Rat Mast Cells Research Articles

Antimicrobial cetylpyridinium chloride suppresses mast cell function by targeting tyrosine phosphorylation of Syk kinase.

Cetylpyridinium chloride (CPC) is a quaternary ammonium antimicrobial used in numerous personal care products, human food, cosmetic products, and cleaning solutions. Yet, there is minimal published data on CPC effects on eukaryotes, immune signaling, and human health. Previously, we showed that low-micromolar CPC inhibits rat mast cell function by inhibiting antigen (Ag)-stimulated Ca 2+ mobilization, microtubule polymerization, and degranulation. In this study, we extend the findings to human mast cells (LAD2) and present data indicating that CPC's mechanism of action centers on its positively-charged quaternary nitrogen in its pyridinium headgroup. CPC's inhibitory effect is independent of signaling platform receptor architecture. Tyrosine phosphorylation events are a trigger of Ca 2+ mobilization necessary for degranulation. CPC inhibits global tyrosine phosphorylation in Ag-stimulated mast cells. Specifically, CPC inhibits tyrosine phosphorylation of specific key players Syk kinase and LAT, a substrate of Syk. In contrast, CPC does not affect Lyn kinase phosphorylation. Thus, CPC's root mechanism is electrostatic disruption of particular tyrosine phosphorylation events essential for signaling. This work outlines the biochemical mechanisms underlying the effects of CPC on immune signaling and allows the prediction of CPC effects on cell types, like T cells, that share similar signaling elements.

Novel IgE crosslinking-induced luciferase expression method using human-rat chimeric IgE receptor-carrying mast cells

BackgroundThe measurement of antigen-specific serum IgE is common in clinical assessments of type I allergies. However, the interaction between antigens and IgE won't invariably trigger mast cell activation. We previously developed the IgE crosslinking-induced luciferase expression (EXiLE) method using the RS-ATL8 mast cell line; however, the method may not be sensitive enough in some cases. MethodsIn this study, we introduced an NF-AT-regulated luciferase reporter gene into the RBL-2H3 rat mast cell line and expressed a chimeric high-affinity IgE receptor (FcεRI) α chain gene, comprising an extracellular domain from humans and transmembrane/intracellular domains from rats. ResultsWe generated multiple clones expressing the chimeric receptor. Based on their responsiveness and proliferation, we selected the HuRa-40 clone. This cell line exhibited significantly elevated human α chain expression compared to RS-ATL8 cells, demonstrating a 10-fold enhancement of antigen-specific reactivity. Reproducibility across different batches and operators was excellent. Moreover, we observed a detectable response inhibition by an anti-allergy drugs (omalizumab and cyclosporin A). ConclusionsHuRa-40 cells—which carry the human-rat chimeric IgE receptor—comprise a valuable reporter cell line for the EXiLE method. Their versatility extends to various applications and facilitates high-throughput screening of anti-allergy drugs.

Suppression of Mast Cell Activation by GPR35: GPR35 Is a Primary Target of Disodium Cromoglycate.

Mast cell stabilizers, including disodium cromoglycate (DSCG), were found to have potential as the agonists of an orphan G protein-coupled receptor, GPR35, although it remains to be determined whether GPR35 is expressed in mast cells and involved in suppression of mast cell degranulation. Our purpose in this study is to verify the expression of GPR35 in mast cells and to clarify how GPR35 modulates the degranulation. We explored the roles of GPR35 using an expression system, a mast cell line constitutively expressing rat GPR35, peritoneal mast cells, and bone marrow-derived cultured mast cells. Immediate allergic responses were assessed using the IgE-mediated passive cutaneous anaphylaxis (PCA) model. Various known GPR35 agonists, including DSCG and newly designed compounds, suppressed IgE-mediated degranulation. GPR35 was expressed in mature mast cells but not in immature bone marrow-derived cultured mast cells and the rat mast cell line. Degranulation induced by antigens was significantly downmodulated in the mast cell line stably expressing GPR35. A GPR35 agonist, zaprinast, induced a transient activation of RhoA and a transient decrease in the amount of filamentous actin. GPR35 agonists suppressed the PCA responses in the wild-type mice but not in the GPR35-/- mice. These findings suggest that GPR35 should prevent mast cells from undergoing degranulation induced by IgE-mediated antigen stimulation and be the primary target of mast cell stabilizers. SIGNIFICANCE STATEMENT: The agonists of an orphan G protein-coupled receptor, GPR35, including disodium cromoglycate, were found to suppress degranulation of rat and mouse mature mast cells, and their antiallergic effects were abrogated in the GPR35-/- mice, indicating that the primary target of mast cell stabilizers should be GPR35.

