Agonist-promoted kappa opioid receptor (KOR) phosphorylation has behavioral endpoint-dependent and sex-specific effects.

We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse kappa opioid receptor (mKOR) in vitro at four residues in the C-terminal domain. In this study, we generated a mutant mouse line in which all the four residues were mutated to Ala (K4A) to examine the in vivo functional significance of agonist-induced KOR phosphorylation. U50,488H promoted KOR phosphorylation in brains of the wildtype (WT), but not K4A, male and female mice. Autoradiography of [3H] 69,593 binding to KOR in brain sections showed that WT and K4A mice had similar KOR distribution and expression levels in brain regions without sex differences. In K4A mice, U50,488H inhibited compound 48/80-induced scratching and attenuated novelty-induced hyperlocomotion to similar extents as in WT mice without sex differences. Interestingly, repeated pretreatment with U50,488H (80 mg/kg, s.c.) resulted in profound tolerance to the anti-scratch effects of U50,488H (5 mg/kg, s.c.) in WT mice of both sexes and female K4A mice, while in male K4A mice tolerance was attenuated. Moreover, U50,488H (2 mg/kg) induced conditioned place aversion (CPA) in WT mice of both sexes and male K4A mice, but not in female K4A mice. In contrast, U50,488H (5 mg/kg) caused CPA in male, but not female, mice, regardless of genotype. Thus, agonist-promoted KOR phosphorylation plays important roles in U50,488H-induced tolerance and CPA in a sex-dependent manner, without affecting acute U50,488H-induced anti-pruritic and hypo-locomotor effects. These results are the first to demonstrate sex differences in the effects of GPCR phosphorylation on the GPCR-mediated behaviors.

Ultramicronized palmitoylethanolamide counteracts the effects of compound 48/80 in a canine skin organ culture model.

BACKGROUND: Ultramicronized palmitoylethanolamide (PEA-um) has been reported to reduce pruritus and skin lesions in dogs with moderate atopic dermatitis and pruritus. HYPOTHESIS/OBJECTIVES: A canine ex vivo skin model was used to investigate the ability of PEA-um to counteract changes induced by compound 48/80, a well-known secretagogue that causes mast cell degranulation. ANIMALS: Normal skin was obtained from three donor dogs subjected to surgery for reasons unrelated to the study. METHODS: Cultured skin biopsy samples in triplicate were treated with 10 and 100 µg/mL compound 48/80, without or with 30 µM PEA-um. Mast cell (MC) degranulation, histamine release into the culture medium, local microvascular dilatation, epidermal thickness, keratinocyte proliferation and epidermal differentiation markers were evaluated. RESULTS: Exposure of the skin organ culture to PEA-um 24 h before and 72 h concomitantly to compound 48/80 resulted in a significant decrease of degranulating MCs. PEA-um also reduced the histamine content in the culture medium by half, although the effect did not reach statistical significance. PEA-um significantly counteracted vasodilation induced by 100 µg/mL compound 48/80. Finally, PEA-um alone did not induce changes in epidermal thickness, differentiation markers, keratinocyte proliferation, MC density and/or degranulation. CONCLUSIONS AND CLINICAL IMPORTANCE: Collectively, these results support the protective action PEA-um on the skin of dogs undergoing allergic changes.

2-Hydroxy-3-methoxybenzoic acid attenuates mast cell-mediated allergic reaction in mice via modulation of the FcεRI signaling pathway.

Mast cells are important effector cells in immunoglobulin (Ig) E-mediated allergic reactions such as asthma, atopic dermatitis and rhinitis. Vanillic acid, a natural product, has shown anti-oxidant and anti-inflammatory activities. In the present study, we investigated the anti-allergic inflammatory effects of ortho-vanillic acid (2-hydroxy-3-methoxybenzoic acid, o-VA) that was a derivative of vanillic acid isolated from Amomum xanthioides. In mouse anaphylaxis models, oral administration of o-VA (2, 10, 50 mg/kg) dose-dependently attenuated ovalbumin-induced active systemic anaphylaxis and IgE-mediated cutaneous allergic reactions such as hypothermia, histamine release, IgE production and vasodilation; administration of o-VA also suppressed the mast cell degranulator compound 48/80-induced anaphylaxis. In cultured mast cell line RBL-2H3 and isolated rat peritoneal mast cells in vitro, pretreatment with o-VA (1-100 µmol/L) dose-dependently inhibited DNP-HSA-induced degranulation of mast cells by decreasing the intracellular free calcium level, and suppressed the expression of pro-inflammatory cytokines TNF-α and IL-4. Pretreatment of RBL-2H3 cells with o-VA suppressed DNP-HSA-induced phosphorylation of Lyn, Syk, Akt, and the nuclear translocation of nuclear factor-κB. In conclusion, o-VA suppresses the mast cell-mediated allergic inflammatory response by blocking the signaling pathways downstream of high affinity IgE receptor (FcεRI) on the surface of mast cells.

