Histamine Stimulation Research Articles

Histamine stimulates human microglia to alter cellular prion protein expression via the HRH2 histamine receptor

Although the cellular prion protein (PrPC) has been evolutionarily conserved, the role of this protein remains elusive. Recent evidence indicates that PrPC may be involved in neuroinflammation and the immune response in the brain, and its expression may be modified via various mechanisms. Histamine is a proinflammatory mediator and neurotransmitter that stimulates numerous cells via interactions with histamine receptors 1-4 (HRH1-4). Since microglia are the innate immune cells of the central nervous system, we hypothesized that histamine-induced stimulation regulates the expression of PrPC in human-derived microglia. The human microglial clone 3 (HMC3) cell line was treated with histamine, and intracellular calcium levels were measured via a calcium flux assay. Cytokine production was monitored by enzyme-linked immunosorbent assay (ELISA). Western blotting and quantitative reverse transcription-polymerase chain reaction were used to determine protein and gene expression of HRH1-4. Flow cytometry and western blotting were used to measure PrPC expression levels. Fluorescence microscopy was used to examine Iba-1 and PrPC localization. HMC3 cells stimulated by histamine exhibited increased intracellular calcium levels and increased release of IL-6 and IL-8, while also modifying PrPC localization. HMC3 stimulated with histamine for 6 and 24 hours exhibited increased surface PrPC expression. Specifically, we found that stimulation of the HRH2 receptor was responsible for changes in surface PrPC. Histamine-induced increases in surface PrPC were attenuated following inhibition of the HRH2 receptor via the HRH2 antagonist ranitidine. These changes were unique to HRH2 activation, as stimulation of HRH1, HRH3, or HRH4 did not alter surface PrPC. Prolonged stimulation of HMC3 decreased PrPC expression following 48 and 72 hours of histamine stimulation. HMC3 cells can be stimulated by histamine to undergo intracellular calcium influx. Surface expression levels of PrPC on HMC3 cells are altered by histamine exposure, primarily mediated by HRH2. While histamine exposure also increases release of IL-6 and IL-8 in these cells, this cytokine release is not fully dependent on PrPC levels, as IL-6 release is only partially reduced and IL-8 release is unchanged under the conditions of HRH2 blockade that prevent PrPC changes. Overall, this suggests that PrPC may play a role in modulating microglial responses.

Sex-dependent differential increase of specialized pro-resolving mediators in extracellular vesicles secreted by human primary conjunctival goblet cells during allergic inflammation

AimsConjunctival epithelium lines the inside of the eyelids and covers the sclera, thus providing stability to the eye surface. Goblet cells in conjunctival epithelium (CjGCs) are well known for their mucin-secretion function, which wet and protect the ocular surface, but other aspects are still not well understood. To expand our understanding beyond their mucin-secreting function, we investigated CjGC-secreted extracellular vesicles (EVs) and lipid mediators therein. Materials and methodsUsing histamine-mediated allergic inflammation in human primary CjGCs (HCjGCs) as a disease model, we quantified using ELISA a proinflammatory mediator PGE2 and two specialized pro-resolving mediators (SPMs) LXA4 and RvD1 in EVs secreted during allergic inflammation. Key findingsAt 18 h post histamine stimulation, the amount of LXA4 and RvD1 in EVs was notably higher compared to those in unstimulated. Interestingly, this increase was only observed in female EVs but not in males. The mean fold increase of LXA4 and RvD1 in female EVs was 3.9 and 3.4, respectively, but it was only 0.9 and 1.0 in male EVs. Supplying docosahexaenoic acid (DHA, the source of RvD1 and other SPMs) to the culture medium during the allergic inflammation resulted in even higher mean fold increase of 5.3 and 6.9 for LXA4 and RvD1 in female EVs, respectively, but it was only 0.5 and 0.8 in male EVs. SignificanceWe conclude that HCjGCs show a clear sex difference in allergic response. Our results may also provide a new insight into the male predisposition to severe forms of allergic conjunctivitis and potential improvement in disease care in the clinic.