Potential Role of Moesin in Regulating Mast Cell Secretion.

Mast cells have existed for millions of years in species that never suffer from allergic reactions. Hence, in addition to allergies, mast cells can play a critical role in homeostasis and inflammation via secretion of numerous vasoactive, pro-inflammatory and neuro-sensitizing mediators. Secretion may utilize different modes that involve the cytoskeleton, but our understanding of the molecular mechanisms regulating secretion is still not well understood. The Ezrin/Radixin/Moesin (ERM) family of proteins is involved in linking cell surface-initiated signaling to the actin cytoskeleton. However, how ERMs may regulate secretion from mast cells is still poorly understood. ERMs contain two functional domains connected through a long α-helix region, the N-terminal FERM (band 4.1 protein-ERM) domain and the C-terminal ERM association domain (C-ERMAD). The FERM domain and the C-ERMAD can bind to each other in a head-to-tail manner, leading to a closed/inactive conformation. Typically, phosphorylation on the C-terminus Thr has been associated with the activation of ERMs, including secretion from macrophages and platelets. It has previously been shown that the ability of the so-called mast cell "stabilizer" disodium cromoglycate (cromolyn) to inhibit secretion from rat mast cells closely paralleled the phosphorylation of a 78 kDa protein, which was subsequently shown to be moesin, a member of ERMs. Interestingly, the phosphorylation of moesin during the inhibition of mast cell secretion was on the N-terminal Ser56/74 and Thr66 residues. This phosphorylation pattern could lock moesin in its inactive state and render it inaccessible to binding to the Soluble NSF attachment protein receptors (SNAREs) and synaptosomal-associated proteins (SNAPs) critical for exocytosis. Using confocal microscopic imaging, we showed moesin was found to colocalize with actin and cluster around secretory granules during inhibition of secretion. In conclusion, the phosphorylation pattern and localization of moesin may be important in the regulation of mast cell secretion and could be targeted for the development of effective inhibitors of secretion of allergic and inflammatory mediators from mast cells.

Ferruginous bodies exert a strong proinflammatory effect

ABSTRACT One of the main problems related to ferruginous-asbestos bodies (ABs) exposure is their potential pathogenetic role in asbestos-related diseases. The aim of this study was to examine whether purified ABs, might stimulate inflammatory cells. ABs were isolated by exploiting their magnetic properties, therefore avoiding the strong chemical treatment usually employed for this purpose. This latter treatment, which is based upon the digestion of organic matter with concentrated hypochlorite, may markedly modify the AB structure and consequently also their “in vivo” manifestations. ABs were found to induce secretion of human neutrophil granular component myeloperoxidase, as well as stimulate rat mast cell degranulation. Data demonstrated that by triggering secretory processes in inflammatory cells, purified ABs may play a role in the pathogenesis of asbestos-related diseases by continuing and enhancing the pro-inflammatory activity of the asbestos fibers.

Effect of electroacupuncture on the morphology of prostate and degranulation of mast cells in rats with chronic nonbacterial prostatitis

