SAR observed in corresponding unlabeled congeners. Compound 31a, both potent and subtype-selective, enabled H1R visualization using confocal microscopy at a concentration of 10 nM. Molecular docking of 31a with the human H1R predicts that the optimized peptide linker makes interactions with key residues in the receptor.

Molecular Signaling and Transcriptional Regulation of Histamine H1 Receptor Gene.

Histamine-activated histamine H1 receptor (H1R) signaling regulates many gene expressions, mainly through the protein kinase C (PKC)/extracellular signal-regulated kinases (ERK) signaling. Involvement of other signaling, including NF-κB, Wnt, RUNX-2, and Rho A signaling was also demonstrated. In addition, cAMP production through the activation of H1R signaling was reported. H1R gene itself is also up-regulated by the activation of H1R signaling with histamine. Here, we review our recent findings in the molecular signaling and transcriptional regulation of the H1R gene. Stimulation with histamine up-regulates H1R gene expression through the activation of H1R in HeLa cells. The PKCδ/ERK/poly(ADP)ribosyl transferase-1 (PARP-1) signaling was involved in this up-regulation. Heat shock protein 90 also plays an important role in regulating PKCδ translocation. Promoter analyses revealed the existence of two promoters in the human H1R gene in HeLa cells. H1R-activated H1R gene up-regulation in response to histamine was also observed in U373 astroglioma cells. However, this up-regulation was mediated not through the PKCδ signaling but possibly through the PKCα signaling. In addition, the promoter region responsible for histamine-induced H1R gene transcription in U373 cells was different from that of HeLa cells. These findings suggest that the molecular signaling and transcriptional regulation of the H1R gene are different between neuronal cells and non-neuronal cells.

Histamine H1 receptor antagonists selectively kill cisplatin-resistant human cancer cells.

Cancer therapy is often hampered by the disease's development of resistance to anticancer drugs. We previously showed that the autonomously upregulated product of fibroblast growth factor 13 gene (FGF13; also known as FGF homologous factor 2 (FHF2)) is responsible for the cisplatin resistance of HeLa cisR cells and that it is likely responsible for the poor prognosis of cervical cancer patients treated with cisplatin. Here we show that cloperastine and two other histamine H1 receptor antagonists selectively kill HeLa cisR cells at concentrations that little affect parental HeLa S cells. The sensitivity of HeLa cisR cells to cloperastine was abolished by knocking down FGF13 expression. Cisplatin-resistant A549 cisR cells were similarly susceptible to cloperastine. H2, H3, and H4 receptor antagonists showed less or no cytotoxicity toward HeLa cisR or A549 cisR cells. These results indicate that histamine H1 receptor antagonists selectively kill cisplatin-resistant human cancer cells and suggest that this effect is exerted through a molecular mechanism involving autocrine histamine activity and high-level expression of FGF13. We think this represents a potential opportunity to utilize H1 receptor antagonists in combination with anticancer agents to treat cancers in which emergent drug-resistance is preventing effective treatment.

Defining the role of mirtazapine in the treatment of refractory pruritus.

BACKGROUND/OBJECTIVE: Mirtazapine has traditionally been used for the treatment of major depressive disorder, with an added benefit in patients who have comorbid insomnia or anxiety. Recent studies describe its usefulness in treating refractory pruritus of various causes as well. Our goal is to better define the use of mirtazapine in the treatment of refractory pruritus. METHOD: Through a thorough literature review of PubMed, we identified all reports of the use of mirtazapine for pruritus. RESULTS: Upon examination of 8 supporting articles, we found mirtazapine has quality evidence for the treatment of intra-thecal morphine-induced pruritus. Mirtazapine may also be effective in treating pruritus related to various other conditions, including psoriasis, atopic dermatitis, cutaneous malignancies (primary or metastatic), hematologic malignancies (lymphomas and leukemias), liver failure, renal failure, cholestasis, as well as pruritus of unknown origin. CONCLUSIONS: Mirtazapine plays a role in treatment for intra-thecel morphine-induced pruritis yet high-quality trials are needed to confirm its efficacy in other dermatologic conditions.