Pretreatment with Evans blue, a stimulator of BK(Ca) channels, inhibits compound 48/80-induced shock, systemic inflammation, and mast cell degranulation in the rat.

The present study demonstrated that intravenous injection of a high dose of compound 48/80 to the rat induced 50% drop, within a few min, in the mean arterial pressure and pulse pressure as well as systemic inflammatory plasma leakage that might lead to circulatory and respiratory failure. We also investigated whether pretreatment with Evans blue, a stimulator of BK(Ca) channels, could exert inhibitory effect against compound C48/80-induced allergic circulatory shock and systemic inflammation. Different groups of Sprague-Dawley rats received an intravenous injection of a dose of Evans blue (0, 5, 10, or 50 mg/kg) just 20 s prior to injection of compound 48/80 (200 µg/kg, over 2 min). The present study found that pretreatment with Evans blue in a dose of 10 or 50 mg/kg exerted acute inhibitory effect on compound 48/80-induced sudden drop in mean arterial and pulse pressures. We also showed that pretreatment with Evans blue in a dose of 5, 10, or 50 mg/kg significantly inhibited compound 48/80-induced extensive plasma extravasation, mast cell degranulation, and edema formation in various organs including the airways, esophagus, and skin. Pretreatment with Evans blue 50 mg/kg 1 h earlier exhibited longer-term inhibitory effect on compound 48/80-induced arterial hypotension and systemic inflammation. We concluded that Evans blue pretreatment prevented rats from compound 48/80-triggered allergic shock and systemic inflammation, possibly mainly through inhibition of mast cell degranulation. Evans blue might be potentially useful in elucidating the mechanism and acting as a therapeutic agent of allergic shock and systemic inflammation.

Methylene blue administration in the compound 48/80-induced anaphylactic shock: hemodynamic study in pigs / Administração de azul de metileno no choque anafilático induzido por composto 48/80: estudo hemodinâmico em suínos

PURPOSE: To verify if the methylene blue (MB) administration prevents and/or reverses the compound 48/80 (C48/80)-induced anaphylactic shock in pigs. METHODS: Female Dalland pigs were anesthetized and had the hemodynamic parameters recorded during the necessary time to administer some drugs and observe their effect. The animals were randomly assigned to one of the five groups: 1) control; 2) MB: the animals received a bolus injection of MB (2 mg/kg) followed by continuous infusion of MB (2.66 mg/Kg/h delivered by syringe infusion pump); 3) C48/80: the animals received a bolus injection of C48/80 (4 mg/kg); 4) C48/80+MB: the animals received a bolus injection of C48/80 (4 mg/kg) and 10 minutes after the C48/80 administration the animals received a bolus injection of MB (2 mg/kg) followed by continuous infusion of MB (2.66 mg/Kg/h delivered by syringe infusion pump); 5) MB+C48/80: the animals received a bolus injection of MB (2 mg/kg) and 3 minutes later they received a bolus injection of C48/80 (4 mg/kg). RESULTS: The intravenous infusion of MB alone caused no changes in the mean arterial pressure (MAP) showing that the administered MB dose was safe in this experimental model. The C48/80 was effective in producing experimental anaphylactic shock since it was observed a decrease in both MAP and cardiac output (CO) after its administration. The MB did not prevent or reverse the C48/80-induced anaphylactic shock in this model. In fact, the MAP of the animals with anaphylactic shock treated with MB decreased even more than the MAP of the animals from the C48/80 group. On the other hand, the C48/80-induced epidermal alterations disappeared after the MB infusion. CONCLUSION: Despite our data, the clinical manifestations improvement brings some optimism and does not allow excluding the MB as a possible therapeutic option in the anaphylactic shock.

