GSK2646264, a spleen tyrosine kinase inhibitor, attenuates the release of histamine in ex vivo human skin.

BACKGROUND AND PURPOSE: Chronic spontaneous urticaria presents as a heterogeneous syndrome characterised by wheals, angioedema, or both for greater than 6 weeks. Spleen tyrosine kinase mediates allergen-induced mast cell degranulation via the IgE signalling pathway, a central component of wheal formation and inflammation. In this study, we investigated the effects of perfused or topically administered GSK2646264 on IgE-mediated histamine release from mast cells in an ex vivo human skin model. EXPERIMENTAL APPROACH: Using a novel SkiP device, ex vivo human skin from mastectomy surgeries was challenged with anti-IgE, complement 5a (C5a), and buffer to induce histamine release from skin mast cells. Histamine was collected via microdialysis fibres and measured fluorometrically. GSK2646264 was delivered via perfusion either using microdialysis fibres or topically in a cream. Drug concentrations in the skin were measured by LC-MS, and a pharmacokinetic/ pharmacodynamic (PK/PD) relationship developed. KEY RESULTS: Perfused GSK2646264 significantly inhibited anti-IgE (but not C5a)-induced histamine release in a concentration-dependent manner. The 0.5, 1, and 3% cream delivered GSK2646264 to the dermis above the IC90 and dose-dependently attenuated anti-IgE-induced histamine release. CONCLUSIONS AND IMPLICATIONS: GSK2646264 administered topically or direct to the dermis blocked histamine release from in situ skin mast cells. A PK/PD relationship curve suggests that dermal concentrations above 6.8 µM should lead to approximately 90% inhibition of histamine release from skin mast cells following activation of the Fc fragment of IgE receptor 1a, implicating a potential use for the compound in skin mast cell diseases such as urticaria.

Effects of Secreted Mast Cell Mediators on Retinal Pigment Epithelial Cells: Focus on Mast Cell Tryptase.

Numerous mast cells are present in the choroid, but the effects of mast cell mediators on retinal pigment epithelial (RPE) cells are not well understood. We investigated the influence of mast cell mediators on RPE cells in vitro, focusing on tryptase. Expression of receptors was examined by the reverse transcription polymerase chain reaction. We also assessed production of interleukin 8 and vascular endothelial growth factor (VEGF) after RPE cells were stimulated with mast cell mediators by using an antibody array and enzyme-linked immunosorbent assay. Furthermore, we investigated the influence of tryptase on RPE cell migration and integrity by the scratch assay and the transepithelial resistance. RPE cells expressed protease-activated receptor 2 (PAR2), histamine receptor 1, tumor necrosis factor-α (TNF-α) receptor 1, and CCR 1, 3, 4, 8, and 11. Tryptase, PAR2 agonists, histamine, and TNF-α all enhanced interleukin 8 production by RPE cells, while only tryptase enhanced VEGF production. Tryptase also enhanced expression of phosphorylated extracellular signal-regulated kinases 1/2, resulting in increased migration of RPE cells. However, tryptase did not alter epithelial integrity or the expression of zonula occludens-1 and junctional adhesion molecule-A by RPE cells. Mast cell mediators, especially tryptase, may influence RPE cell inflammation.

Research Resource: A Chromogranin A Reporter for Serotonin and Histamine Secreting Enteroendocrine Cells.

Chromogranin A (ChgA) is an acidic protein found in large dense-core secretory vesicles and generally considered to be expressed in all enteroendocrine cells of the gastrointestinal (GI) tract. Here, we characterize a novel reporter mouse for ChgA, ChgA-humanized Renilla reniformis (hr)GFP. The hrGFP reporter was found in the monoamine-storing chromaffin cells of the adrenal medulla, where ChgA was originally discovered. hrGFP also was expressed in enteroendocrine cells throughout the GI tract, faithfully after the expression of ChgA, as characterized by immunohistochemistry and quantitative PCR analysis of fluorescence-activated cell sorting-purified cells, although the expression in the small intestine was weak compared with that of the stomach and colon. In the stomach, hrGFP was highly expressed in almost all histamine-storing enterochromaffin (EC)-like cells, at a lower level in the majority of serotonin-storing EC cells and ghrelin cells, in a small fraction of somatostatin cells, but was absent from gastrin cells. In the small intestine, the hrGFP reporter was selectively, but weakly expressed in EC cells, although not in any peptide-storing enteroendocrine cells. In the colon, hrGFP was exclusively expressed in EC cells but absent from the peptide-storing enteroendocrine cells. In contrast, in the pancreas, hrGFP was expressed in ß-cells, α-cells, and a fraction of pancreatic polypeptide cells. It is concluded that ChgA-hrGFP in the GI tract functions as an effective reporter, particularly for the large populations of still poorly characterized monoamine-storing enteroendocrine cells. Furthermore, our findings substantiate the potential function of ChgA as a monoamine-binding protein that facilitates the regulated endocrine secretion of large amounts of monoamines from enteroendocrine cells.