Histamine augments collagen content via H1 receptor stimulation in cultures of myofibroblasts taken from wound granulation tissue.

The inflammatory reaction influences the deposition of collagen within wound granulation tissue. The aim of the present study is to determine whether histamine acting directly on myofibroblasts derived from wound granulation tissue may influence collagen deposition. It also identifies the histamine receptor involved in this process. The experiments were carried out on cells isolated from the granulation tissue of a wound model (a polypropylene net inserted subcutaneously to rats) or intact rat skin. Collagen content was measured following the addition of different concentrations of histamine and treatment with histamine receptor antagonists (ketotifen - H1 inhibitor, ranitidine - H2 inhibitor) and a histamine receptor H1 agonist (2-pyridylethylamine dihydrochloride).The cells were identified as myofibroblasts: alpha-smooth muscle actin, vimentin, and desmin positive in all experimental conditions. Histamine increased the collagen level within both cell cultures, i.e., those isolated from granulation tissue or intact skin. It did not, however, influence the expression of either the collagen type I or III genes within the cultured myofibroblasts. Histamine activity was reduced by ketotifen (the H1 receptor inhibitor) and increased by the H1 receptor agonist, as demonstrated by changes in the levels of collagen in the myofibroblast culture. Histamine increased collagen content within the cultures, acting directly on myofibroblasts via H1 receptor stimulation.

Agonism of Histaminergic-H1 Receptors in Ischemic Postconditioning During Cerebral Ischemia-Reperfusion Injury is Protective.

BACKGROUND: Stroke is associated with cerebral ischemia/reperfusion (I/R) injury. Ischemic postconditioning (IPoC) reduces cerebral ischemic injury in rats and offers neuroprotection. The central histaminergic pathway possesses a crucial role in the pathogenesis of cerebral I/R, but its neuroprotective role in IPoC is still unidentified. OBJECTIVE: This research explored the role of the histaminergic in IPoC during cerebral I/R injury in the rat. METHODS: Global cerebral ischemia/reperfusion (GCI/R) injury in Wistar albino rats was induced by occluding the bilateral carotid arteries for 10 minutes, followed by reperfusion. IPoC was provided by giving three episodes of I/R post GCI (10 min), after which of reperfusion was permitted. Inclined- beam-walk, hanging-wire, lateral-push, and rota-rod tests were employed to assess motor functions, and Morris water maze (MWM) was used to assess spatial learning as well as memory in animals. Cerebral oxidative markers (thiobarbituric acid reactive species-TBARS, reduced glutathione- GSH), inflammatory markers (myeloperoxidase-MPO), acetylcholinesterase activity- AChE, infarct size, and histopathological changes were also assessed. L-histidine and chlorpheniramine were used as histaminergic agonists and antagonists. RESULTS: I/R animals showed a reduction in memory and motor function, and an increase in cerebral oxidative stress, inflammation, AChE activity, infarct size and histopathological changes. Episodes of IPoC post-ischemia attenuated the deleterious effects of I/R injury. Pretreatment (30 min before cerebral ischemia) with L-histidine mimicked the neuroprotective effects of IPoC. However, neuroprotection produced by IPoC was abolished by pretreatment with chlorpheniramine (histaminergic- H1 receptor antagonist). CONCLUSION: IPoC may provide neuroprotection against cerebral I/R induced brain injury by modulating the histaminergic-H1-receptor pathway.

Histamine receptor 1 is expressed in leukaemic cells and affects differentiation sensitivity.

Despite the success of immunotherapy in several haematological neoplasms, the effectiveness in acute myeloid leukaemia (AML) is still controversial, partially due to the lack of knowledge regarding immune-related processes in this disease and similar neoplasias. In this study, we analysed the role and expression of histamine receptor 1 (HRH1) in haematological malignancies. Although the histamine receptor type 1 was widely expressed in healthy and malignant haematopoiesis, especially along the myeloid lineage, HRH1 lacked a relevant role in survival/proliferation and chemoresistance of AML cells, as analysed by HRH1 knockdown (KD) and pharmacological modulation. However, HRH1-mediated signalling was critical for the activation of the differentiation process induced by several agents including all-trans retinoic acid, establishing a role for HRH1 in myeloid differentiation. Pharmacological activation of Erk was able to partially restore differentiation capacity in HRH1 KD AML cells, suggesting that HRH1 signalling acts upstream MAPK-Erk pathway. As an indirect consequence of our results, treatment-related histamine release is not expected to confer a proliferative advantage in leukaemic cells.