Immunological disturbance effect of exogenous histamine towards key immune cells

Histamine in food has attracted widespread attention due to the potential toxicity and associated health risk. However, its influences on immunological components, especially the function of key immune cells, are still poorly known. In this work, we explored the effects of exogenous histamine on the function of key immune cells such as intestinal epithelial cells, dendritic cells, and T cells. The results showed that histamine could affect the expression of allergy-related genes in CMT93 cells at a high dose of histamine. Moreover, it’s found that histamine could cause an imbalance in the levels of relevant immune factors secreted by dendritic cells and T cells, especially those related to allergy. At the same time, the proportion of MHC class II-positive dendritic cells and the proportion of T helper 2 (Th2) cells in CD4+ T cells increased after histamine stimulation. We concluded that the presence of a certain level of histamine in food may affect the expression of allergy-related cytokines, disrupt the balance of the immune homeostasis, and potentially lead to adverse immune reactions. This work demonstrated the importance of including the estimation of histamine's immune safety in aquatic products rather than merely considering the potential risk of food poisoning.

ADAMTS13-Mediated Proteolytic Cleavage of Unusually Large von Willebrand Factor Polymers on Endothelial Cells in the Absence of Fluid Shear Stress

To investigate the molecular mechanism of proteolytic cleavage of unusually large von Willebrand Factor(ULVWF) on endothelial cells by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) in the absence of fluid shear stress, so as to provide a theoretical basis for the pathogenesis of thrombotic thrombocytopenic purpura (TTP) and other thrombotic disorders. The ADAMTS13-mediated proteolysis of ULVWF on the surface of endothelial cells in the absence of fluid shear stress was observed through immunofluorescence microscopy. The variation in VWF antigen levels in the conditioned media were determined by ELISA assay. The levels of VWF and the proteolytic fragments released into the conditioned media were determined by ELISA assay and Western blot in the absence and presence of fluid shear stress or FVIII. The effect of ADAMTS13-mediated ULVWF cleavage on the normal distribution of plasma VWF multimers was evaluated by multimer analysis. Histamine stimulated human umbilical vein endothelial cells (HUVECs) were incubated with ADAMTS13 and various N- and C-terminally truncated mutants. Then the ULVWF that maintained binding to the cells were observed through immunofluorescence microscopy and the soluble ULVWF released from endothelial cells was determined by ELISA, so as to demonstrate the domains of ADAMTS13 required for proteolysis of ULVWF on endothelial cells. The ULVWF strings on the endothelial cell surface were rapidly proteolyzed by recombinant and plasma ADAMTS13 in the absence of fluid shear stress. This proteolytic processing of ULVWF depended on incubation time and ADAMTS13 concentration, but not shear stress and FVIII. The distribution of VWF releaseded by ADAMTS13-mediated proteolysis was quite similar to that secreted by endothelial cells under histamine stimulation, suggesting the ULVWF cleavage occured at the cell surface. The proteolysis of the ULVWF on endothelial cells required the Cys-rich(CysR) and spacer domains, but not the TSP1 2-8 and CUB domains of ADAMTS13. The ULVWF polymers on endothelial cells are sensitive to ADAMTS13-mediated cleavage even in the absence of fluid shear stress. The findings provide novel insight into the molecular mechanism of ADAMTS13-mediated ULVWF cleavage at the cellular level and may contribute to understanding of the pathogenesis of TTP and other thrombotic disorders.

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.

Prediction of gastroesophageal reflux episodes by smooth muscle electromyography: A translational study in rats and adolescents

AimsOur aim was to measure the myoelectric modifications during gastric acid secretion along with the gastric pH in a rat model and to detect the gastrointestinal (GI) myoelectric changes in adolescents suffering from gastroesophageal reflux disease (GERD) along with the esophageal pH measurement. Main methodsIn anesthetized rats, gastric acid secretion was initiated with intragastric histamine (50 mg/kg), and gastric pH, GI myoelectric activity and mechanical GI contractions were measured with intragastric pH electrode, subcutaneously implanted smooth muscle electromyography (SMEMG) electrodes and organ implanted strain gauges, respectively. In the clinical study, esophageal pH and GI myoelectric activity were measured in adolescents suffering from GERD with intraesophageal pH electrode and SMEMG electrodes placed on the abdominal surface, respectively. The SMEMG records were analyzed by fast Fourier transformation (FFT) and power spectrum density maximum (PsDmax) values were calculated for the GI segments. Key findingsIn rats, histamine initiated an immediate increase in gastric PsDmax, which preceded the significant reduction in gastric pH by 75 min. The myoelectric change was independent of mechanical GI contractions. In adolescents, the GERD episodes were preceded by a significant increase in gastric PsDmax 45 min earlier. These changes were independent of motion or meals. SignificanceIncreased gastric myoelectric activity during histamine stimulation or GERD might be linked to the enhanced activity of the gastric proton pump, indicating a link between gastric acid secretion and GERD episodes. It is supposed that SMEMG might be a tool for predicting forthcoming reflux episodes in GERD.