Effects of different routes of nicotine administration on gastric morphology and hormonal secretion in rats.

NEW FINDINGS: What is the central question of this study? Does chronic administration of nicotine by different routes affect gastric hormonal secretions and morphology in rats? What is the main finding and its importance? Chronic nicotine administration increased levels of gastrin, ghrelin and histamine but decreased prostaglandin E2 . Nicotine administered orally and by inhalation had a marked negative impact on the histological structure of the gastric mucosa compared with intraperitoneal administration. The negative impact of nicotine administration on gastric structure was associated with an increased concentration of gastrin and decreased prostaglandin E2 , which might be the cause of gastric/peptic ulcers in heavy smokers. The increase in ghrelin concentration and its effect following chronic nicotine administration needs further investigation. The aim was to assess the effects of different routes of chronic nicotine administration on gastric morphology and hormonal secretion; mainly gastrin, ghrelin, histamine and prostaglandin E2 (PGE2 ). Forty adult male albino rats were randomly assigned into four groups (10 rats per group), treated for 21 days as follows: control group (given standard rat pellets and water only); oral nicotine-treated group [50 µg (ml drinking water)(-1) ]; intraperitoneal nicotine-treated group [0.5 mg (kg body weight)(-1) ]; and inhaled nicotine-treated group [0.5 mg (kg body weight)(-1) ]. Concentrations of gastrin, ghrelin, PGE2 and histamine in serum and gastric tissue homogenates were assessed using ELISA kits. Stomach fundus was processed for histopathology and immunohistochemistry using light and electron microscopy. Different routes of chronic nicotine administration resulted in a significant increase in serum and gastric homogenate gastrin and ghrelin concentrations and a significant decrease in serum and homogenate PGE2 concentrations compared with the control group. Moreover, nicotine administration via oral and inhalation routes caused gastric erosion, transformation of peptic cells into the mucous variety, a significant increase in parietal cell numbers and an increase in expression of gastrin. In conclusion, the negative impact of nicotine administration on gastric structure that is associated with an increased concentration of gastrin and decreased concentration PGE2 might be the leading cause of gastric/peptic ulcers in heavy smokers. The increased ghrelin concentration and its effect following nicotine chronic administration needs further investigation. Based on these findings, we suggest that the alteration in gastric structure following chronic administration of nicotine can be prevented by reducing gastrin secretion and/or targeting its receptors.

Histamine H4 and H1 receptors contribute to postinflammatory visceral hypersensitivity.

OBJECTIVES: Substantial evidence implicates mast cells and their main constituent histamine in the pathogenesis of visceral hypersensitivity. We explored the specific contribution of histamine H4 (H4R) and H1 (H1R) receptors to visceral hypersensitivity in a postinflammatory rat model. DESIGN: Trinitrobenzenesulfonic acid (TNBS)-colitis was monitored individually by colonoscopy: first on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence and determine the exact timepoint of endoscopic healing in each rat. Experiments were performed 3 days after endoscopic resolution of colitis. Visceral sensitivity was assessed by quantifying visceromotor responses (VMRs) to colorectal distension. Colonic mast cell numbers, histamine release and H4R and H1R mRNA expression were quantified. JNJ7777120 (H4R antagonist) and/or levocetirizine (H1R antagonist) were administered 30 min prior to VMR assessment or histamine release assay. RESULTS: Postcolitis rats displayed a higher number of colonic mast cells, excessive histamine release and significantly enhanced VMRs. Heightened VMRs were dose-dependently reduced by JNJ7777120 and levocetirizine; combined administration of JNJ7777120 and levocetirizine potentiated the antinociceptive effect. In the colon, both H4R and H1R mRNA were present; in the dorsal root ganglia, only H1R mRNA was found. Only colonic H4R mRNA expression was increased in postcolitis rats. Excessive histamine release in postcolitis rats was attenuated by the highest dose of JNJ7777120. CONCLUSIONS: H4R and H1R antagonists dose-dependently reduce and even normalise postinflammatory visceral hypersensitivity via different underlying mechanisms but with a synergistic effect. Both receptor subtypes represent promising targets for the treatment of postinflammatory visceral hypersensitivity.