To observe the effect of electroacupuncture (EA) on the morphology of prostate and degranulation of mast cells in prostate of rats with chronic nonbacterial prostatitis (CNP). Male SD rats were randomly divided into sham operation group, model group and EA group, with 8 rats in each group. CNP model was established by surgical castration combined with 17-β estradiol injection once daily for 30 days. EA was applied to "Zhongji" (CV3), "Guanyuan" (CV4) and bilateral "Dahe" (KI12) for 20 min, once daily for 8 days. The mechanical pain threshold of scrotum skin area was tested before modeling, after modeling and after intervention. The pathological morphology of the prostate was observed by HE staining. Collagenous fiber was observed by Masson staining. The infiltration of mast cells was observed by toluidine blue staining. The contents of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in prostate were determined by ELISA. The protein expression levels of tryptase and transforming growth factor β1 (TGF-β1) in prostate were detected by Western blot. A normal appearance with little inflammatory cell infiltration was observed in the prostate of the sham operation group. Gland atrophy, epithelial destruction, hyperemia and edema, diffuse inflammatory cell infiltration and visible collagen fiber deposition were observed in prostate of the model group. The degree of infiltration of inflammatory cells and collagen fiber deposition were reduced in the EA group. Compared with the sham operation group, mechanical pain threshold was decreased (P<0.01), while the collagen volu-me fraction (CVF) of prostate, the degranulated rate of mast cells, the protein expression levels of tryptase and TGF-β1, and the contents of IL-6 and TNF-α were increased (P<0.01) in the model group. Following EA intervention, compared with the model group, the mechanical pain threshold was increased (P<0.01), CVF of the prostate, the degranulated rate of mast cells, the protein expression levels of tryptase and TGF-β1, and the contents of IL-6 and TNF-α were decreased (P<0.05, P<0.01) in the EA group. EA can relieve pain and reduce inflammation and fibrosis of prostate in CNP rats, which may be related to inhibiting the degranulation of prostate mast cells and down-regulating the expression of inflammatory factors and TGF-β1.

Cannabinoid non-cannabidiol site modulation of TRPV2 structure and function

TRPV2 is a ligand-operated temperature sensor with poorly defined pharmacology. Here, we combine calcium imaging and patch-clamp electrophysiology with cryo-electron microscopy (cryo-EM) to explore how TRPV2 activity is modulated by the phytocannabinoid Δ9-tetrahydrocannabiorcol (C16) and by probenecid. C16 and probenecid act in concert to stimulate TRPV2 responses including histamine release from rat and human mast cells. Each ligand causes distinct conformational changes in TRPV2 as revealed by cryo-EM. Although the binding for probenecid remains elusive, C16 associates within the vanilloid pocket. As such, the C16 binding location is distinct from that of cannabidiol, partially overlapping with the binding site of the TRPV2 inhibitor piperlongumine. Taken together, we discover a new cannabinoid binding site in TRPV2 that is under the influence of allosteric control by probenecid. This molecular insight into ligand modulation enhances our understanding of TRPV2 in normal and pathophysiology.

Effect of melatonin on degranulation of dura mater mast cells in Wistar rats

Introduction Migraine is a highly prevalent neurological disorder that affects about 15% of the world's population. Involved in migraine pain, the cranial dura mater is a richly vascularized and innervated membrane, where we also find mast cells, and immune cells that help in the formation of the inflammatory process. As one of the treatments for migraine, we have melatonin, a hormone produced in the pineal gland and with properties such as circadian cycle control and antioxidant action. Capsaicin, a bioactive compound found in peppers and with a pungent and burning effect, also has an action in the painful process and serves as a tool in the study of physiopathogenic processes. Objectives To evaluate the degranulation of dura mater mast cells from Wistar rats stimulated in situ with capsaicin and synthetic interstitial fluid (SIF) and previously treated with melatonin. Methods Twenty-five male Wistar rats were used, obtained from the Department of Antibiotics - Danti of the Federal University of Pernambuco - UFPE and with use authorized by the Ethics Committee in the Use of Animals - CEUA of UFPE, according to protocol nº 0084/2019. During the adaptation period, the animals were acclimatized under standard laboratory conditions, with water and food ad libitum. After this period, they were separated into two groups: a control group (CG) (n = 12) and a melatonin group (GM) (n = 13), and underwent a daily treatment for 10 days with the intraperitoneal application. The GM received a dosage of 10 mg/kg of the animal weight of melatonin diluted in a saline solution at 0.9% and the GC was treated only with the vehicle. After the treatment, the animals were anesthetized and submitted to an experimental surgery... (To see the complet abstract, please, check out the PDF.)