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.

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.

Monitoring of histamine-induced calcium channel activity of a single cell using semiconducting carbon nanotube transistors

A method using transistors based on semiconducting carbon nanotubes were developed for the real-time monitoring of the electrophysiological responses of individual cells to histamine stimulation. Transistors with one or three floating electrodes were utilized to evaluate histamine-induced Ca2+ influx into Hela cells via the recording of the conductance changes of the transistors. The Ca2+ influx resulted from the activation of histamine H1 receptors embedded on the cell membranes by histamine, which generated a temporary negative potential at the gap between the cell and the transistor. Moreover, the antihistamine effects of chlorpheniramine on histamine-induced Ca2+ influx were also investigated by using a transistor including three floating electrodes. Especially, only a single transistor was applied to repeat the measurements of the responses of multiple Hela cells pretreated with chlorpheniramine to histamine stimulation. This allows us to acquire data without being suffered from device-to-device variations, implying our method would be a simple but powerful method for applications of nanoscale biosensors to electrophysiological studies.

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.

Effects of rosmarinic acid, carnosic acid, rosmanol, carnosol, and ursolic acid on the pathogenesis of respiratory diseases.

This review aimed to identify preclinical and clinical studies examining the effects of rosmarinic acid (RA), carnosic acid (CaA), rosmanol (RO), carnosol (CA), and ursolic acid (UA) against allergic and immunologic disorders. Various online databases, including PubMed, Science Direct, EMBASE, Web of Sciences, Cochrane trials, and Scopus, were searched from inception until October 2022. Due to the suppression of the nuclear factor-κB (NF-κB) pathway, the main factor in allergic asthma, RA may be a promising candidate for the treatment of asthma. The other ingredients comprising CA and UA reduce the expression of interleukin (IL)-4, IL-5, and IL-13 and improve airway inflammation. Rosemary's anti-cancer effect is mediated by several mechanisms, including DNA fragmentation, apoptosis induction, inhibition of astrocyte-upregulated gene-1 expression, and obstruction of cell cycle progression in the G1 phase. The compounds, essentially found in Rosemary essential oil, prevent smooth muscle contraction through its calcium antagonistic effects, inhibiting acetylcholine (ACH), histamine, and norepinephrine stimulation. Additionally, CA exhibits a substantially greater interaction with the nicotinic ACH receptor than a family of medications that relax the smooth muscles, making it a potent antispasmodic treatment. The components have demonstrated therapeutic effects on the immune, allergy, and respiratory disorders.

Lamin-A/C Is Modulated by the Involvement of Histamine-Mediated Calcium/Calmodulin-Dependent Kinase II in Lung Cancer Cells.

Lamins are nuclear envelope proteins involved in various cellular functions, such as DNA modulation, cellular differentiation, and development. In this study, we investigate the role of histamine in lung cancer biology. Since it is known that lamin-A/C is negatively regulated in lung cancer, we hypothesize that histamine signaling is related to nuclear lamin-A/C regulation and cancer progression. Our findings reveal that histamine stimulation enhances lamin-A/C expression in lung cancer cells. Lamin-A/C expression is dependent on histamine-mediated intracellular calcium signaling and subsequent calcium/calmodulin-dependent kinase II (Ca/CaMKII) activation. The nuclear protein nestin, which stabilizes lamin-A/C expression, is also modulated by Ca/CaMKII. However, histamine-mediated lamin-A/C expression is independent of Akt/focal adhesion kinase or autophagy signaling. Histamine stimulation attenuates lung cancer motility in the presence of enhanced lamin-A/C expression. In conclusion, we propose a regulatory mechanism that accounts for the modulation of lamin-A/C levels through the involvement of Ca/CaMKII in cancer cells and provides molecular evidence of histamine signaling in lamin-A/C biology.

