M. Mio

Analysis of the mechanism of histamine release induced by substance P

Substance P causes release of histamine from rat peritoneal mast cells; the structure-activity relationship shows that N-terminal residue is essential and the hydrophobic region of C-terminal plays an important role. Electrical conductivity of black lipid membrane containing phosphatidic acid was augmented by substance P. In this case, N-terminal residues and C-terminal hydrophobicity were also unavoidable. The partitioning of substance P into the organic phase increased in the presence of phosphatidic acid. The CD spectrum of substance P was changed from the unordered form to beta-form by coexistence of phosphatidic acid/PC liposomes in the medium. The addition of calcium or magnesium in the test solution is effective to prevent either of these phenomena. These findings indicate that substance P probably binds to negatively charged sites of membrane lipids, and subsequent penetration of C-terminal into the hydrophobic core of lipid bilayer may induce an increase of membrane permeability and the following histamine release.

Anti-allergic constituents in the culture medium of Ganoderma lucidum. (I) Inhibitory effect of oleic acid on histamine release

The chloroform extract fromGanoderma lucidum broth markedly inhibited histamine release from rat peritoneal mast cells. From the active fractions, palmitic acid, stearic acid, oleic acid and linoleic acid were isolated. Oleic acid dose-dependently inhibited the histamine release and45Ca uptake into mast cells induced by compound 48/80 and A-23187 at concentrations of 5 to 50 μM and 0.5 to 5 μM, respectively. Saturated fatty acids, however, had only a weak inhibitory effect on histamine release. Although linoleic acid and linolenic acid effectively prevented this release, these two compounds caused marked release at concentrations higher than 10 μM and 20 μM, respectively. Oleic acid induces membrane-stabilization in model membrane systems. It was concluded that one of the effective constituents obtainable from the chloroform extract ofG. lucidum-cultured broth is oleic acid.

Inhibitory effects of oxatomide on intracellular Ca mobilization, Ca uptake and histamine release, using rat peritoneal mast cells

Oxatomide at concentrations of 0.01-10 microM inhibited not only an increase in 45Ca uptake but also the intracellular Ca2+ release induced by compound 48/80 in rat peritoneal mast cells. At higher concentrations, ketotifen or other calcium antagonists caused similar inhibitory effects. However, the inhibitory effect of oxatomide on the 45Ca uptake into rat neonatal heart cells was much weaker than that of verapamil. Through image processing of quin 2-stained mast cells, it was revealed that oxatomide inhibited Ca2+ release from the intracellular store. Although oxatomide alone did not affect cAMP and cGMP contents in sensitized guinea pig lung samples, the drug effectively prevented changes in the nucleotide contents evoked by antigen challenge. These results suggest that the inhibitory effect of oxatomide on histamine release may be caused by a combination of prevention of Ca uptake, which is highly selective toward mast cells; inhibition of Ca2+ release from the intracellular Ca store, and elevation of the cAMP content in mast cells.

Antiallergic Effects of Terfenadine on Immediate Type Hypersensitivity Reactions

Terfenadine dose-dependently inhibited rat homologous PCA (2.5-10 mg/kg, p.o.) and experimentally-induced asthma in guinea pigs (0.5-5 mg/kg, p.o.). Similarly, metabolites I and II dose-dependently inhibited experimentally-induced asthma but their respective potencies were approximately 1/2 and 1/15th that of terfenadine. These results suggest that the metabolites contribute to the antiallergic effects of terfenadine. In ex vivo, terfenadine (5-20 mg/kg, p.o.) also inhibited the release of both antigen-induced histamine and SRS-A from sensitized guinea pig lung samples and that of histamine from rat peritoneal mast cells. Terfenadine dose-dependently increased the cAMP content in rat mast cells and in the lungs; in the latter, the augmented cAMP is associated with an increase in adenylate cyclase activity, but not with the inhibition of phosphodiesterase activity. The above evidence indicates that the inhibitory effects of terfenadine on mediator release from mast cells are in some way related to its antiallergic effects, and that an elevated cAMP content may be effective to enhance mediator release inhibition.

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.

Histamine release inhibition and prevention of the decrease in membrane fluidity induced by certain anti-allergic drugs: Analysis of the inhibitory mechanisms of NCO-650

NCO-650 exerted a dose-dependent inhibition of histamine release from isolated rat mast cells without affecting the Ca-uptake and intracellular cAMP levels. When the membrane stabilizing action of NCO-650 was investigated with the liposomes prepared by various phospholipids with or without cholesterol, it became clear that compound 48/80 decreases the order parameter of liposomes and NCO-650 inhibits the decrease of order parameter. Histamine release from histamine-containing liposomes was brought about by compound 48/80 at concentrations higher than 0.5 μg/ml, and pretreatment of the liposomes with NCO-650 inhibited histamine release due to compound 48/80. The incorporation of NCO-650 in the liposomes was effective in abolishing the phase transition of the lipid bilayer membranes from crystalline gel to liquid crystalline phases, and decreased the phase transition temperature. Those changes in the lipid bilayer membranes correspond to those displayed by incorporation of cholesterol in the liposomes. An increase of permeability of lipid membrane by compound 48/80 may be due to partial loosening of the lipid order; NCO-650 rigidifled the liposome membrane and as a result of this, it decreased histamine release.

