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.

Lysergic acid diethylamide stimulates cardiac human H2 histamine and cardiac human 5-HT4-serotonin receptors.

Lysergic acid diethylamide (LSD) is an artificial hallucinogenic drug. Thus, we hypothesized that LSD might act 5-HT4 serotonin receptors and/or H2 histamine receptors. We studied isolated electrically stimulated left atrial preparations, spontaneously beating right atrial preparations, and spontaneously beating Langendorff-perfused hearts from transgenic mice with cardiomyocyte-specific overexpression of the human 5-HT4 receptor (5-HT4-TG) or of the H2-histamine receptor (H2-TG). For comparison, we used wild type littermate mice (WT). Finally, we measured isometric force of contraction in isolated electrically stimulated muscle strips from the human right atrium obtained from patients during bypass surgery. LSD (up to 10 µM) concentration dependently increased force of contraction and beating rate in left or right atrial preparations from 5-HT4-TG (n = 6, p < 0.05) in 5-HT4-TG atrial preparations. The inotropic and chronotropic effects of LSD were antagonized by 10 µM tropisetron in 5-HT4-TG. In contrast, LSD (10 µM) increased force of contraction and beating rate in left or right atrial preparations, from H2-TG. After pre-stimulation with cilostamide (1 µM), LSD (10 µM) increased force of contraction in human atrial preparations (n = 6, p < 0.05). The contractile effects of LSD in human atrial preparations could be antagonized by 10 µM cimetidine and 1 µM GR 125487. LSD leads to H2-histamine receptor and 5-HT4-receptor mediated cardiac effects in humans.

Exogenous histamine and H 2 receptor activation and H 3 receptor inhibition in nucleus accumbens modulate formalin-induced orofacial nociception through opioid receptors.

It has been demonstrated that the nucleus accumbens (NAc) plays an important role in modulation of nociception due to its extensive connections with different regions of the brain. In addition, this nucleus receives histaminergic projections from tuberomammillary nucleus. Considering the role of the central histaminergic system in nociception, the effect of histamine and its H 2 and H 3 receptors agonist and antagonist microinjections into the NAc on orofacial formalin nociception was investigated. In male Wistar rats, using stereotaxic surgery, two guide cannulas were bilaterally implanted into the right and left sides of the NAc. Diluted formalin solution (1.5%, 50 µl) injection into the vibrissa pad led to orofacial nociception. Immediately after injection, face rubbing was observed at 3-min blocks for 45 min. Orofacial formalin nociception was characterized by a biphasic nociceptive response (first phase: 0-3 min and second phase: 15-33 min). Microinjections of histamine (0.5 and 1 µg/site), dimaprit (1 µg/site, H 2 receptor agonist) and thioperamide (2 µg/site, H 3 receptor antagonist) attenuated both phases of formalin orofacial nociception. Prior microinjection of famotidine (2 µg/site) inhibited the antinociceptive effects of dimaprit (1 µg/site). Furthermore, comicroinjection of thioperamide (2 µg/site) and immepip (1 µg/site) prevented thioperamide (2 µg/site)-induced antinociception. Naloxone (2 µg/site) also prevented histamine, dimaprit- and thioperamide-induced antinociception. The results of this study demonstrate that at the level of the NAc, histamine and its H 2 and H 3 receptors are probably involved in the modulation of orofacial nociception with an opioid system-dependent mechanism.

Effectiveness of natural biomaterials in the protection and healing of experimentally induced gastric mucosa Ulcer in rats.

BACKGROUND: A gastric ulcer is a painful lesion of the gastric mucosa that can be debilitating or even fatal. The effectiveness of several plant extracts in the therapy of this illness has been demonstrated in traditional pharmacopoeias. AIM: this study was aimed to see if propolis, ginseng in normal or nano form, and amygdalin might help in preventing the ulcerative effects of absolute ethanol. METHODS: Gastroprotective properties of pretreatments before ethanol gavage in rats were compared to omeprazole. The ulcer and stomach parameters (ulcerated regions) were measured (mm2), ulcer inhibition percentage, the stomachs were assessed macroscopically with gastric biopsy histological examinations. RESULTS: Amygdalin, normal and nano ginseng, nano propolis followed by propolis all showed great efficacy in protecting the cyto-architecture and function of the gastric mucosa. The number of ulcerated sites was greatly reduced, and the percentage of stomach protection was increased. Histopathological examination had confirmed great protective effects of the nanoformulations followed by amygdalin. The protection and healing rate was completed to about 100% in all tested materials while ulcer areas were still partially unhealed in normal propolis and omeprazole. Quantitative assay of the m-RNA levels Enothelin 1(ET-1), leukotriene4 (LT-4), and caspase 3(Cas-3) genes and Histamine were done and revealed significant up-regulations in ethanol group and the maximum protective effect was reported with ginseng nano, moreover the histamine content was significantly decreased with nano- formulated extracts. CONCLUSION: Amygdalin and the nanoformulated ginseng and propolis had exhibited a marked protective effect against the ulcerative toxic effects of ethanol.