Adenine nucleotides transport across the inner mitochondrial membrane in cancer cells: role of ANTs and SCaMCs

Introduction: The regulation of adenine nucleotide levels in the mitochondria and the cytosol is essential for cell metabolism. To sustain mitochondrial respiration, ATP generated by ATP synthase must be exported to the cytosol, whereas mitochondria must be refilled with ADP and Pi. Adenine Nucleotide Translocases (ANTs), which catalyze the electrogenic ATP4-/ADP3- exchange, have been proposed to work together with ATP synthase and the mitochondrial phosphate carrier to support mitochondrial oxidative phosphorylation by forming the so-called ATP synthasome. Nevertheless, disruption of the main ANT isoform or pharmacological inhibition of ANTs in cancer cells does not decrease oxidative phosphorylation. Furthermore, the import of cytosolic ATP to the mitochondria when oxidative phosphorylation is inhibited (required, for example, for the maintenance of the ΔΨm) is neither mediated by ANTs in cancer cells. Here, we study the possibility that calcium-regulated mitochondrial ATP-Mg2+/Pi or ADP/Pi carriers, (also called SCaMCs), might participate in the mitochondrial transport of adenine nucleotides in cancer cells. Material and methods: To study the function of SCaMC-1, the main isoform in cancer cells, we have generated SCaMC-1 KO HeLa cells using CRISPR-Cas9 genome editing. These cells have been characterized in terms of OCR (with Seahorse XF24), mitochondrial membrane potential (with TMRM) and mitochondrial ATP levels (with mitoGoATeam genetic probe) in basal conditions and in the presence of ETC inhibitors. Results and conclusions: We show that SCaMC-1, the isoform that is abundant in tumor cells, mediates ATP import to the mitochondria after histamine stimulation of HeLa cells, probably to buffer calcium entry to the mitochondria. However, SCaMC-1 does not participate in the transport of cytosolic ATP for the maintenance of ΔΨm after OXPHOS inhibition, neither decreases mitochondrial respiration. Future studies based on the deletion of the other SCaMCs isoforms (SCaMC-2, SCaMC-3, SCaMC-3 like) together with simultaneous deletion of ANTs and SCaMCs will be required to shed light on the control of adenine nucleotides transport between the mitochondria and the cytosol in cancer cells.

In vivo visualization and analysis of ciliary motion in allergic rhinitis models induced by ovalbumin.

Due to the lack of an assessment approach, the image of in vivo nasal ciliary motion of allergic rhinitis (AR) has never been captured and analyzed to date. Here, we have used an optimized approach to analyze the nasal ciliary function in vivo in AR rats. The digital microscopy system, a method for direct observation of ciliary motion in a living AR rat model, was applied to visualize and measure ciliary motion in vivo, including ciliary beat frequency (CBF) and ciliary beat distance (CBD). The AR rat model was established by ovalbumin sensitization. Comparisons of nasal ciliary motion in vivo between the experimental group (ovalbumin sensitization, allergen, or histamine) and the control group were analyzed. In the living rat model of allergic rhinitis, CBF and CBD decreased to 57.8 and 73.1% of the control group, respectively, but were restored after administration of chlorpheniramine maleate. Ovalbumin (OVA) significantly inhibited the ciliary motion of normal mucosa in vivo. However, responding to the OVA challenge, the ciliary motion of OVA-sensitized mucosa would not decrease further and stay at a stable level. Histamine stimulated in vivo ciliary motion quickly within 30 min, but afterward, the ciliary motion gradually decreased below the baseline. These results have clarified that in vivo ciliary motion was impaired by nasal mucosal sensitization, and this impairment was most likely related to allergen challenge and histamine. In addition, the short-term stimulation and long-term inhibition effects of histamine on in vivo ciliary motion were first reported in this study.