Extracellular Ca2+ aggravates IgE-induced allergic reaction in mast cells through GPRC6A, a novel family C G-protein-coupled receptor

AimsAs an essential indicator of allergic reactions, mast cell (MC) activation involves FcεRI-mediated signaling and the release of allergic mediators. In FcεRI signaling, Ca2+ is located at the intersection of multiple cellular signaling pathways. However, the effect of extracellular Ca2+ (exCa2+) on MCs during anaphylaxis remains unclear, along with its exact mechanisms. Therefore, we sought to determine whether and how elevated exCa2+ amplifies allergic reactions. Main methodsIn vitro experiments used immunoglobulin E (IgE)/antigen (Ag)-induced activation of rat and mouse MCs in vitro. The levels of MC degranulation mediators were used to evaluate the effect of exCa2+. In vivo experiments used MC-mediated passive systemic anaphylaxis (PCA) Balb/c mice. After stimulation, anaphylaxis indexes such as rectal temperature and allergic symptom score were detected. Key findingsIn vitro experiments revealed that exCa2+ is a stimulus signal for the aggravation of allergic reactions in MCs. When antagonists or siRNA inhibited GPRC6, MCs released fewer inflammatory mediators. Moreover, in vivo experiments confirmed in vitro results. Allergic symptoms were alleviated by antagonists NPS2143 in PCA mice, demonstrating that exCa2+ aggravates allergic reactions through GPRC6A. SignificanceOur study provides an essential theoretical basis for targeting Ca2+ and GPRC6A as therapeutic options for allergies.

Mast Cell Population and Histamine Content in Hypothyroid Rat Tissues.

Simple SummaryIn this study, we investigated the putative regulatory role of the pituitary–thyroid axis on mast cells (MCs). We found that hypothyroidism resulted in a significant increase in number of MCs and the histamine content in the skin and exorbital lacrimal gland of Wistar rats, Rattus norvegicus. Furthermore, an increase in the percentage of degranulating MCs suggests that the thyroid status also influences the activation state of these cells.The morphological features and relative number of mast cells (MCs) were studied in the skin and exorbital lacrimal glands of hypothyroid Wistar rats, Rattus norvegicus. Hypothyroidism significantly increased the number of MCs (up to 4.5-fold) and histamine content (up to 50%) in the examined tissues. The magnitude of the increase in the number of MCs was greater in the cheek skin and exorbital lacrimal glands than in the back skin. In the skin, the MCs were mainly located within the hypodermis and closely associated with the blood vessels, nerve fascicles, and adipocytes. In the exorbital lacrimal gland, which is a seromucous gland located lateral to the cheek below the ear, the MCs were distributed in the connective tissue surrounding the acini. The secretory granules of MCs showed histochemical characteristics of connective tissue MCs. They were metachromatic with Toluidine blue and safranin positive with the Alcian blue/safranin reactions. Finally, a significant increase in degranulating MCs was observed in hypothyroid tissues, relative to euthyroid tissues. At the ultrastructural level, the MCs of euthyroid rats were predominantly non-degranulating (Stage I). In hypothyroid animals, numerous MCs showed partial degranulation (Stage II–III) or were in a stage of complete degranulation. Our results concerning the skin and exorbital lacrimal gland suggested that the thyroid status might be involved in regulating the frequency and activation state of MCs.

Effects of bladder perfusion of pentosan polysulfate sodium and hyaluronic acid on mucosal mast cells of rats with interstitial cystitis.

article: Effects of bladder perfusion of pentosan polysulfate sodium and hyaluronic acid on mucosal mast cells of rats with interstitial cystitis - Minerva Surgery 2022 Jun 16 - Minerva Medica - Journals

Effects of Nitric Oxide on the Electrical Activity of the Rat Trigeminal Nerve and Mast Cell Morphology

Effects of Nitric Oxide on the Electrical Activity of the Rat Trigeminal Nerve and Mast Cell Morphology

Effect of L-Glutamine on Chylomicron Formation and Fat-Induced Activation of Intestinal Mucosal Mast Cells in Sprague-Dawley Rats.