OBJETIVO: Verificar se a administração de azul de metileno (AM) previne e/ou reverte o choque anafilático induzido por composto 48/80 (C48/80) em suínos. MÉTODOS: Porcos fêmeas Dalland foram anestesiados e tiveram os parâmetros hemodinâmicos registados durante o tempo necessário para administrar algumas drogas e observar seu efeito. Os animais foram aleatoriamente destribuídos em um dos cinco grupos: 1) controle, 2) AM: os animais receberam uma injeção em bolus de AM (2mg/kg), seguido de infusão contínua de AM (2,66mg/Kg /h por bomba de infusão de seringa); 3) C48/80: os animais receberam uma injeção em bolus de C48/80 (4mg/kg); 4) C48/80 + AM: os animais receberam uma injeção em bolus de C48/80 (4mg/kg) e 10 minutos após a administração de C48/80 os animais receberam uma injeção em bolus de AM (2mg/kg), seguido de infusão contínua de AM (2,66mg/kg/h por bomba de infusão de seringa); 5) AM+C48/80: os animais receberam uma injeção em bolus de AM (2mg/kg) e três minutos depois, receberam uma injeção em bolus de C48/80 (4mg/kg). RESULTADOS: A infusão intravenosa de AM não causou mudanças na pressão arterial média (PAM), mostrando que a dose de AM administrada foi segura neste modelo experimental. O C48/80 foi eficaz na indução do choque anafilático experimental, uma vez que foi observada redução na PAM e débito cardíaco (DC), após a sua administração. O AM não preveniu ou reverte o choque anafilático induzido por C48/80 neste modelo. Na verdade, a PAM dos animais com choque anafilático tratados com AM diminuiu mais do que o PAM dos animais do grupo C48/80. Por outro lado, as alterações epidérmicas induzidas pelo C48/80 desapareceu após a infusão do AM. CONCLUSÃO: Apesar dos resultados a melhora clínica das manifestações anafiláticas permite considerar a possibilidade do azul de metileno como opção terapêutica no tratamento do choque anafilático.

Methylene blue administration in the compound 48/80-induced anaphylactic shock: hemodynamic study in pigs.

PURPOSE: To verify if the methylene blue (MB) administration prevents and/or reverses the compound 48/80 (C48/80)-induced anaphylactic shock in pigs. METHODS: Female Dalland pigs were anesthetized and had the hemodynamic parameters recorded during the necessary time to administer some drugs and observe their effect. The animals were randomly assigned to one of the five groups: 1) control; 2) MB: the animals received a bolus injection of MB (2 mg/kg) followed by continuous infusion of MB (2.66 mg/Kg/h delivered by syringe infusion pump); 3) C48/80: the animals received a bolus injection of C48/80 (4 mg/kg); 4) C48/80+MB: the animals received a bolus injection of C48/80 (4 mg/kg) and 10 minutes after the C48/80 administration the animals received a bolus injection of MB (2 mg/kg) followed by continuous infusion of MB (2.66 mg/Kg/h delivered by syringe infusion pump); 5) MB+C48/80: the animals received a bolus injection of MB (2 mg/kg) and 3 minutes later they received a bolus injection of C48/80 (4 mg/kg). RESULTS: The intravenous infusion of MB alone caused no changes in the mean arterial pressure (MAP) showing that the administered MB dose was safe in this experimental model. The C48/80 was effective in producing experimental anaphylactic shock since it was observed a decrease in both MAP and cardiac output (CO) after its administration. The MB did not prevent or reverse the C48/80-induced anaphylactic shock in this model. In fact, the MAP of the animals with anaphylactic shock treated with MB decreased even more than the MAP of the animals from the C48/80 group. On the other hand, the C48/80-induced epidermal alterations disappeared after the MB infusion. CONCLUSION: Despite our data, the clinical manifestations improvement brings some optimism and does not allow excluding the MB as a possible therapeutic option in the anaphylactic shock.

Effects of aegeline, a main alkaloid of Aegle Marmelos Correa leaves, on the histamine release from mast cells.