Unexpected Ca2+-mobilization of oxaliplatin via H1 histamine receptors.

Oxaliplatin is a widely used chemotherapeutic drug and represents the cornerstone of colorectal cancer therapy, in combination with 5-fluorouracil and folinic acid. As with many chemotherapeutic agents, its use is associated with a number of side effects, ranging from hypersensitivity reactions to haematological dyscrasias. Oxaliplatin also induces acute and chronic peripheral neuropathy. While it is likely that the haematological side effects are associated with its anti-proliferative effects and with the ability to form DNA adducts, the molecular mechanisms underlying peripheral neuropathy and hypersensitivity reactions are poorly understood, and therefore the choice of adequate supportive therapies is largely empirical. Here we show that an acute low dose oxaliplatin application on DRG neurons is able to induce an increase in intracellular calcium that is dependent on the Histamine 1 receptor (H1). Oxaliplatin-induced intracellular calcium rises are blocked by two selective H1 antagonist, as well as by U73122, a PLC inhibitor, and by 2-APB, a non-specific IP3 receptor blocker. Moreover, expression of the H1 receptor on HEK293 t cells unmasks an oxaliplatin-induced Ca2+-rise. Last, activation of H1 via either histamine or oxaliplatin activates TRPV1 receptors, a mechanism that has been associated with itch. These data, together with literature data that has shown that anti-histamine agents reduce the incidence of oxaliplatin-induced hypersensitivity, may provide a molecular mechanism of this side effect in oncological patients.

Histamine Induces Microglia Activation and the Release of Proinflammatory Mediators in Rat Brain Via H1R or H4R.

Histamine is a major peripheral inflammatory mediator and a neurotransmitter in the central nervous system. We have reported that histamine induces microglia activation and releases proinflammatory factors in primary cultured microglia. Whether histamine has similar effects in vivo is unknown. In the present study, we aimed to investigate the role of histamine and its receptors in the release of inflammatory mediators and activation of microglia in rat brain. We site-directed injected histamine, histamine receptor agonists or histamine receptor antagonists in the rat lateral ventricle using stereotaxic techniques. Flow cytometry was employed to determine histamine receptor expression in rat microglia. Microglia activation was assessed by Iba1 immunohistochemistry. The levels of tumour necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß) and interleukin-10 (IL-10) were measured with commercial enzyme-linked immunosorbent assay (ELISA) kits, TNF-α, IL-1ß and IL-10 mRNA expressions were determined with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). We found that all four types of histamine receptors were expressed in rat brain microglia. Histamine was able to induce microglia activation and subsequent production of the inflammatory factors TNF-α, IL-1ß and IL-10, and these effects were partially abolished by H1R and H4R antagonists. However, H2R and H3R antagonists significantly increased production of TNF-α and IL-1ß, and decreased IL-10 levels. The H1R or H4R agonists stimulated the production of TNF-α and IL-1ß, while the H2R or H3R agonists increased IL-10 release. Our results demonstrate that histamine induces microglia activation and the release of both proinflammatory and anti-inflammatory factors in rat brain, thus contributing to the development of inflammation in the brain. Graphical Abstract Histamine induces microglia activation and the release of both proinflammatory (TNF-α and IL-1ß) and anti-inflammatory factors (IL-10) in rat brain, thus contributing to the development of inflammation in the brain.

Ameliorative Effect of Hesperidin Against Motion Sickness by Modulating Histamine and Histamine H1 Receptor Expression.