Glutamine (Gln) is required for intestinal mucosal homeostasis, and it can promote triglyceride absorption. The intestinal mucosal mast cells (MMCs) are activated during fat absorption. This study investigated the potential role of Gln on fat absorption-induced activation of MMCs in rats. Lymph fistula rats (n = 24) were studied after an overnight recovery with the infusion of saline only, saline plus 85 mM L-glutamine (L-Gln) or 85 mM D-glutamine (D-Gln), respectively. On the test day, rats (n = 8/group) were given an intraduodenal bolus of 20% Intralipid contained either saline only (vehicle group), 85 mM L-Gln (L-Gln group), or 85 mM D-Gln (D-Gln group). Lymph was collected hourly for up to 6 h for analyses. The results showed that intestinal lymph from rats given L-Gln had increased levels of apolipoprotein B (ApoB) and A-I (ApoA-I), concomitant with an increased spectrum of smaller chylomicron particles. Unexpectedly, L-Gln also increased levels of rat mucosal mast cell protease II (RMCPII), as well as histamine and prostaglandin D2 (PGD2) in response to dietary lipid. However, these effects were not observed in rats treated with 85 mM of the stereoisomer D-Gln. Our results showed that L-glutamine could specifically activate MMCs to degranulate and release MMC mediators to the lymph during fat absorption. This observation is potentially important clinically since L-glutamine is often used to promote gut health and repair leaky gut.

SAHA Alleviates Diarrhea-Predominant Irritable Bowel Syndrome Through Regulation of the p-STAT3/SERT/5-HT Signaling Pathway.

ObjectiveIrritable bowel syndrome (IBS) is characterized by abdominal pain, bloating, and stool irregularity. However, its pathophysiological mechanisms, which trigger intestinal motility disorders and diarrhea leading to diarrhea-predominant IBS (D-IBS), remain largely unknown.MethodsIn the present study, we established a D-IBS rat model by mother–infant separation combined with restraint stress. Then we exposed the modelled rats to suberoylanilide hydroxamic acid (SAHA) treatment, followed by determination of their visceral sensitivity. Toluidine blue staining served to reveal the effects of SAHA treatment on mast cells of D-IBS model rats. Then we measured the expression of serotonin (5-hydroxytryptamine; 5-HT) and its receptors by ELISA.ResultsConstruction of short hairpin RNA (sh)-serotonin transporter (SERT) lentivirus vectors verified the regulation of the 5-HT signaling pathway by phosphorylated (p)-STAT/SERT. SAHA treatment of D-IBS model rats reduced the fecal water content, electromyography integral change rate, abdominal withdrawal reflex score, and number of mast cells, as well as the expression of 5-HT type 3A (5-HT3AR), 3B receptor (5-HT3BR), and 4 receptor (5-HT4R) receptors. The treatment also elevated the expression of signal transducer and activator for transcription 3 (STAT3) and SERT. Activation of p-STAT3 may reverse the inhibitory effect of SAHA on the elevated visceral sensitivity of D-IBS model rats. Moreover, SAHA promoted the transcription of SERT through repression of the p-STAT3/5-HT signaling, thereby inhibiting the visceral sensitivity of D-IBS model rats.ConclusionThis study highlights that SAHA treatment can alleviate D-IBS through regulation of the p-STAT3/SERT/5-HT signaling pathway.

Guiding Chemically Synthesized Peptide Drug Lead Optimization by Derisking Mast Cell Degranulation-Related Toxicities of a NaV1.7 Peptide Inhibitor.

Studies have shown that some peptides and small molecules can induce non IgE-mediated anaphylactoid reactions through mast cell activation. Upon activation, mast cells degranulate and release vasoactive and proinflammatory mediators, from cytoplasmic granules into the extracellular environment which can induce a cascade of severe adverse reactions. This study describes a lead optimization strategy to select NaV1.7 inhibitor peptides that minimize acute mast cell degranulation (MCD) toxicities. Various in vitro, in vivo, and PKPD models were used to screen candidates and guide peptide chemical modifications to mitigate this risk. Anesthetized rats dosed with peptides demonstrated treatment-related decreases in blood pressure and increases in plasma histamine concentrations which were reversible with a mast cell stabilizer, supporting the MCD mechanism. In vitro testing in rat mast cells with NaV1.7 peptides demonstrated a concentration-dependent increase in histamine. Pharmacodynamic modeling facilitated establishing an in vitro to in vivo correlation for histamine as a biomarker for blood pressure decline via the MCD mechanism. These models enabled assessment of structure-activity relationship (SAR) to identify substructures that contribute to peptide-mediated MCD. Peptides with hydrophobic and cationic characteristics were determined to have an elevated risk for MCD, which could be reduced or avoided by incorporating anionic residues into the protoxin II scaffold. Our analyses support that in vitro MCD assessment in combination with PKPD modeling can guide SAR to improve peptide lead optimization and ensure an acceptable early in vivo tolerability profile with reduced resources, cycle time, and animal use.