Role of meningeal mast cells in intrathecal morphine-evoked granuloma formation.

BACKGROUND: Intrathecal morphine forms granulomas that arise from the adjacent arachnoid membrane. The authors propose that these inflammatory cells exit the meningeal vasculature secondary to meningeal mast cell degranulation. METHODS: Three sets of experiments were accomplished in dogs: (1) ex vivo meningeal mast cell degranulation (histamine release was measured ex vivo from canine dura incubated with opiates); (2) in vivo cutaneous mast cell degranulation (flare areas on the dog abdomen were measured after subcutaneous opiates); and (3) in vivo granuloma pharmacology. Dogs with lumbar intrathecal catheters received infusion of intrathecal saline or intrathecal morphine. Intrathecal morphine dogs received (1) no other treatment (control); (2) twice-daily subcutaneous naltrexone; (3) intrathecal co-infusion of cromolyn; or (4) twice-daily subcutaneous cromolyn for the 24- to 28-day study course. RESULTS: Morphine but not fentanyl evoked dural histamine release, which was blocked by cromolyn but not naloxone. Wheal/flare was produced by subcutaneous morphine, methadone, hydromorphone, but not fentanyl, and was unaffected by naltrexone but prevented by cromolyn. Granulomas occurred in all dogs receiving intrathecal morphine (15 of 15); subcutaneous naltrexone had no effect on granulomas (six of six) but was reduced by concurrent intrathecal cromolyn (zero of five) or twice-daily subcutaneous cromolyn (one of five). CONCLUSIONS: The pharmacology of cutaneous/dural mast cell degranulation and intrathecal granulomas are comparable, not mediated by opioid receptors, and reduced by agents preventing mast cell degranulation. If an agent produces cutaneous mast cell degranulation at concentrations produced by intrathecal delivery, the agent may initiate granulomas.

MCP-1-induced histamine release from mast cells is associated with development of interstitial cystitis/bladder pain syndrome in rat models.

OBJECTIVE: Interstitial cystitis/bladder pain syndrome (IC/BPS) is characterized by overexpression of monocyte chemoattractant protein-1 (MCP-1) in bladder tissues and induction of mast cell (MC) degranulation. This study was undertaken to explore the mechanism of action of MCP-1 in the development of IC/BPS. METHODS: A rat model of IC/BPS was developed by perfusing bladders of nine SPF- grade female Sprague-Dawley rats with protamine sulfate and lipopolysaccharide (PS+LPS). MCP-1 and histamine levels in bladder tissue and urine were detected by immunohistochemistry and ELISA. MC degranulation was measured by immunofluorescence techniques and chemokine (C-C motif) receptor 2 (CCR2) was assayed by flow cytometry. RESULTS: Increased MCP-1 expression in bladder tissue and elevated MCP-1 and histamine levels were observed in the urine of LS+LPS-treated rats. This was accompanied by the expression of CCR2 on MC surfaces, suggesting MCP-1 may induce MC degranulation through CCR2. Exposure to LPS stimulated MCP-1 expression in bladder epithelial cells, and exposure to MCP-1 induced histamine release from MCs. CONCLUSIONS: MCP-1 upregulation in IC/BPS is one of possible contributing factors inducing histamine release from MCs. CCR2 is involved in the process of mast cell degranulation in bladder tissues. These changes may contribute to the development of symptoms of IC/BPS.

Impact of RANTES, MCP-1 and IL-8 in mast cells.

Chemokines are cytokines with chemotactic properties on inflammatory cells and other cell types. RANTES, MCP-1 and related molecules, constitute the C-C class of chemokine supergene family and a group of cytokines produced by hematopoietic cells, while IL-8 constitute the C-X-C class. The roles of most of these chemokines are not well known, although members of the chemokine family are inflammatory agents. The C-C chemokine plays a role in regulating Th-cell cytokine production and leukocyte trafficking. In this study we clearly show that RANTES and MCP-1 are mediators of acute inflammatory responses. Our report describes additional biological activities for RANTES, MCP-1, and IL-8, suggesting that these chemokines play a fundamental role in histamine and serotonin generation and cell function in mast cells.

Diverse effects of bacterial cell wall components on mast cell degranulation, cysteinyl leukotriene generation and migration.