Motion sickness (MS) is the visceral discomfort caused due to contradicting visual and vestibular inputs to the brain leading to nausea and vomiting. Sensory conflict theory which proves histamine elevations as the primary reason for MS provides a path for an effective pharmaco-therapy. We aimed to evaluate the anti-MS effect of hesperidin (HSP) by modulating histamine and histamine receptor H1 (HRH1) expression. The inhibitory effect of HSP on histamine release was studied in KU812 cells treated with 10 µM calcium ionophore. The in vivo anti-MS effect of HSP was evaluated in Balb/c mice. Thirty six mice were divided into six groups namely, normal control (NC, no rotation), hesperidin at 80 mg/kg body weight control (HSP80, no rotation), motion sickness (MS, rotation induced), dimenhydrinate (Standard drug) at 20 mg/kg body weight + rotation (STD + MS), hesperidin at 40 mg/kg body weight + rotation (HSP40 + MS) and hesperidin at 80 mg/kg body weight + rotation (HSP80 + MS). Hypothalamus and brainstem samples were analysed for histamine levels and HRH1 expression by RT-PCR, Western blot and immunohistochemistry analysis. Calcium ionophore treated KU812 cells significantly increased histamine release when compared to control cells. Pre-treatment with HSP inhibited histamine, HRH1 mRNA and protein expression. Histamine, HRH1 mRNA and protein expression in hypothalamus and brainstem samples of MS group increased significantly when compared to the NC group. Pre-treatment with HSP significantly reduced histamine, HRH1 mRNA and protein expression. Thus, indicating that HSP has a potent anti- MS effect by decreasing the elevated levels of histamine, HRH1 mRNA and protein expression in hypothalamus and brainstem regions.

Histamine induces peripheral and central hypersensitivity to bladder distension via the histamine H1 receptor and TRPV1.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a common chronic pelvic disorder with sensory symptoms of urinary urgency, frequency, and pain, indicating a key role for hypersensitivity of bladder-innervating sensory neurons. The inflammatory mast cell mediator histamine has long been implicated in IC/BPS, yet the direct interactions between histamine and bladder afferents remain unclear. In the present study, we show, using a mouse ex vivo bladder afferent preparation, that intravesical histamine enhanced the mechanosensitivity of subpopulations of afferents to bladder distension. Histamine also recruited "silent afferents" that were previously unresponsive to bladder distension. Furthermore, in vivo intravesical histamine enhanced activation of dorsal horn neurons within the lumbosacral spinal cord, indicating increased afferent signaling in the central nervous system. Quantitative RT-PCR revealed significant expression of histamine receptor subtypes (Hrh1-Hrh3) in mouse lumbosacral dorsal root ganglia (DRG), bladder detrusor smooth muscle, mucosa, and isolated urothelial cells. In DRG, Hrh1 was the most abundantly expressed. Acute histamine exposure evoked Ca2+ influx in select populations of DRG neurons but did not elicit calcium transients in isolated primary urothelial cells. Histamine-induced mechanical hypersensitivity ex vivo was abolished in the presence of the histamine H1 receptor antagonist pyrilamine and was not present in preparations from mice lacking transient receptor potential vanilloid 1 (TRPV1). Together, these results indicate that histamine enhances the sensitivity of bladder afferents to distension via interactions with histamine H1 receptor and TRPV1. This hypersensitivity translates to increased sensory input and activation in the spinal cord, which may underlie the symptoms of bladder hypersensitivity and pain experienced in IC/BPS.

Histamine H1 and H3 receptor activation increases the expression of Glucose Transporter 1 (GLUT-1) in rat cerebro-cortical astrocytes in primary culture.