Aegeline or N-[2-hydroxy-2(4-methoxyphenyl) ethyl]-3-phenyl-2-propenamide is a main alkaloid isolated from Aegle marmelos Correa collected in Yogyakarta Indonesia. In our study, we investigated the effects of aegeline on the histamine release from mast cell. The study was performed by using (1) rat basophilic leukemia (RBL-2H3) cell line, and (2) rat peritoneal mast cells (RPMCs). DNP(24)-BSA, thapsigargin, ionomycin, compound 48/80 and PMA were used as inducers for histamine release from mast cell. In our study, aegeline inhibited the histamine release from RBL-2H3 cells induced by DNP(24)-BSA. Indeed, aegeline showed strong inhibition when RBL-2H3 cells induced by Ca(2+) stimulants such as thapsigargin and ionomycin. Aegeline is suggested to influence the intracellular Ca(2+) pool only since could not inhibit the (45)Ca(2+) influx into RBL-2H3 cells. Aegeline showed weak inhibitory effects on the histamine release from RPMCs, even though still succeed to inhibit when the histamine release induced by thapsigargin. These findings indicate that aegeline altered the signaling pathway related to the intracellular Ca(2+) pool in which thapsigargin acts. Based on the results, the inhibitory effects of aegeline on the histamine release from mast cells depended on the type of mast cell and also involved some mechanisms related to intracellular Ca(2+) signaling events via the same target of the action of thapsigargin or downstream process of intracellular Ca(2+) signaling in mast cells.

Nalfurafine prevents 5'-guanidinonaltrindole- and compound 48/80-induced spinal c-fos expression and attenuates 5'-guanidinonaltrindole-elicited scratching behavior in mice.

The aims of the present study were to establish if nalfurafine, a kappa opioid agonist, inhibits compulsive scratching in mice elicited by the s.c. administration (behind the neck) of 5'-guanidinonaltrindole (GNTI), a kappa opioid antagonist; to assess if nalfurafine prevents c-fos expression provoked by GNTI or compound 48/80, two chemically diverse pruritogens; and to distinguish on the basis of neuroanatomy, those neurons in the brainstem activated by either GNTI-induced itch or formalin-induced pain (both compounds given s.c. to the right cheek). Pretreatment of mice with nalfurafine (0.001-0.03 mg/kg s.c.) attenuated GNTI (0.3 mg/kg)-evoked scratching dose-dependently. A standard antiscratch dose of nalfurafine (0.02 mg/kg) had no marked effect on the spontaneous locomotion of mice. Tolerance did not develop to the antiscratch activity of nalfurafine. Both GNTI and compound 48/80 provoked c-fos expression on the lateral side of the superficial layer of the dorsal horn of the cervical spinal cord and pretreating mice with nalfurafine inhibited c-fos expression induced by both pruritogens. In contrast to formalin, GNTI did not induce c-fos expression in the trigeminal nucleus suggesting that pain and itch sensations are projected differently along the sensory trigeminal pathway. Our data indicate that the kappa opioid system is involved, at least in part, in the pathogenesis of itch; and that nalfurafine attenuates excessive scratching and prevents scratch-induced neuronal activity at the spinal level. On the basis of our results, nalfurafine holds promise as a potentially useful antipruritic in human conditions involving itch.

Anti-allergic effects of Teucrium japonicum on mast cell-mediated allergy model.

The mast cell-mediated immediate-type allergic reaction is involved in many allergic diseases such as asthma, allergic rhinitis, and sinusitis. Stimulation of mast cells starts the process of degranulation resulting in release of mediators such as histamine and an array of inflammatory cytokines. In this report, we investigated the effect of aqueous extract of Teucrium japonicum Houttuyn (Labiatae) (AXTJ) on the mast cell-mediated allergy model and studied its possible mechanisms of action. AXTJ inhibited compound 48/80-induced systemic reactions and serum histamine release in mice. AXTJ decreased immunoglobulin E-mediated passive cutaneous anaphylaxis reaction. AXTJ reduced histamine release and intracellular calcium from rat peritoneal mast cells activated by compound 48/80. In addition, AXTJ attenuated activation of nuclear factor (NF)-kappaB, and downstream tumor necrosis factor (TNF)-alpha expression in phorbol 12-myristate 13-acetate and calcium ionophore A23187-stimulated human mast cells. Our findings provide evidence that AXTJ inhibits mast cell-derived allergic reactions and involvement of intracellular calcium, TNF-alpha, and NF-kappaB in these effects.

