Keiji Izushi

Anti-allergic constituents in the culture medium ofGanoderma lucidum. (II) The inhibitory effect of cyclooctasulfur on histamine release

For centuries,Ganoderma lucidum has been used in Oriental medicine for the treatment of chronic bronchitis. Sequential fractions of the culture medium of this plant revealed that one of the active constitutents was cyclooctasulfur. The latter effectively inhibited hsitamine release from rat peritoneal mast cells and impeded45Ca uptake into these cells without affecting the cyclic AMP content. SDS-PAGE analysis indicated that cyclooctasulfur induced some changes in protein bands obtained from the membrane fraction of mast cells, suggesting that this compound interacts with membrane proteins so as to inhibit45Ca uptake, and that this may be the main cause of histamine release inhibition.

Antiallergic Effect of Epinastine (WAL 801 CL) on Immediate Hypersensitivity Reactions: (I) Elucidation of the Mechanism for Histamine Release Inhibition

Epinastine caused an inhibition of histamine release from rat peritoneal mast cells induced by both antigen-antibody reaction and compound 48/80. Epinastine was similarly effective in inhibiting compound 48/80-induced histamine release not only from isolated rat peritoneal mast cells but also from rat mesenterial pieces. Also, histamine release from lung pieces obtained from actively sensitized guinea pigs after exposure to antigen challenge was markedly inhibited by epinastine. The drug was effective in inhibiting not only Ca2+ uptake into lung mast cells in actively sensitized guinea pigs but also Ca2+ release from the intracellular Ca store of rat peritoneal mast cells exposed to both compound 48/80 and substance P. No significant changes were observed in phosphodiesterase activity in rat peritoneal mast cells treated with epinastine, while adenylate cyclase activity was augmented by epinastine. Epinastine has no inhibitory effect on histamine release induced by Ca2+ or IP3 from permeabilized mast cells. However, the drug significantly and dose-dependently suppressed calmodulin activity suggesting that histamine release inhibition due to epinastine may be partly attributable to Ca(2+)-calmodulin dependent process(es). The drug caused no visible changes in thermodynamic behavior of lipids, either in order parameter or in differential scanning calorimetry, indicating that the drug has no influence on membrane fluidity.

Histamine release form β-escin-permeabilized rat peritoneal mast cells and its inhibition by intracellular Ca2+ blockers, calmodulin inhibitors and cAMP

In order to study the intracellular events leading to histamine release, rat peritoneal mast cells were permeabilized using beta-escin, a triterpenoid. IP3 (0.5-10 microM) dose-dependently elicited significant histamine release from permeabilized mast cells in a Ca2+-free medium, as did GTP-gamma-S at concentrations higher than 5 microM, GTP-gamma-S induced IP3 production dose-dependently in association with histamine release. Histamine release induced by IP3 and GTP-gamma-S was inhibited by pretreatment with TMB-8, while neomycin pretreatment suppressed histamine release caused by GTP-gamma-S but not that caused by IP3.IP3 may cause Ca2+ release from the intracellular Ca2+ store as the key event leading to histamine release. At concentrations higher than 0.1 microM, Ca2+ dose-dependently induced histamine release. Both IP3-and Ca2+-induced histamine release from permeabilized mast cells was inhibited by pretreatment with calmodulin inhibitors (W-7 and calmidazolium), or with cytochalasin D or colchicine. Pretreatment with cAMP also inhibited the histamine release induced by either IP3 or Ca2+. From the present study, it can be assumed that (1) Ca2+ may act on a calmodulin-associated process(es) and cytoskeletal systems, in the process leading to histamine release, and (2) cAMP seems to inhibit histamine release, even in the stages following Ca2+ release from the intracellular Ca2+ store.

Substance P-induced histamine release from rat peritoneal mast cells and its inhibition by antiallergic agents and calmodulin inhibitors

Substance P-induced histamine release and Ca2+ release from the intracellular Ca store of rat peritoneal mast cells were inhibited by both antiallergic drugs and microtubule inhibiting agents. It was found that in the case of antiallergic compounds, histamine release inhibition may be intimately related to the inhibition of Ca2+ release from the intracellular store in which the microtubules play an important role. When mast cells were pretreated with either theophylline or dibutyryl cAMP, the inhibition of histamine release was closely related to the inhibition of Ca2+ release from the intracellular Ca store. Calmodulin inhibitors were also effective in inhibiting histamine release from mast cells induced by substance P. The inhibitory potencies of calmodulin inhibitors on histamine release from mast cells were closely correlated with those exerted on calmodulin activity.