Characterizing the Luminal Microenvironment of Human Organoids for Studies of Gastric pH Regulation

The stomach is notorious for having the steepest proton gradient in the body, which pathogenic Helicobacter pylori must traverse to infect the epithelium. This gradient across the 200‐300 μm‐thick gastric mucus layer enables the stomach to maintain a near‐neutral pH at the epithelial surface and a strongly acidic environment in the stomach lumen. In this study, we use human gastric organoids to study the maintenance and regulation of this pH gradient. Human gastric organoids are 3D cellular constructs derived from patient biopsies that mimic the microanatomy and physiology of the stomach, as they contain both mucus and gastric acid. Organoids have been used to investigate H. pylori‐induced gastritis, peptic ulcers, and stomach cancer, which claims 700,000 lives each year. Gastric mucus is critical for protection against H. pylori infection, which is the leading cause of gastric cancer. It is presently unclear how the gastric mucosal pH gradient is maintained, and how closely organoid models can mimic this microenvironment. First, we detected mucus within human gastric organoids using histological staining and bead microrheology. We also used a FITC‐conjugated lectin to visualize the mucosal matrix inside of the organoids. These data were supported by our observation that a clear viscous material was produced on the apical side of two‐dimensional, organoid‐derived cell monolayers. Additionally, we developed a novel method for the measurement of intraluminal pH of the gastric organoids. Using a micromanipulator and stereomicroscope to maneuver a glass microelectrode into the organoid lumen, we measured and manipulated luminal pH using histamine stimulation. We determined that pH remains consistent across organoid lines, and that the intraluminal space is more acidic than the surrounding environment. In ongoing studies, we will develop the spatial resolution of our organoid microprofiling technique to fully characterize the proton gradient within the mucus‐filled lumens gastric organoids. Our studies are the first to utilize pH microsensors in these models, and will lead to improved molecular and biophysical understanding of gastric physiology and potential new treatment approaches for disease conditions (such as peptic ulcers and gastric cancer) that involve dysregulated gastric acid secretion.

Elucidating the Bitter Taste Signaling Paradox in Airway Smooth Muscle Relaxation

RATIONALEAirway smooth muscle (ASM) hypersensitivity and hypercontractility play a pivotal role in the pathophysiology of obstructive airway diseases such as asthma. Furthermore, over half of affected individuals are unable to reliably control these disorders using the current standard of care, β‐(adrenergic) agonists. Previously, our group discovered the expression of a novel group of G protein‐coupled receptors (GPCRs), type 2 taste receptors (TAS2Rs), in the human airway which evoke ASM relaxation. Previous work in our lab suggests that TAS2R agonists evoke relaxation in a G protein beta‐gamma subunit‐, phospholipase C beta‐ and calcium‐dependent manner. We hypothesized that the downstream molecular targets of contractile‐related signaling components are differentially regulated by TAS2Rs compared to other GPCRs. The goal of this study was to establish the molecular mechanism by which TAS2Rs induce ASM relaxation.METHODSWe employed a phosphoproteomic approach, involving dual‐mass spectrometry, to determine differences in the phosphorylation status of contractile‐related proteins in ASM following the stimulation of TAS2Rs and other GPCRs. Healthy human ASM cells were treated with the TAS2R agonist chloroquine or β‐agonist isoproterenol alone or in combination with the bronchoconstrictive agonist histamine to study the ability of each relaxant compound to attenuate the histamine‐mediated phosphorylation profile of contractile‐related proteins. These phosphoproteomic profiles were compared and a select set of targets were validated using western blot analysis.RESULTSOur phosphoproteomic data demonstrated that histamine stimulation results in increased phosphorylation of Ras homolog family member A (RhoA) guanine nucleotide exchange factor (ARHGEF) 12 and A kinase anchoring proteins (AKAP) 2 and 12, as well as decreased phosphorylation of RhoA GTPase‐activating protein (ARHGAP) 6. Furthermore, chloroquine treatment attenuated histamine‐induced phosphorylation of ARHGEF12 but not AKAP2 or AKAP12. Interestingly, isoproterenol attenuated histamine‐induced phosphorylation of AKAP2 and AKAP12 but not ARHGEF12. Validation studies using western blot analysis revealed chloroquine mediated inhibition of histamine‐induced RhoA activation and myosin phosphatase target subunit 1 (MYPT1) phosphorylation at the T853 residue, which was not observed with isoproterenol.CONCLUSIONOur findings were able to show that contractile‐related proteins are phosphorylated differentially by chloroquine and isoproterenol to elicit ASM relaxation. Furthermore, our studies strongly suggest the importance of RhoA and the T853 residue on MYPT1 as points of mechanistic divergence between TAS2R and other GPCR pathways. These findings provide further insight into the mechanism by which TAS2R signaling reduces airway contractility and its potential as a novel therapy for obstructive airway disease.