Trafficking of carbonic anhydrase 12 and bicarbonate transporters by histamine stimulation mediates intracellular acidic scenario in lung cancer cells.

Carbonic anhydrase 12 is considered an oncogenic and acidic microenvironmental factor in cancer cells. To verify the role of histamine signalling as an anti-cancer signal, we determined the roles of CA12 and its associated bicarbonate transporters. In this study, histamine stimulation mediated mislocalization of CA12 in lung cancer cells. Histamine receptor activation-mediated CA12 endocytosis and pH were restored by CaMKII inhibition. CA12-associated AE2 expression was enhanced, whereas NBCn1 expression and its activity were reduced by histamine stimulation. Histamine receptor activation-mediated acidification was induced by internalised CA12 and NBCn1 and, at the same time by increased bicarbonate efflux through enhanced AE2 expression. Inhibition of protein trafficking by bafilomycin restored CA12 and AE2 localisation and diminished cellular acidosis. Thus, we verified that histamine stimulation induced an acidic scenario, which revealed trafficking of CA12 and its associated bicarbonate transporters in lung cancer cells and its dysregulated pH modulation may be involved in the histamine signalling-mediated anti-cancer process.

Anti-Inflammatory and Pro-Resolving Actions of the N-Terminal Peptides Ac2-26, Ac2-12, and Ac9-25 of Annexin A1 on Conjunctival Goblet Cell Function.

Annexin A1 (AnxA1) is the primary mediator of the anti-inflammatory actions of glucocorticoids. AnxA1 functions as a pro-resolving mediator in cultured rat conjunctival goblet cells to ensure tissue homeostasis through stimulation of intracellular [Ca2+] ([Ca2+]i) and mucin secretion. AnxA1 has several N-terminal peptides with anti-inflammatory properties of their own, including Ac2-26, Ac2-12, and Ac9-25. The increase in [Ca2+]i caused by AnxA1 and its N-terminal peptides in goblet cells was measured to determine the formyl peptide receptors used by the compounds and the action of the peptides on histamine stimulation. Changes in [Ca2+]i were determined by using a fluorescent Ca2+ indicator. AnxA1 and its peptides each activated formyl peptide receptors in goblet cells. AnxA1 and Ac2-26 at 10-12 mol/L and Ac2-12 at 10-9 mol/L inhibited the histamine-stimulated increase in [Ca2+]i, as did resolvin D1 and lipoxin A4 at 10-12 mol/L, whereas Ac9-25 did not. AnxA1 and Ac2-26 counter-regulated the H1 receptor through the p42/p44 mitogen-activated protein kinase/extracellular regulated kinase 1/2, ß-adrenergic receptor kinase, and protein kinase C pathways, whereas Ac2-12 counter-regulated only through ß-adrenergic receptor kinase. In conclusion, current data show that the N-terminal peptides Ac2-26 and Ac2-12, but not Ac9-25, share multiple functions with the full-length AnxA1 in goblet cells, including inhibition of histamine-stimulated increase in [Ca2+]i and counter-regulation of the H1 receptor. These actions suggest a potential pharmaceutical application of the AnxA1 N-terminal peptides Ac2-26 and Ac2-12 in homeostasis and ocular inflammatory diseases.

Prorelaxant E-type Prostanoid Receptors Functionally Partition to Different Procontractile Receptors in Airway Smooth Muscle.

Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1-EP4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate the ASM contractile state remains subject to debate. We used EP2 and EP4 subtype-specific agonists (ONO-259 and ONO-329, respectively) in cell- and tissue-based models of human ASM contraction-magnetic twisting cytometry (MTC), and precision-cut lung slices (PCLSs), respectively-to study the EP2 and EP4 regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (<0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC50]: 4.9 × 10-7 vs. 2.2 × 10-6; maximal bronchodilation ± SE, 35 ± 2% vs. 15 ± 2%). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLSs. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329- and ONO-259-induced phosphorylation of the protein kinase A substrates VASP and HSP20, with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh-induced phosphorylation of the regulatory myosin light chain (pMLC20) and the F-actin/G-actin ratio (F/G-actin ratio) while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and the F/G-actin ratio with both MCh and histamine. Thus, the contractile-agonist-dependent differential effects are not explained by changes in the global levels of phosphorylated protein kinase A substrates but are reflected in the regulation of pMLC20 (cross-bridge cycling) and F/G-actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes.

Effect of progesterone administration on tissue mast cell population and histamine content in mice uterus after ovulation induction.

OBJECTIVE: Mast cell population and histamine affect on blastocyst implantation. This study aimed to evaluate the effects of progesterone administration after induction of ovulation on the uterine tissue mast cell population and histamine content in mice. METHODS: We ran an experimental study on three groups of mice; control group, ovulation induction (induction group), and ovulation induction along with progesterone administration (progesterone group). Mast cells were counted using toluidine blue staining, and the histamine level was measured through spectrophotometry. RESULTS: According to the analysis of variance (ANOVA), there was no difference in mast cell population in endometrium (p=0.138) nor in myometrium (p=0.611). The ratio of mast cells in the myometrium per endometrium increased in the progesterone group in comparison to the control group based on a generalized linear model (p=0.041). The uterine histamine level was different between the groups, based on the ANOVA (p=0.039), in which the progesterone group had lower amounts of histamine. CONCLUSIONS: Progesterone administration after ovulation induction did not decrease the number of endometrial mast cells and could have increased the ratio of myometrium mast cells per endometrium mast cell. The histamine level in uterus decreased by the administration of progesterone in the ovulation-induced mice.

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.

Differential regulation of histamine H1 receptor-mediated ERK phosphorylation by Gq proteins and arrestins.

Gq protein-coupled histamine H1 receptors play crucial roles in allergic and inflammatory reactions, in which the phosphorylation of extracellular signal-regulated kinase (ERK) appears to mediate the production of inflammatory cytokines. ERK phosphorylation is regulated by G protein- and arrestin-mediated signal transduction pathways. Here, we aimed to explore how H1 receptor-mediated processes of ERK phosphorylation might be differentially regulated by Gq proteins and arrestins. For this purpose, we evaluated the regulatory mechanism(s) of H1 receptor-mediated ERK phosphorylation in Chinese hamster ovary cells expressing Gq protein- and arrestin-biased mutants of human H1 receptors, S487TR and S487A, in which the Ser487 residue in the C-terminal was truncated and mutated to alanine, respectively. Immunoblotting analysis indicated that histamine-induced ERK phosphorylation was prompt and transient in cells expressing Gq protein-biased S487TR, whereas it was slow and sustained in cells expressing arrestin-biased S487A. Inhibitors of Gq proteins (YM-254890) and protein kinase C (PKC) (GF109203X), and an intracellular Ca2+ chelator (BAPTA-AM) suppressed histamine-induced ERK phosphorylation in cells expressing S487TR, but not those expressing S487A. Conversely, inhibitors of G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), clathrin (hypertonic sucrose), Raf (LY3009120), and MEK (U0126) suppressed histamine-induced ERK phosphorylation in cells expressing S487A, but not those expressing S487TR. These results suggest that H1 receptor-mediated ERK phosphorylation might be differentially regulated by the Gq protein/Ca2+/PKC and GRK/arrestin/clathrin/Raf/MEK pathways to potentially determine the early and late phases of histamine-induced allergic and inflammatory responses, respectively.

P19-derived neuronal cells express H1, H2, and H3 histamine receptors: a biopharmaceutical approach to evaluate antihistamine agents.