Nowadays there is more and more evidence that mast cells take part in antibacterial defence. Mast cells have the ability to kill bacteria via phagocytose-dependent or phagocytose-independent ways and express antimicrobial peptides that can directly kill pathogens at their site of entry. What is more, mast cells are capable of processing bacterial antigens for presentation through class I and II MHC molecules. Some data indicate that these cells can release various proinflammatory mediators in response to activation with bacteria and/or their products, however this information is still far from complete. Therefore, in this study we examined the ability of PGN from Staphylococcus aureus, LPS from Eschericha coli and LAM from Mycobacterium smegmatis to stimulate mature rat mast cell degranulation as well as cysteinyl LT generation. We also studied the influence of these bacterial components on mast cell migration. We found that PGN, LPS and LAM all failed to induce mast cell degranulation and histamine release. At the same time, activation of mast cells with these bacterial antigens resulted in generation and release of significant amounts of LT. Moreover, we documented that, even in the presence of laminin, none of the bacterial antigens used stimulated mast cell migration. However, PGN did induce migration of RANTES-primed mast cells, and LPS did stimulate mast cell migratory response after priming with IL-6. Our results show that PGN, LPS and LAM might be among the important bacterial antigens involved in mast cell activation during bacterial infection.

The long-acting beta-adrenoceptor agonist, indacaterol, inhibits IgE-dependent responses of human lung mast cells.

BACKGROUND AND PURPOSE: The long-acting beta(2)-adrenoceptor agonist, indacaterol, has been developed as a bronchodilator for the therapeutic management of respiratory diseases. The aim of the present study was to determine whether indacaterol has any anti-inflammatory activity. To this end, the effects of indacaterol on human lung mast cell responses were investigated. EXPERIMENTAL APPROACH: The effects of indacaterol, and the alternative long-acting beta-agonists formoterol and salmeterol, were investigated on the IgE-dependent release and generation of histamine, cysteinyl-leukotrienes and prostaglandin D(2) from human lung mast cells. Moreover, the extent to which long-term (24-72 h) incubation of mast cells with long-acting beta-agonists impaired the subsequent ability of beta-agonists to inhibit mast cell responses was assessed. KEY RESULTS: Indacaterol was as potent and as efficacious as the full agonist, isoprenaline (EC(50), approximately 4 nmol x L(-1)), at inhibiting the IgE-dependent release of histamine from mast cells. Formoterol was a full agonist whereas salmeterol was a partial agonist as inhibitors of histamine release. All three long-acting beta-agonists were effective inhibitors of the IgE-dependent generation of cysteinyl-leukotrienes and prostaglandin D(2). Long-term incubation of mast cells with long-acting beta-agonists led to a reduction in the subsequent ability of beta-agonists to stabilize mast cell responses. This tendency to induce functional desensitization was least evident for indacaterol. CONCLUSIONS AND IMPLICATIONS: Indacaterol is an effective inhibitor of the release of mediators from human lung mast cells. This suggests that, as well as bronchodilation, mast cell stabilization may constitute an additional therapeutic benefit of indacaterol.

Activation of CD47 receptors causes histamine secretion from mast cells.

Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcepsilonRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the betagamma dimer of a G(i) protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G(i) protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G(i) protein complex.

Differential activation of mast cells by antigens from Trichinella spiralis muscle larvae, adults, and newborn larvae.

Mast cell (MC) hyperplasia and activation are prominent features in Trichinella spiralis infection. Indeed a temporal correlation has been shown between the kinetics of intestinal mastocytosis, release of inflammatory mediators from MC, and adult worm loss, which constitutes a major component of the defense against T. spiralis infection. It is well known that during the intestinal phase of trichinellosis, muscle larvae (ML) and adult worms (AD) enter into contact with the host; however, interaction with MC may also occur during migration of newborn larvae (NBL). Therefore, it is plausible that antigens from these developmental stages could activate MC. We have previously demonstrated by in vitro assays that T. spiralis muscle larval (TSL-1) antigens activate MC through an Ig-independent mechanism leading to the release of histamine, MC protease 5, IL-4 and TNF alpha. In this work we evaluated whether total antigens from AD or NBL could activate unsensitized MC and we compared this activation with the activation seen when MC are stimulated with TSL-1 antigens. MC activation was also tested with affinity chromatography purified antigens from NBL using the monoclonal antibody CE-4 that recognizes NBL surface components. The results obtained in this study showed that AD total extracts and TSL-1 antigens induced the release of histamine but not beta-hexosaminidase from unsensitized MC, suggesting a selective secretion of MC mediators. In contrast, NBL total extracts or purified NBL antigens did not induce the release of either histamine or beta-hexosaminidase from MC. Interestingly, AD and ML are the stages that interact with the host during the intestinal phase of infection. The mechanisms involved in TSL-1 and AD activation of unsensitized MC may function together with other mechanisms of MC activation in host protection against T. spiralis.