The role of histamine H1 receptors in late-phase reaction of allergic conjunctivitis

The role of histamine H(1) receptors in the late-phase reaction of allergic conjunctivitis was studied using histamine H(1) receptor-deficient mice. To clarify the eosinophil infiltration, which is a reliable indicator of late-phase reaction, eosinophil peroxidase activity in the conjunctiva was measured. Mice were actively immunized with ovalbumin, and conjunctivitis was induced by topical instillation of ovalbumin. A significantly high eosinophil peroxidase level in the conjunctiva was observed in sensitized wild-type mice, whereas sensitized histamine H(1) receptor-deficient mice showed no significant increase in the conjunctival eosinophil peroxidase level. In addition, the elevation of eosinophil peroxidase level observed in sensitized wild-type mice was significantly antagonized by pretreatment with anti-P-selectin antibody. From these findings, it was concluded that eosinophil infiltration into the conjunctival tissue in late-phase reaction of allergic conjunctivitis is mediated by P-selectin stored in endothelial cells via histamine H(1) receptors.

Vascular permeability in allergic conjunctivitis in mice lacking histamine H1 receptors.

To clarify the role of histamine H1 receptors in allergic conjunctivitis, changes in vascular permeability of the conjunctiva were measured in histamine H1 receptor deficient mice. Wild-type mice showed a significant increase in vascular permeability of the conjunctiva induced by histamine. However, no such increase was found in histamine H1 receptor deficient mice. On the other hand, no differences were observed between wild-type and histamine H1 receptor deficient mice in response to serotonin. A significant increase in vascular permeability was observed in actively sensitized wild-type mice, whereas no increase was observed in histamine H1 receptor deficient mice. Similar findings were noted in passively sensitized animals. Histamine contents of the conjunctiva were significantly decreased by topical application of antigen in both wild-type and histamine H1 receptor deficient mice after active sensitization with antigen. These findings suggested that vascular permeability in the conjunctiva in allergic conjunctivitis is entirely regulated through histamine H1 receptor.

Essential Role of ATP and Possibility of Activation of Protein Kinase C in Ca2+-Dependent Histamine Release from Permeabilized Rat Peritoneal Mast Cells

To elucidate the role of ATP in histamine release, the present study was performed using beta-escin-permeabilized rat peritoneal mast cells. Ca(2+)-induced histamine release from permeabilized cells is totally dependent upon exogenous ATP in the medium. In the presence of Ca2+, ATP caused histamine release concentration-dependently at concentrations ranging from 0.01 to 5 mmol/l. The maximum release was achieved at 3 mmol/l of ATP in the medium. When the other adenosine nucleotides (AMP, ADP), or nonhydrolyzable ATP analogues (adenylylimidodiphosphate, beta, gamma-methylene ATP) were added in place to ATP, no histamine release took place. Other ribonucleoside triphosphates (GTP, ITP, UTP and CTP) had little effect at the same concentration range. When the ribonucleoside triphosphate content of mast cells was determined by means of HPLC, ITP and CTP were not detectable. A millimolar range of the ATP content was determined in mast cells, but the amounts of other ribonucleoside triphosphates (GTP and UTP) were remarkably lower than that of ATP. These results seem to indicate that the ATP molecule plays a crucial role in histamine release from rat mast cells in association with its concurrent hydrolysis. Furthermore, 12-O-tetradecanoylphorbol-13-acetate and 1-oleoyl-2-acetylglycerol enhanced histamine release elicited in the presence of Ca2+ (0.1 mumol/l) and ATP (3 mmol/l). Calphostin C, a potent inhibitor of protein kinase C, inhibited Ca2+/ATP-dependent histamine release by approximately 60%. At the same concentration, calphostin C inhibited by 95% protein kinase C activity in the crude extract obtained from rat mast cells. It was suggested that protein kinase C activation took place in the Ca2+/ATP-dependent histamine release from permeabilized rat mast cells.