Histamine is a biogenic amine implicated in various biological and pathological processes. Convenient cellular models are needed to screen and develop new antihistamine agents. This report aimed to characterize the response of neurons differentiated from mouse P19 embryonal carcinoma cells to histamine treatment, and to investigate the modulation of this response by antihistamine drugs, vegetal diamine oxidase, and catalase. The exposure of P19 neurons to histamine reduced cell viability to 65% maximally. This effect involves specific histamine receptors, since it was prevented by treatment with desloratadine and cimetidine, respectively, H1 and H2 antagonists, but not by the H3 antagonist ciproxifan. RT-PCR analysis showed that P19 neurons express H1 and H2 receptors, and the H3 receptor, although it seemed not involved in the histamine effect on these cells. The H4 receptor was not expressed. H1 and H2 antagonists as well as vegetal diamine oxidase diminished the intracellular Ca2+ mobilization triggered by histamine. The treatment with vegetal diamine oxidase or catalase protected against mortality and a significant reduction of H2O2 level, generated from the cells under the histamine action, was found upon treatments with desloratadine, cimetidine, vegetal diamine oxidase, or catalase. Overall, the results indicate the expression of functional histamine receptors and open the possibility of using P19 neurons as model system to study the roles of histamine and related drugs in neuronal pathogenesis. This model is less expensive to operate and can be easily implemented by current laboratories of analysis and by Contract Research Organizations.

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.

Temperature alters the inotropic, chronotropic and proarrhythmic effects of histamine in atrial muscle preparations from humans and H2-receptor overexpressing mice.

We investigated whether hypothermia and hyperthermia can alter the efficacy and potency of histamine at increasing the force of cardiac contractions in mice that overexpress the human H2 receptor only in their cardiac myocytes (labelled H2-TG). Contractile studies were performed in an organ bath on isolated, electrically driven (1 Hz) left atrial preparations and spontaneously beating right atrial preparations from H2-TG mice and wild-type (WT) littermate control mice. The basal beating rate in the right atrial preparations from H2-TG mice was lowered by hypothermia (23 °C) and elevated by hyperthermia (42 °C). Furthermore, the efficacy of histamine (0.01-100 µM) at exerting positive inotropic effects was more severely attenuated in the left and right H2-TG mouse atria under hypothermia and hyperthermia than under normothermia (37 °C). Similarly, the inotropic response to histamine was attenuated under hypothermia and hyperthermia in isolated electrically stimulated (1 Hz) right atrial preparations obtained from humans undergoing cardiac surgery. The phosphorylation state of phospholamban at serine 16 at 23 °C was inferior to that at 37 °C in left atrial preparations from H2-TG mice in the presence of 10 µM histamine. In contrast, in human atrial preparations, the phosphorylation state of phospholamban at serine 16 in the presence of 100 µM histamine was lower at 42 °C than at 37 °C. Finally, under hyperthermia, we recorded more and longer lasting arrhythmias in right atrial preparations from H2-TG mice than in those from WT mice. We conclude that the inotropic effects of histamine in H2-TG mice and in human atrial preparations, as well as the chronotropic effects of histamine in H2-TG mice, are temperature dependent. Furthermore, we observed that, even without stimulation of the H2 receptors by exogenous agonists, temperature elevation can increase arrhythmias in isolated right atrial preparations from H2-TG mice. We propose that H2 receptors play a role in hyperthermia-induced supraventricular arrhythmias in human patients.

Ceefourin-1, a MRP4/ABCC4 inhibitor, induces apoptosis in AML cells enhanced by histamine.

BACKGROUND: Ceefourin-1 is a specific MRP4/ABCC4 inhibitor with potential antileukemic activity. In this study, we evaluate the ability of ceefourin-1 alone or in combination with histamine, an approved antileukemic agent, to induce cell differentiation or apoptosis in human acute myeloid leukemic cells. We also examine ceefourin-1 toxicity in mice. METHODS: U937, HL-60, and KG1a cells were used as models for human acute myeloid leukemia. Cyclic AMP efflux was estimated by measuring intracellular and extracellular cAMP levels. Cell differentiation was assessed by levels of CD14 and CD11b by FACS, and CD88 by western blot, and by cell morphology. Apoptosis was evaluated by cleavage of caspase-3 and PARP by western blot, and by annexin V binding assay. Subacute toxicity study of ceefourin-1 was carried out in BALB/c mice. RESULTS: Ceefourin-1 inhibits cAMP exclusion in AML cells and promotes intracellular signaling via CREB. Ceefourin-1 leads AML cells to apoptosis and histamine potentiates this effect, without evidence of cell differentiation. Intraperitoneal administration of ceefourin-1 shows no important alterations in mice blood parameters, hepatic, and renal functions, nor signs of histologic damage. CONCLUSIONS: These results show that ceefourin-1 promotes apoptosis in AML cells that is enhanced by histamine. GENERAL SIGNIFICANCE: This work indicates that ceefourin-1 represents a promising molecule that could be used alone or in combination with histamine for in vivo evaluation in acute myeloid leukemia malignancies.