Histamine suppresses fibulin-5 and insulin-like growth factor-II receptor expression in melanoma.

We previously showed that transgenic enhancement of histamine production in B16-F10 melanomas strongly supports tumor growth in C57BL/6 mice. In the present study, gene expression profiles of transgenic mouse melanomas, secreting different amounts of histamine, were compared by whole genome microarrays. Array results were validated by real-time PCR, and genes showing histamine-affected behavior were further analyzed by immunohistochemistry. Regulation of histamine-coupled genes was investigated by checking the presence and functional integrity of all four known histamine receptors in experimental melanomas and by administering histamine H1 receptor (H1R) and H2 receptor (H2R) antagonists to tumor-bearing mice. Finally, an attempt was made to integrate histamine-affected genes in known gene regulatory circuits by in silico pathway analysis. Our results show that histamine enhances melanoma growth via H1R rather than through H2R. We show that H1R activation suppresses RNA-level expression of the tumor suppressor insulin-like growth factor II receptor (IGF-IIR) and the antiangiogenic matrix protein fibulin-5 (FBLN5), decreases their intracellular protein levels, and also reduces their availability in the plasma membrane and extracellular matrix, respectively. Pathway analysis suggests that because plasma membrane-bound IGF-IIR is required to activate matrix-bound, latent transforming growth factor-beta1, a factor suggested to sustain FBLN5 expression, the data can be integrated in a known antineoplastic regulatory pathway that is suppressed by H1R. On the other hand, we show that engagement of H2R also reduces intracellular protein pools of IGF-IIR and FBLN5, but being a downstream acting posttranslational effect with minimal consequences on exported IGF-IIR and FBLN5 protein levels, H2R is rather irrelevant compared with H1R in melanoma.

Distinct characteristics of signal transduction events by histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced priming and activation of human basophils.

We previously identified a negative correlation between histamine release to histamine releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of the Src homology 2 domain-containing inositol 5' phosphatase (SHIP) in basophils. We have also demonstrated that HRF/TCTP primes basophils to release mediators. The purpose of this study was to begin characterization of signal transduction events directly induced by HRF/TCTP and to investigate these events when HRF/TCTP is used as a priming agent for human basophil histamine release. Highly purified human basophils were examined for surface expression of bound HRF/TCTP, changes in calcium, and phosphorylation of Akt, mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), Syk, and FcepsilonRIgamma. Results showed that basophils from all donors bound HRF/TCTP. There was a biphasic calcium response to HRF/TCTP, which corresponded to the magnitude of histamine release. Furthermore, those donors who have direct histamine release when exposed to HRF/TCTP (HRF/TCTP responder [HRF/TCTP-R] donors) have phosphorylation of Syk, Akt, MEK, and ERK. Remarkably, basophils from HRF/TCTP-nonresponder (HRF/TCTP-NR) donors do not show phosphorylation of these molecules. This finding is different from IL-3, which also primes basophils for histamine release, but does show phosphorylation of these events. We conclude that priming induced by HRF/TCTP is distinct from that induced by IL-3.

STI571 (Glivec) affects histamine release and intracellular pH after alkalinisation in HMC-1560, 816.