Ginger-derived compounds exert in vivo and in vitro anti-asthmatic effects by inhibiting the T-helper 2 cell-mediated allergic response.

6-Shogaol (SHO) and 6-gingerol (GIN), naturally derived compounds of ginger (Zingiber officinale Roscoe), have been found to have anti-allergic effects on dermatitis-like skin lesions and rhinitis. Although SHO and GIN have demonstrated a potential in various inflammatory diseases, their efficacy and mechanism in asthma have not been largely examined. Therefore, the present study demonstrated the anti-asthmatic effects of SHO and GIN on the T-helper (Th) 2 cell-mediated allergic response pathway in an ovalbumin (OVA)-induced asthma mouse model. The asthma mouse model was established with an intraperitoneal (i.p.) injection of 50 µg OVA and 1 mg aluminum hydroxide with or without an i.p. injection of SHO and GIN (10 mg/kg) before treatment with OVA. In addition, the current study assessed mast cell degranulation in antigen-stimulated RBL-2H3 cells under different treatment conditions (SHO or GIN at 0, 10, 25, 50 and 100 nM) and determined the mRNA and protein levels of anti-oxidative enzymes [superoxide dismutase (SOD)1, SOD2, glutathione peroxidase-1/2, catalase] in lung tissues. SHO and GIN inhibited eosinophilia in the bronchoalveolar lavage fluids and H&E-stained lung tissues. Both factors also decreased mucus production in periodic acid-Schiff-stained lung tissues and the levels of Th2 cytokines in these tissues. GIN attenuated oxidative stress by upregulating the expression levels of anti-oxidative proteins. In an in vitro experiment, the degranulation of RBL-2H3 rat mast cells was significantly decreased. It was found that SHO and GIN effectively suppressed the allergic response in the mouse model by inhibiting eosinophilia and Th2 cytokine production. Collectively, it was suggested that SHO can inhibit lung inflammation by attenuating the Th2 cell-mediated allergic response signals, and that GIN can inhibit lung inflammation and epithelial cell remodeling by repressing oxidative stress. Therefore, SHO and GIN could be used therapeutically for allergic and eosinophilic asthma.

The aggravation of allergic airway inflammation with dibutyl phthalate involved in Nrf2-mediated activation of the mast cells

Dibutyl phthalate (DBP)-an organic pollutant-is ubiquitous in the environment. DBP as an immune adjuvant is related to the development of multiple allergic diseases. However, the current research involving DBP-induced pulmonary toxicity remains poorly understood. Therefore, this research aimed to explore the adverse effect and potential mechanism of DBP exposure on the lungs in rats. In our study, ovalbumin was used to build a rat model of allergic airway inflammation to study any harmful effect of DBP exposure on lung tissues. Rats were treated by intragastric administration of DBP (500 mg kg−1 or 750 mg kg−1) and/or subcutaneous injection of SFN (4 mg kg−1). The results of histopathological analysis, cell count, and myeloperoxidase showed that DBP promoted the inflammatory damage of lungs. In the lung tissues, the detection of terminal deoxynucleotidyl transferase dUNT nick end labeling and oxidative stress indices showed that DBP significantly increased the level of apoptosis and oxidative stress. Western blot analysis indicated that DBP raised the expression level of thymic stromal lymphopoietin and reduced the nuclear expression level of nuclear factor-erythroid-2-related factor 2 (Nrf2), which was further verified by quantitative real-time PCR. Meanwhile, DBP treatment markedly up-regulated the inflammatory cytokines such as IL-4 and IL-13, and rat mast cell protease-2, a marker secreted by mast cells (MCs). Conversely, sulforaphane, a Nrf2 inducer, ameliorated the pulmonary damage induced by DBP in the above. Altogether, our data provides a new insight into the impacts of the activation of MCs on the DBP-induced pulmonary toxicity as well as the safety evaluation of DBP.