DPP4 Inhibitor Sitagliptin Reduces Inflammatory Responses and Mast Cell Activation in Allergic Rhinitis.

INTRODUCTION: DPP4 is thought to be involved in certain immune processes and plays an important role in allergic reactions in the lungs. The effect of the DPP4 inhibitor sitagliptin on the effector phase of allergic rhinitis (AR) in ovalbumin (OVA)-sensitized mice and on mast cell degranulation in vitro was assessed. METHODS: The AR mouse model was established by intraperitoneal injection combined with OVA intranasal method. OVA was injected intraperitoneally 3 times for the first 2 weeks, and the mice were subsequently given DPP4 inhibitors by oral gavage, accompanied by an OVA intranasal challenge. The impacts of DPP4 inhibitors on DPP4 levels in mouse model were determined. Nasal mucosa tissue was collected for H&E staining and toluidine blue staining. Immunoglobulin E (IgE) levels and histamine levels were analyzed, and IL-4, IL-5, and IL-12 as well as IFN-γ levels were assessed. Following the treatment of dinitrophenol (DNP)-IgE or DNP-IgE plus sitagliptin in RBL-2H3 cells, ß-hexosaminidase activity was analyzed and toluidine blue staining was performed. RESULTS: DPP4 level was reduced in AR patients, as well as in AR mouse models. Nasal allergic symptoms such as sneezing and nose-scratching showed high frequency in OVA-induced mice. Sitagliptin treatment during the intranasal challenge of OVA decreased DPP4 levels, suppressed allergic symptoms, eosinophil infiltration, IgE levels, mast cell infiltration, as well as the levels of inflammatory cytokines. We further found that sitagliptin inhibited mast cell activation and histamine levels in vitro. CONCLUSION: Sitagliptin suppresses the effector phase of AR, and this mechanism is partly attributed to the suppression of inflammatory response and mast cell degranulation.

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.

α-Asarone alleviates allergic asthma by stabilizing mast cells through inhibition of ERK/JAK2-STAT3 pathway.

Asthma is a heterogeneous disease related to numerous inflammatory cells, among which mast cells play an important role in the early stages of asthma. Therefore, treatment of asthma targeting mast cells is of great research value. α-Asarone is an important anti-inflammatory component of the traditional Chinese medicine Acorus calamus L, which has a variety of medicinal values. To investigate whether α-asarone can alleviate asthma symptoms and its mechanism. In this study, we investigated the effect of α-asarone on mast cell activation in vivo and in vitro. The release of chemokines or cytokines, AHR (airway hyperresponsiveness), and mast cell activation were examined in a mast cell-dependent asthma model. Western blot was performed to determine the underlying pathway. α-Asarone inhibited the degranulation of LAD2 (laboratory allergic disease 2) cells and decreased IL-8, MCP-1, histamine, and TNF-α in vitro. α-Asarone reduced paw swelling and leakage of Evans blue, as well as serum histamine, CCL2, and TNF-α in vivo. In the asthma model, α-asarone showed an inhibitory effect on AHR, inflammation, mast cells activation, infiltration of inflammatory cells, and the release of IL-5 and IL-13 in lung tissue. α-Asarone decreased the levels of phosphorylated JAK2, phosphorylated ERK, and phosphorylated STAT3 induced by C48/80. Our findings suggest that α-asarone alleviates allergic asthma by inhibiting mast cell activation through the ERK/JAK2-STAT3 pathway.

Accumbal Histamine Signaling Engages Discrete Interneuron Microcircuits.