Tea catechins have dual effect on mast cell degranulation induced by compound 48/80.

Green tea catechins are emerging as one of the most efficient and safest ingredient in health promoting food. We investigated catechin's effects on intracellular ROS generation in mast cell activation and degranulation. Compound 48/80, receptor mimetic basic secretagogues for mast cell, induced ROS generation dose-dependently with bell-shaped degranulation pattern in canine cutaneous mastocytoma cells (CM-MC). When intracellular ROS level was relatively low, catechins decreased both ROS and the degranulation. However, when intracellular ROS level was remarkably high, catechins decreased ROS level but increased the degranulation paradoxically. Gallocatechins showed the stronger effects than non-gallated catechins. Exogenous H(2)O(2) also shows dual effect on degranulation dose-dependently. EGCG shows the dual effect on the tyrosine and threonine phosphorylation depending on the concentration of compound 48/80. Particularly, 60 kDa protein tyrosine-phosphorylated by EGCG with 3 microg/ml of compound 48/80 might be a negative regulator for the degranulation. Taken together, there is an optimal level of ROS for the degranulation, and the catechins have a dual function by controlling ROS level.

Inhibition of basic secretagogue-induced signaling in mast cells by cell permeable G alpha i-derived peptides.

BACKGROUND: Basic secretagogues of connective tissue mast cells act as receptor mimetic agents that trigger mast cells by activating G proteins. This leads to simultaneous propagation of two signaling pathways: one that culminates in exocytosis, while the other involves protein tyrosine phosphorylation and leads to release of arachidonic acid metabolites. We have previously shown that introduction of a peptide that comprises the C-terminal end of G alpha i3 into permeabilized mast cells inhibits basic secretagogue-induced exocytosis [Aridor et al., Science 1993;262:1569-1572]. We investigated whether cell-permeable peptides, composed of the C-terminus of G alpha i3 fused with importation sequences, affect mast cell function. METHODS: Following preincubation with the fused peptides, rat peritoneal mast cells were activated by compound 48/80 and analyzed for histamine and prostaglandin D2 release and protein tyrosine phosphorylations. RESULTS: We demonstrate that out of three importation sequences tested only G alpha i3 peptide fused with the Kaposi fibroblast growth factor importation sequence (ALL1) inhibited release of histamine. ALL1 as well as a cell-permeable peptide that corresponds to G alpha i2 also blocked compound 48/80-stimulated protein tyrosine phosphorylation, though the latter did not block histamine release. ALL1 effect was G protein-specific, as it was incapable of blocking protein tyrosine phosphorylation stimulated by pervanadate. CONCLUSION: ALL1, a transducible G alpha i3-corresponding peptide, blocks the two signaling pathways in mast cells: histamine release and protein tyrosine phosphorylation. Cell permeable peptides that block these two signaling cascades may constitute a novel approach for preventing the onset of the allergic reaction.

Characteristics of scratching behavior induced by some chemical mediators in hairless mice.

To find the characteristics of scratching behavior in hairless mice (HR-1), compound 48/80 and some putative chemical mediators of allergic reaction were injected intradermally into the backs of mice, and the number of scratching behaviors was measured. As reference mice, NC/Nga, ICR, and ddY mice were used. Scratching behavior in HR-1 and ICR mice was increased dose-dependently by compound 48/80. The same result was also observed with NC/Nga and ddY mice. However, the response in NC/Nga and ddY mice was far less than those of HR-1 and ICR mice. Similar to NC/Nga and ddY mice, HR-1 mice showed less sensitivity to histamine than ICR mice. On the other hand, the HR-1 mice showed a high response to serotonin compared with those of the NC/Nga and ddY mice. The scratching behavior in HR-1 mice induced by substance P was increased, but the effect was less potent than those in NC/Nga, ICR, and ddY mice. These results suggest that the scratching behavior induced by compound 48/80 in HR-1 mice is mainly attributable to serotonin.

Caffeic acid inhibits compound 48/80-induced allergic symptoms in mice.