Astrocytes take up glucose via the 45 kDa isoform of the Glucose Transporter 1 (GLUT-1), and in this work we have investigated whether histamine regulates GLUT-1 expression in rat cerebro-cortical astrocytes in primary culture. Cultured astrocytes expressed histamine H1 and H3 receptors (H1Rs and H3Rs) as evaluated by radioligand binding. Receptor functionality was confirmed by the increase in the intracellular concentration of Ca2+ (H1R) and the inhibition of forskolin-induced cAMP accumulation (H3R). Quantitative RT-PCR showed that histamine and selective H1R and H3R agonists (1 h incubation) significantly increased GLUT-1 mRNA to 153 ±â€¯7, 163 ±â€¯2 and 168 ±â€¯13% of control values, respectively. In immunoblot assays, incubation (3 h) with histamine or H1R and H3R agonists increased GLUT-1 protein levels to 224 ±â€¯12, 305 ±â€¯11 and 193 ±â€¯13% of control values, respectively, an action confirmed by inmunocytochemistry. The effects of H1R and H3R agonists were blocked by the selective antagonists mepyramine (H1R) and clobenpropit (H3R). The pharmacological inhibition of protein kinase C (PKC) prevented the increase in GLUT-1 protein induced by either H1R or H3R activation. Furthermore, histamine increased ERK-1/2 phosphorylation, and the effect of H1R and H3R activation on GLUT-1 protein levels was reduced or prevented, respectively, by MEK-1/2 inhibition. These results indicate that by activating H1Rs and H3Rs histamine regulates the expression of GLUT-1 by astrocytes. The effect appears to involve the phospholipase C (PLC) → diacylglycerol (DAG)/Ca2+→ PKC and PLC → DAG/Ca2+ → PKC → MAPK pathways.

Identification of inhibitors of the RGS homology domain of GRK2 by docking-based virtual screening.

AIMS: G protein-coupled receptor (GPCR) kinases (GRKs) are mainly involved in the desensitization of GPCRs. Among them, GRK2 has been described to be upregulated in many pathological conditions and its crucial role in cardiac hypertrophy, hypertension, and heart failure promoted the search for pharmacological inhibitors of its activity. There have been several reports of potent and selective inhibitors of GRK2, most of them directed to the kinase domain of the protein. However, the homologous to the regulator of G protein signaling (RH) domain of GRK2 has also been shown to regulate GPCRs signaling. Herein, we searched for potential inhibitors of receptor desensitization mediated by RH domain of GRK2. MATERIALS AND METHODS: We performed a docking-based virtual screening utilizing the crystal structure of GRK2 to search for potential inhibitors of the interaction between GRK2 and Gαq protein. To evaluate the biological activity of compounds we measured, calcium response of histamine H1 receptor (H1R) using Fura-2AM dye and H1R internalization by saturation binding experiments in A549 cells. GRK2(45-178)GFP translocation was determined in HeLa cells through confocal fluorescence imaging. KEY FINDINGS: We identified inhibitors of GRK2 able to reduce the RH mediated desensitization of the histamine H1 receptor and GRK2 translocation to plasma membrane. Also candidates presented adequate lipophilia and cytotoxicity profile. SIGNIFICANCE: We obtained compounds with the ability of reducing RH mediated actions of GRK2 that can be useful as a starting point in the development of novel drug candidates aimed to treat pathologies were GRK2 plays a key role.

The effects of histamine H1 type receptor (H1R) in regulating osteoblastic cell differentiation and mineralization.

Osteoblastic bone formation is important for maintaining the balance of bone turnover. However, the underlying mechanisms are still needed to be elucidated. Histamine H1 type receptor (H1R) is a major subtype of histamine membrane receptors family, which has displayed diverse biological functions in various tissues and cells. In the current study, we have identified a novel physiological function of H1R in regulating osteoblastic differentiation and mineralization of the MC3T3-E1 cells. We found that H1R is expressed in the MC3T3-E1 cells. Interestingly, H1R is up-regulated in the process of differentiation and mineralization of the MC3T3-E1cells induced by osteogenic medium (OM). Blockage of H1R using its specific antagonist Loratadine prevented differentiation and mineralization of the MC3T3-E1 cells by reducing ALP activity, bone matrix deposition, and the expressions of osteogenic marker genes including ALP, OCN, Osx, and type I collagen as well as the transcriptional factor RUNX-2, which is a central regulator of osteoblastogenesis. In contrast, we found that activation of H1R with Histamine exerts opposite actions by increasing the expressions of RUNX-2. Finally, we found that the effects of H1R in osteoblastic differentiation and mineralization are mediated by the AMPK/eNOS signaling. Based on these findings, we concluded that H1R might be an important therapeutic target for the treatment of skeletal disorders.