Ca uptake and Ca releasing properties of the endoplasmic reticulum in rat peritoneal mast cells

In order to study the characteristics of the intracellular Ca store of mast cells, organelles of rat peritoneal mast cells were fractionated. The binding of 45Ca was at its peak in the fractions where the highest activity of glucose-6-phosphatase, the marker enzyme for the endoplasmic reticulum (ER), was measured. The ER-rich fraction exhibited an ATP-dependent uptake of 45Ca and this uptake was inhibited by pretreatment with ATPase inhibitors such as LaCl3 or Na3VO4. When inositol 1,4,5-trisphosphate (IP3) was added to a medium containing the 45Ca-loaded ER fraction, it caused a dose-dependent release of 45Ca at concentrations higher than 0.5 microM, while inositol 1-monophosphate and inositol 1,4-bisphosphate were not effective even at higher concentrations. The results of a binding assay using 3H-labeled IP3 indicated that there exist two kinds of IP3 binding site in the ER: one is of high affinity but low capacity while the other is of low affinity and high capacity. IP3-induced 45Ca release was dose-dependently inhibited by pretreatment with c-AMP. The present study supports the assumption that the intracellular Ca store associated with histamine release from the mast cell is the ER.

Role of Microfilaments in the Exocytosis of Rat Peritoneal Mast Cells

When rat peritoneal mast cells were treated with the potent histamine releaser compound 48/80 in the presence of tetramethylrhodamine-labeled G-actin, the fluorescent G-actin particles were bound to the surface of extruded granules and to the cell surface. When rhodamine-phalloidin was incorporated into permeabilized rat mast cells in a Ca2+-free medium, rhodamine fluorescence was observed on the cell surface accompanied by serpentine ridges which appeared in the resting state. After perfusion with a cytosol-like solution containing Ca2+, rhodamine fluorescence appeared on the cell surface as a distinct network formation. In some cases, circular fluorescences which appeared to surround the extruded pores were observed in the cell membrane. These findings indicate the existence of actin filaments in the cell membrane and/or subplasmalemmal network. In whole-mount preparations, the granules were surrounded very densely with microfilaments of various widths. After exposure to compound 48/80, granules protruding through the cell membrane were wrapped in many filaments. The extruded granules located in the periphery of the cells were connected by many filamentous structures and disruptions in the middle of these connections were occasionally observed. In some cases, circular configurations of microfilaments were observed at the bottom of the extruded granules and in others dense gatherings of microfilaments were seen just beneath the granules, as if the latter were being pushed up and out of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of intracellular Ca2+ in the degranulation of skinned mast cells

The intracellular pH of rat peritoneal mast cells was slightly acidic and compound 48/80 induced a decrease in the cytoplasmic pH of these cells. By means of chemical skinning, it was revealed that perfusion with Ca2+ or inositol 1,4,5-trisphosphate (IP3) induced degranulation dose-dependently in mast cells at concentrations higher than 10 μM and 0.1 μM, respectively. Na+ was essential for the release of histamine from mast cells. An assay based on the binding of45Ca to mast cell fragments revealed that the intracellular Ca store of the mast cell is located in the endoplasmic reticulum. IP3 liberated Ca from the endoplasmic reticulum.

Changes in intracellular Ca2+ distribution of rat peritoneal mast cells before and after histamine release.

Rat peritoneal mast cells were stained with quin 2, a fluorescent Ca2+ chelator. By means of a fluorescence microscope and real time image processer, it was revealed that the fluorescence derived from the Ca-quin 2 complex was weak in the area occupied by the nucleus and distributed unevenly in the cytoplasm of the resting cells so as to encompass the individual granules. When compound 48/80 or substance P was added in a Ca-free medium, the fluorescence intensity of quin 2 increased markedly all over the cell, suggesting that a large amount of Ca2+ was released from intracellular Ca stores. The increase in the fluorescence intensity produced by compound 48/80 or substance P in a Ca-free medium was inhibited by pretreatment with certain drugs eliciting an increase of c-AMP levels, such as dibutyryl c-AMP and theophylline, or by some anti-allergic drugs providing a membrane stabilizing action.