The effect of caffeic acid on scratching behavior and vascular permeability changes induced by compound 48/80 in ICR mice were investigated. An oral dose of 500 mg/kg of caffeic acid significantly inhibited scratching behavior and vascular permeability induced by compound 48/80. The inhibitory effects of daily administration of lower doses of caffeic acid, 100 and 200 mg/kg, were also investigated; and it was found that 200 mg/kg significantly inhibited compound 48/80-induced scratching behavior after the second week of consecutive administration. The effect of 200 mg/kg of caffeic acid on scratching behavior was observed up to the third week of the treatment. The decrease in histamine content induced by compound 48/80 was significantly antagonized by 200 mg/kg. The findings suggest that caffeic acid may be effective for treating itch and edema in allergic dermatitis.

Inhibitory effects of epigallocatechin gallate on compound 48/80-induced mast cell activation and passive cutaneous anaphylaxis.

Epigallocatechin gallate (EGCG) is a principle phenolic antioxidant found in a variety of plants, including green and black tea. The anti-allergic effect of EGCG is unknown. The purpose of this study is to investigate the effects of EGCG on compound 48/80-induced mast cell activation and passive cutaneous anaphylaxis. For this, the influences of EGCG on the compound 48/80-induced cutaneous reaction were measured in vivo and the effects of EGCG on the compound 48/80-induced mast cell activations were examined in vitro. Results are below: as 1) EGCG significantly inhibited compound 48/80-induced passive cutaneous anaphylaxis, 2) the compound 48/80-induced degranulation, calcium influx and histamine release of rat peritoneal mast cells (RPMCs) were significantly inhibited by the pretreatment with EGCG, and 3) the compound 48/80-mediated inhibition of cAMP level in RPMCs was significantly increased by the pretreatment with EGCG. These results suggested that EGCG, the most abundant polyphenol in green tea, inhibits the compound 48/80-induced mast cell activation and the increase of vascular permeability, and potentially serve as effective therapeutic tools for allergic diseases.

Inhibitory effects of Okbyungpoong-Gamhmi on anaphylactic responses.

We investigated the effect of a herbal formulation Okbyungpoong-Gamhmi (OG) on mast cell-dependent anaphylactic reactions by intra-rectal administration. OG concentration dependently inhibited compound 48/80-induced anaphylaxis-like response and ear swelling response with doses of 0.01-1g/kg. OG also inhibited the passive cutaneous anaphylaxis at the same concentrations. The histamine release induced by compound 48/80 or IgE from the rat peritoneal mast cells was reduced by 64.2 and 63.6%, respectively, at 1g/l. These results provide evidence that intra-rectal therapy of OG may be beneficial in the treatment of anaphylactic response.

Desloratadine prevents compound 48/80-induced mast cell degranulation: visualization using a vital fluorescent dye technique.

BACKGROUND: Desloratadine is a selective H1-antihistamine used in the treatment of allergic rhinitis and chronic idiopathic urticaria. Desloratadine inhibits the release of allergic inflammatory mediators in vitro. We studied the impact of desloratadine on mast cell degranulation due to activation and re-activation by the secretagogue, compound 48/80. METHODS: Rat peritoneal eluate containing 5-6% mast cells were activated by a low concentration of compound 48/80 in a medium containing the vital fluorescent dye, Sulforhodamine-B (SFRM-B, 200 microg/ml), which is engulfed by activated mast cells. The fluorescent image of activated mast cells was captured digitally and the total fluorescent area was analyzed when desloratadine was applied before or after compound 48/80. RESULTS: Mast cells were not activated by desloratadine (10(-4) M), SFRM-B (200 microg/ml), or diluent alone. A low concentration of compound 48/80 (0.125 microg/ml) induced fluorescence, while mast cells lost fluorescent images due to further degranulation on re-exposure to compound 48/80. Desloratadine (10(-8)-10(-4) M), inhibited compound 48/80-induced mast cell degranulation in a concentration-dependent manner. Desloratadine also reduced the loss of fluorescent images due to re-exposure to compound 48/80. CONCLUSIONS: Desloratadine may have a mast cell stabilizing effect at low concentrations in response to repeated mast cell activation in vitro.