BACKGROUND: Central histamine (HA) signaling modulates diverse cortical and subcortical circuits throughout the brain, including the nucleus accumbens (NAc). The NAc, a key striatal subregion directing reward-related behavior, expresses diverse HA receptor subtypes that elicit cellular and synaptic plasticity. However, the neuromodulatory capacity of HA within interneuron microcircuits in the NAc remains unknown. METHODS: We combined electrophysiology, pharmacology, voltammetry, and optogenetics in male transgenic reporter mice to determine how HA influences microcircuit motifs controlled by parvalbumin-expressing fast-spiking interneurons (PV-INs) and tonically active cholinergic interneurons (CINs) in the NAc shell. RESULTS: HA enhanced CIN output through an H2 receptor (H2R)-dependent effector pathway requiring Ca2+-activated small-conductance K+ channels, with a small but discernible contribution from H1Rs and synaptic H3Rs. While PV-IN excitability was unaffected by HA, presynaptic H3Rs decreased feedforward drive onto PV-INs via AC-cAMP-PKA (adenylyl cyclase-cyclic adenosine monophosphate-protein kinase A) signaling. H3R-dependent plasticity was differentially expressed at mediodorsal thalamus and prefrontal cortex synapses onto PV-INs, with mediodorsal thalamus synapses undergoing HA-induced long-term depression. These effects triggered downstream shifts in PV-IN- and CIN-controlled microcircuits, including near-complete collapse of mediodorsal thalamus-evoked feedforward inhibition and increased mesoaccumbens dopamine release. CONCLUSIONS: HA targets H1R, H2R, and H3Rs in the NAc shell to engage synapse- and cell type-specific mechanisms that bidirectionally regulate PV-IN and CIN microcircuit activity. These findings extend the current conceptual framework of HA signaling and offer critical insight into the modulatory potential of HA in the brain.

PD-1+ mast cell enhanced by PD-1 blocking therapy associated with resistance to immunotherapy.

BACKGROUND: Programmed cell death protein 1 (PD-1) antibody has been approved for a variety of tumors, but its effective rate is unsatisfactory. New evidence suggests that mast cells are an important component of the tumor microenvironment and are associated with resistance to immunotherapy, but the underlying mechanism is not clear. METHODS: Bioinformatics analysis of patients with melanoma in TCGA-SKCM and GSE91061 was used to determine the prognostic value of mast cells and their association with anti-PD-1 immunotherapy. HMC-1 cells (mast cell line) and bone marrow-derived mast cells (BMMCs) were used to verify the effect of PD-1 antibody and cromolyn sodium in vitro. The mouse subcutaneous melanoma model was used to verify the effect of the PD-1 antibody on mast cells in vivo. RESULTS: Bioinformatics analysis showed that mast cells were a poor prognostic factor associated with resistance to anti-PD-1 immunotherapy. PD-1 was expressed on the mast cell membrane. The PD-1 antibody promoted the release of histamine and cytokines from mast cells via the PI3K/AKT pathway and calcium signaling pathway. The activation of mast cells induced by PD-1 antibody could be partially inhibited by cromolyn sodium. In vivo, cromolyn sodium increased the efficacy of PD-1 antibody and decreased the infiltration of mast cells and the density of microvessels. CONCLUSION: PD-1+ mast cell activated by PD-1 antibody plays a negative role in the tumor microenvironment via the enhanced function of releasing histamine and cytokines. Inhibition of mast cell may provide a new solution to solve the low response rate of anti-PD-1 immunotherapy.

Differential endothelial hydrogen peroxide signaling via Nox isoforms: Critical roles for Rac1 and modulation by statins.

Statins have manifold protective effects on the cardiovascular system. In addition to lowering LDL cholesterol levels, statins also have antioxidant effects on cardiovascular tissues involving intracellular redox pathways that are incompletely understood. Inhibition of HMG-CoA reductase by statins not only modulates cholesterol synthesis, but also blocks the synthesis of lipids necessary for the post-translational modification of signaling proteins, including the GTPase Rac1. Here we studied the mechanisms whereby Rac1 and statins modulate the intracellular oxidant hydrogen peroxide (H2O2) via NADPH oxidase (Nox) isoforms. In live-cell imaging experiments using the H2O2 biosensor HyPer7, we observed robust H2O2 generation in human umbilical vein endothelial cells (HUVEC) following activation of cell surface receptors for histamine or vascular endothelial growth factor (VEGF). Both VEGF- and histamine-stimulated H2O2 responses were abrogated by siRNA-mediated knockdown of Rac1. VEGF responses required the Nox isoforms Nox2 and Nox4, while histamine-stimulated H2O2 signals are independent of Nox4 but still required Nox2. Endothelial H2O2 responses to both histamine and VEGF were completely inhibited by simvastatin. In resting endothelial cells, Rac1 is targeted to the cell membrane and cytoplasm, but simvastatin treatment promotes translocation of Rac1 to the cell nucleus. The effects of simvastatin both on receptor-dependent H2O2 production and Rac1 translocation are rescued by treatment of cells with mevalonic acid, which is the enzymatic product of the HMG-CoA reductase that is inhibited by statins. Taken together, these studies establish that receptor-modulated H2O2 responses to histamine and VEGF involve distinct Nox isoforms, both of which are completely dependent on Rac1 prenylation.