Role of cytoskeletons on Ca2+ release from the intracellular Ca store of rat peritoneal mast cells

Cytochalasin D, colchicine or vinblastine effectively inhibited both histamine release and 45Ca uptake induced by compound 48/80 in rat mast cells. The inhibitory effects of cytochalasin D or colchicine on histamine release were exerted more remarkably when permeabilized mast cells were stimulated with either Ca2+ or inositol-1,4,5-trisphosphate (IP3). Since colchicine, vinblastine or cytochalasin D were not effective in inhibiting IP3 formation, it was assumed that microtubules or microfilaments may not participate in the initial stages of the membrane events leading to histamine release. By contrast, in Ca2+ release from the intracellular Ca store both colchicine and vinblastine (but not cytochalasin D) were effective in inhibiting Ca2+ mobilization, indicating that microtubules, rather than microfilaments, are intimately related to Ca2+ release from the endoplasmic reticulum (ER). By means of a fluorescence microscope, it was revealed that colchicine decreased the fluorescence intensity of FITC-labeled anti-tubulin antibody in the mast cells, while the amount of tubulin polymer in mast cells increased after exposure to compound 48/80. The findings indicate that colchicine simply suppressed polymerization of tubulin, while the rearrangement of microtubules so as to increase the polymerization took place after exposure to compound 48/80. Using an electron microscope in combination with potassium antimonate technique, Ca-antimonate dots were clearly observed in a cluster on the surface of the ER and a distinct connection between the ER and microtubules was also observed. It was concluded that microtubules play an important role in the processes leading to Ca2+ release from the intracellular Ca store and in subsequent histamine release.

Ca2+-Induced Translocation of Protein Kinase C during Ca2+-Dependent Histamine Release from Beta-Escin-Permeabilized Rat Mast Cells

When rat mast cells were cultured for a short period in plastic dishes and adhering cells were permeabilized with β-escin and exposed to Ca2+ at concentrations higher than 10-7 mol/l, histamine release was induced dose-dependently. Protein kinase C (PKC) activity in the crude extracts obtained from adhering mast cells was induced in the presence of Ca2+, phospholipid and diacylglycerol. The apparent Km value of PKC for Ca2+ was 0.33 µmol/l, and this Ca2+ concentration was equivalent to that which can elicit half the maximum of the Ca2+-induced histamine release. After permeabilization, approximately 80% of the total PKC activity remained in the cytosolic fraction. In the resting state, 95 % of the total PKC activity was detected in the soluble fraction, and the rest was detected in the membrane fraction. When permeabilized mast cells were incubated with Ca2+ at micromolar concentrations, which are effective in releasing histamine, the total PKC activity did not change. However, the translocation of PKC took place from the cytosolic fraction to the membrane fraction, corresponding to Ca2+ concentrations in the medium. When the crude PKC extract of mast cells was incubated with phospholipid vesicles and centrifuged, the PKC activity in the supernatant was diminished; the amount of PKC binding to the vesicles was dependent upon Ca2+ concentrations in the medium. Calphostin C, a potent PKC inhibitor, interacts with PKC in a noncompetitive manner, and it does not inhibit Ca2+-induced translocation of PKC. It can be concluded that PKC is translocated into the cell membrane along with an increase in intracellular Ca2+ concentrations and the subsequent activation of PKC may be crucial for the process leading to histamine release.

Phosphorylation of smg p21B in rat peritoneal mast cells in association with histamine release inhibition by dibutyryl-cAMP

IP3 formation and histamine release from rat peritoneal mast cells stimulated by compound 48/80 were dose‐dependently inhibited by Bt2cAMP. These inhibitions were restored to the control level in the presence of H‐8, a protein kinase A inhibitor. The 22 kDa protein in mast cells was revealed as a markedly phosphorylated protein by incubating with Bt2cAMP, and this phosphorylation was also diminished by H‐8. The 22 kDa phosphoprotein of rat mast cells comigrated with phosphorylated smg p21B, purified from human platelets and phosphorylated by protein kinase A in cell‐free system, in both one‐ and two‐dimensional PAGE analysis. Moreover, 22 kDa protein in mast cells was identified as smg p21B by immunoblot analysis using an antibody against smg p21B. From the present study, it became clear that smg p21B is phosphorylated by means or protein kinase A system in rat peritoneal mast cells, and it was assumed that phosphorylated smg p21B plays some important role in the suppression of IP3 formation and histamine release from rat peritoneal mast cells.