Inhibitory effects of epigallocatechin gallate on compound 48/80-inducedmast cell activation and passive cutaneous anaphylaxis

Epigallocatechin gallate (EGCG) is a principle phenolic antioxidant found in a variety of plants, including green and black tea. The anti-allergic effect of EGCG is unknown. The purpose of this study is to investigate the effects of EGCG on compound 48/80-induced mast cell activation and passive cutaneous anaphylaxis. For this, the influences of EGCG on the compound 48/80-induced cutaneous reaction were measured in vivo and the effects of EGCG on the compound 48/80-induced mast cell activations were examined in vitro. Results are below: as 1) EGCG significantly inhibited compound 48/80-induced passive cutaneous anaphylaxis, 2) the compound 48/80-induced degranulation, calcium influx and histamine release of rat peritoneal mast cells (RPMCs) were significantly inhibited by the pretreatment with EGCG, and 3) the compound 48/80-mediated inhibition of cAMP level in RPMCs was significantly increased by the pretreatment with EGCG. These results suggested that EGCG, the most abundant polyphenol in green tea, inhibits the compound 48/80-induced mast cell activation and the increase of vascular permeability, and potentially serve as effective therapeutic tools for allergic diseases.

Inhibitory activity of Chrysanthemi sibirici herba extract on RBL-2H3 mast cells and compound 48/80-induced anaphylaxis.

The effects of extracts from various oriental medicinal herbs on mast cell-mediated allergic reaction were investigated. Among them, Chrysanthemi sibirici herba ethanol extract exerted the potent inhibitory activity on antigen-induced degranulation in RBL-2H3 mast cells. Chrysanthemi sibirici herba dose-dependently inhibited DNP-BSA or compound 48/80-induced degranulation in RBL-2H3 mast cells, with IC(50) values of approximately 49 microg/ml and 76 microg/ml, respectively. This extract strongly suppressed compound 48/80-induced systemic anaphylaxis by 48.7% at a dose of 300 mg/kg in mice. Chrysanthemi sibirici herba also inhibited the expression of TNF-alpha and the activation of the MAP kinase, ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of activating phosphorylation of ERK1/2. These results lead us to conclude that Chrysanthemi sibirici herba may be used clinically to treat various allergic diseases.

Rubus croceacanthus Leveille inhibits mast cell-mediated anaphylactic-like reaction and tumor necrosis factor-alpha secretion.

This work aims at examining the effect of the concentrated methanol extract of Rubus croceacanthus Leveille (RCL) on mast cell-mediated anaphylactic-like reaction in a murine model. RCL inhibited compound 48/80-induced systemic anaphylactic-like reaction. When RCL was given as pre-treatment at concentrations ranging from 0.01 to 1 mg/ml, the histamine release from rat peritoneal mast cells induced by compound 48/80 or anti-dinitrophenyl (DNP) immunoglobulin E (IgE) was reduced in a dose-dependent manner. RCL also inhibited passive cutaneous anaphylaxis activated by anti-DNP IgE. In addition, RCL inhibited phorbol 12-myristate 13-acetate and A23187-induced tumor necrosis factor-alpha secretion from human mast cell line HMC-1 cells. These results indicate that RCL may possess a strong anti-anaphylactic activity.

Degranulation of mast cells located in median eminence in response to compound 48/80 evokes adrenocortical secretion via histamine and CRF in dogs.

The effect of intracerebroventricular infusion of compound 48/80 (C48/80), a mast cell secretagogue, on adrenal cortisol secretion was investigated in dogs under pentobarbital sodium anesthesia. A marked increase in adrenal cortisol secretion was elicited by C48/80 along with a concomitant increase in the plasma levels of cortisol and immunoreactive ACTH, but neither arterial blood pressure and heart rate nor the plasma histamine level altered significantly. Pretreatment with either anti-CRF antiserum or pyrilamine maleate (H(1) histamine-receptor antagonist) significantly attenuated the C48/80-evoked increase in cortisol secretion, but pretreatment with metiamide (H(2)-receptor antagonist) significantly potentiated it. Significant attenuation of the C48/80-evoked increase in cortisol also occurred in dogs given ketotifen, a mast cell stabilizing drug, before pharmacologic challenge. In the pars tuberalis and median eminence (ME), mast cells were highly concentrated in close association with the primary plexus of the hypophysial portal system. Degranulated mast cells were extensively found in the ME of C48/80-treated animals. These results suggest that mast cells located in these regions liberated histamine within the brain as a result of degranulation induced by C48/80 and that this led to activation of the hypothalamic-pituitary-adrenocortical axis.