Light‐driven Activation of NADP(H) Oxidase 5

Ultraviolet band A (UVA) light irradiation has been shown to induce cytosolic calcium oscillations in the isolated rat peritoneal mast cells via activation of NADPH oxidase (NOX). It has been clarified that UVA-triggered calcium oscillations in mast cells (isolated rat peritoneal mast cells, rat mast cell line RBL-2H3, mouse bone marrow-derived mast cells) was due to NOX2 activation. To elucidate whether UVA light irradiation would also activate NOX5, a monomeric isoform of the NOX family of oxidases not requiring transmembrane p22 or cytosolic subunits for activity and not endogenously expressed in rodents, NOX5 was expressed ectopically in CHO-K1 cells, and the effect of UVA irradiation or photodynamic action on cytosolic calcium concentration was monitored with Fura-2 fluorescent calcium imaging. It was found that NOX5 expressed ectopically in CHO-K1 cells was activated by UVA (LED 380 nm) light irradiation to induce persistent cytosolic calcium oscillations. Blue LED (450 nm) light irradiation of CHO-K1 cells expressing protein photosensitizer miniSOG-NOX5 fusion protein triggered also persistent cytosolic calcium oscillations which were completely blocked by NOX inhibitor diphenyleneiodonium (DPI), and inhibited by singlet oxygen quencher Trolox C. Sulphonated aluminium phthalocyanine (SALPC) photodynamically activated the heterologously expressed NOX5 in CHO-K1 cells to trigger similarly cytosolic calcium oscillations, which were completely inhibited by DPI. These data suggest that both UVA light irradiation and 1O2 generation with photodynamic action could activate NOX5, to ultimately trigger calcium oscillations. It is therefore possible now to realize light-driven NOX5 activation for cell-type or subcellular organelle specific generation of superoxide anion.

COMPARATIVE STUDY OF HISTOLOGICAL AND HISTOCHEMICAL PROPERTIES OF PERITONEAL MAST CELLS OF ALBINO RAT USING TWO TYPES OF FIXATIVES

Objectives: To make a comparative study of histological and histochemical properties of peritoneal mast cells in albino rat using neutral buffered formalin and formal alcohol as fixatives.&#x0D; Methods: The stretch preparation from Albino rat peritoneum was made on chemically clean glass slides and immediately placed into the fixatives.&#x0D; Results: With toluidine blue (pH 4.4), the mast cells stained deep purple and similar reaction was obtained with toluidine blue at pH 2. These staining reactions indicate the presence of substantial amount of heparin trisulfate, small amount of heparin monosulfate, and neutral mucopolysaccharide.&#x0D; Conclusion: Heparin trisulfate is an active form of heparin and is the main constituent of the mast cell granules of the albino rat.

Authentic and Ectopically Expressed MRGPRX2 Elicit Similar Mechanisms to Stimulate Degranulation of Mast Cells.

The identification of the Mas-related G-protein-coupled receptors (Mrgpr) as targets of diverse stimuli of mast cells (MCs), including neuropeptides and pseudo-allergy causing drugs, has placed these receptors at a prime position in MC research. However, the species-dependent diversity of these receptors raises the need for an adequate model for investigating the human MRGPRX2 receptor. RBL-2H3 cells, stably transfected with MRGPRX2 (RBL-MRGPRX2), are increasingly used for this purpose. Therefore, we investigated whether ectopically expressed MRGPRX2, in rat MCs, recapitulates its authentic signaling. To this purpose, we performed a broad comparative study of the responses of human LAD-2 MCs that express MRGPRX2 endogenously, and RBL-MRGPRX2 cells to compound 48/80, substance P and vancomycin, three proto-type ligands of MRGPRX2. We demonstrate that both models share similar dose–response relationships, kinetics and sensitivities to a wide range of signaling targeting drugs. Therefore, our results indicate that ectopically expressed MRGPRX2 preserves the signaling pathways employed to evoke human MC degranulation, which we show to rely on ERK1/2 MAP kinases, phospholipase C (PLC) and autophagy-related signaling. Importantly, we also show that the underlying mechanisms of MRGPRX2-triggered MC degranulation in either LAD-2 or RBL-MRGPRX2 cells are different from those elicited by its rodent orthologs.