The human mast cell line (HMC-1(560, 816)) was used to study the effect of the tyrosine kinase inhibitor STI571 (Glivec) on exocytosis, intracellular Ca(2+) and pH changes, because STI571 inhibits the proliferation of HMC-1(560) and induces its apoptosis. This drug does not have these effects on HMC-1(560, 816). Exocytosis in HMC-1(560, 816) cells can be stimulated by alkalinisation with NH(4)Cl as well as with ionomycin. Surprisingly 24-h pre-incubation with STI571 decreases spontaneous histamine release of HMC-1(560, 816) cells, but increases the histamine response after alkalinisation and not after ionomycin-stimulation. After addition of NH(4)Cl, pH(i) has a higher increase in STI571 pre-incubated cells, without changing intracellular Ca(2+) concentration. Activation of PKC in combination with tyrosine kinase inhibition increases also histamine release in HMC-1(560, 816) cells. Strangely, STI571 pre-incubated cells with PKC inhibited by rottlerin show the same effects. In these cells, cytosolic pH increases more than in control cells. This is the first report of STI571 effect in HMC-1(560, 816) cells. It seems that different pathways modulate signals for proliferation and exocytosis. STI571 does not only inhibit KIT TyrK, but may also influence cytosolic pH after alkalinisation in both cell lines, HMC-1(560) and HMC-1(560, 816), and this ends in induced histamine release. This work is important since HMC-1(560, 816) cells are reported in 80% of aggressive systemic mastocytosis cases and the understanding of some signalling pathways involved in mast cell response could facilitate drug targeting.

Degranulation of mast cells and histamine release involved in rat pain-related behaviors and edema induced by scorpion Buthus martensi Karch venom.

In the present study, it was investigated whether the degranulation of mast cells and histamine release were involved in rat pain-related behaviors and edema induced by the venom of scorpion Buthus martensi Karch (BmK) or not. It was found that the obvious degranulation of mast cells could be triggered in rat hindpaw skin by BmK venom. The chronic degranulation of mast cells using compound 48/80 relieved the spontaneous nociceptive responses, the primary thermal and bilateral mechanical hyperalgesia and the rat paw edema, as well as partially reduced c-Fos expression in superficial layers (laminae I-II) of bilateral spinal cord induced by BmK venom. In addition, individual peripheral co-administration of either 100 nmol chlorpheniramine or 100 nmol pyrilamine (histamine H(1) receptor antagonist) or 500 nmol cimetidine (histamine H(2) receptor antagonist) and BmK venom suppressed the spontaneous nociceptive responses, partially the primary thermal and bilateral mechanical hyperalgesia and rat paw edema induced by BmK venom. Thus, these results suggest that the peripheral cellular incidents of mast cells degranulation and histamine release are involved in BmK venom-induced pain-related behaviors and inflammation.

Mast cell-derived mediators protect against oxygen-glucose deprivation-induced injury in PC12 cells and neurons.

Recent reports and our previous study suggest that mast cells play a crucial role in the pathological processes that follow cerebral ischemia. In this study, the effect of mast cells on neuron injury after cerebral ischemia was determined by adding in vitro ischemia-induced supernatant from mast cells to neurons and PC12 cells under the same conditions (oxygen-glucose deprivation, OGD). The degree of cell injury was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-dipheny-ltetrazolium bromide (MTT) assay. Mast cell-derived supernatant protected against OGD-induced injury of PC12 cells and neurons, and this protection was reversed by a histamine H1 antagonist and by anti-histamine serum, but not by an H2 antagonist. However, histamine and nerve growth factor (NGF) added separately or together did not have protective effects against OGD-induced injury. These results indicate that mast cell-derived protection during in vitro ischemia is histamine-dependent, and involves cooperation with other mediators, but not NGF.

Fasting-induced changes in ECL cell gene expression.

Gastric enterochromaffin-like (ECL) cells release histamine in response to food because of elevation of gastrin and neural release of pituitary adenylate cyclase-activating peptide (PACAP). Acid secretion is at a basal level in the absence of food but is rapidly stimulated with feeding. Rats fasted for 24 h showed a significant decrease of mucosal histamine despite steady-state expression of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Comparative transcriptomal analysis using gene expression oligonucleotide microarrays of 95% pure ECL cells from fed and 24-h fasted rats, thereby eliminating mRNA contamination from other gastric mucosal cell types, identified significantly increased gene expression of the enzymes histidase and urocanase catabolizing the HDC substrate L-histidine but significantly decreased expression of the cellular L-histidine uptake transporter SN2 and of the vesicular monoamine transporter 2 (VMAT-2) responsible for histamine uptake into secretory vesicles. This was confirmed by reverse transcriptase-quantitative polymerase chain reaction of gastric fundic mucosal samples from fed and 24-h fasted rats. The decrease of VMAT-2 gene expression was also shown by a decrease in VMAT-2 protein content in protein extracts from fed and 24-h fasted rats compared with equal amounts of HDC protein and Na-K-ATPase alpha(1)-subunit protein content. These results indicate that rat gastric ECL cells regulate their histamine content during 24-h fasting not by a change in HDC gene or protein expression but by regulation of substrate concentration for HDC and a decreased histamine secretory pool.