Single-cell imaging of ERK and Akt activation dynamics and heterogeneity induced by G-protein-coupled receptors.

ABSTRACTKinases play key roles in signaling networks that are activated by G-protein-coupled receptors (GPCRs). Kinase activities are generally inferred from cell lysates, hiding cell-to-cell variability. To study the dynamics and heterogeneity of ERK and Akt proteins, we employed high-content biosensor imaging with kinase translocation reporters. The kinases were activated with GPCR ligands. We observed ligand concentration-dependent response kinetics to histamine, α2-adrenergic and S1P receptor stimulation. By using G-protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to α2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single-cell level, especially for ERK. Cluster analysis of time series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs.

Semiconducting Carbon Nanotube-Based Nanodevices for Monitoring the Effects of Chlorphenamine on the Activities of Intracellular Ca2+ Stores.

We report a flexible and noninvasive method based on field-effect transistors hybridizing semiconducting single-walled carbon nanotubes for monitoring the effects of histamine on Ca2+ release from the intracellular stores of a nonexcitable cell. These nanodevices allowed us to evaluate the real-time electrophysiological activities of HeLa cells under the stimulation of histamine via the recording of the conductance changes of the devices. These changes resulted from the binding of histamine to its receptor type 1 on the HeLa cell membrane. Moreover, the effects of chlorphenamine, an antihistamine, on the electrophysiological activities of a single HeLa cell were also evaluated, indicating that the pretreatment of the cell with chlorpheniramine decreased intracellular Ca2+ release. Significantly, we only utilized a single nanodevice to perform the measurements for multiple cells pretreated with various concentrations of chlorphenamine. This enabled the statistically meaningful analysis of drug effects on cells without errors from device variations. Obtained results indicated the novel advantages of our method such as real-time monitoring and quantitative capability. Our devices, therefore, can be efficient tools for biomedical applications such as electrophysiology research and drug screening.

Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells

Weibel–Palade bodies (WPB) are endothelial cell-specific storage granules that regulate vascular hemostasis by releasing the platelet adhesion receptor von Willebrand factor (VWF) following stimulation. Fusion of WPB with the plasma membrane is accompanied by the formation of actin rings or coats that support the expulsion of large multimeric VWF fibers. However, factor(s) organizing these actin ring structures have remained elusive. We now identify the actin-binding proteins Spire1 and Myosin Vc (MyoVc) as cytosolic factors that associate with WPB and are involved in actin ring formation at WPB-plasma membrane fusion sites. We show that both, Spire1 and MyoVc localize only to mature WPB and that upon Ca2+ evoked exocytosis of WPB, Spire1 and MyoVc together with F-actin concentrate in ring-like structures at the fusion sites. Depletion of Spire1 or MyoVc reduces the number of these actin rings and decreases the amount of VWF externalized to the cell surface after histamine stimulation.

Histamine enhances ATP-induced itching and responsiveness to ATP in keratinocytes

Mechanical stimulation of cultured keratinocytes and a living epidermis increases intracellular calcium ion concentrations ([Ca2+]i) in stimulated cells. This action propagates a Ca2+ wave to neighboring keratinocytes via ATP/P2Y2 receptors. Recent behavioral, pharmacological studies revealed that exogenous ATP induces itching via P2X3 receptors in mice. We previously showed that alloknesis occurs when an external stimulus is applied to the skin with increased epidermal histamine in the absence of spontaneous pruritus. Based on these results, we investigated the effects of histamine at a concentration that does not cause itching on ATP-induced itching. The mean number of scratching events induced by the mixture of ATP and histamine increased by 28% over the sum of that induced by histamine alone or ATP alone. A317491, a P2X3 receptor antagonist, suppressed the mixture-induced scratching more often than the ATP-induced scratching. Next, we examined the ATP-induced [Ca2+]i change before and after histamine stimulation using normal human epidermal keratinocytes. Some cells did not respond to ATP before histamine stimulation but responded to ATP afterward, the phenomenon suppressed by chlorpheniramine maleate. These findings suggest that histamine enhances ATP-induced itching and that a potential mechanism could involve increased responsiveness to ATP in keratinocytes.