Induced Serotonin Release Research Articles

Biological role of different antibody classes.

Antibodies are divided into different classes and subclasses (isotypes) according to structural differences in the constant region of the heavy polypeptide chains. The different isotypes mediate specific biologic functions that are important for the response to pathogens and in the pathogenesis of immunological diseases such as allergies. The numbers of different isotypes vary in different species, humans have 9 different Ig classes and subclasses. Of these 9, only IgM, IgG1, IgG2 and IgG3 activate the classical pathway of complement. All four IgG and both IgA subclasses, but not IgM, IgE and IgD, bind to Fc receptors on neutrophils, and induce the release of granule enzymes. All four IgG subclasses but no other Ig isotype induce serotonin release from platelets. In man, only IgE binds to the high-affinity Fc receptors (Fc epsilon RI) on mast cells and basophils and induces histamine and leukotriene release. IgE also binds to low-affinity Fc receptors (Fc epsilon RII) on lymphocytes, monocytes and eosinophils; however, the functions of Fc epsilon RII on these cells are not fully established. Monocytes from patients with atopic dermatitis that express more Fc epsilon RII than monocytes from normals do not release more LTC4 than monocytes from nonallergic healthy humans after activation with aggregated IgE. IgG and IgA are more efficient than IgE in inducing the release of mediators of inflammation from monocytes. The antibody response to certain antigens such as carbohydrates and allergens are often restricted to IgG2 and IgG1, IgG4 and IgE, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and partial characterization of a T-cell-derived antigen-binding factor from mice infected with the intestinal helminth Trichinella spiralis.

Immunochemical and biological characterization was performed of an antigen-binding factor derived from culture supernatants of T cells from mice infected 4 days previously with the intestinal helminth Trichinella spiralis. Affinity chromatography with T. spiralis antigen resulted in the purification of a protein, provisionally designated Trichinella factor (Tric-F), that shared antigenic and other properties with a known T-cell-derived antigen-binding factor of different antigenic specificity, picryl chloride factor, which mediates an early 2-hour component of contact sensitivity. Tric-F lacked determinants of immunoglobulins and possessed determinants shared by other antigen-specific T cell factors, as determined by ELISA and antibody affinity chromatography. Biological activity of Tric-F was assayed in vivo and in vitro. Mice injected intravenously with Tric-F developed an antigen-specific early 2-hour ear swelling response following local challenge with T. spiralis antigen. These results corresponded to delayed-type hypersensitivity responses in the ears of T. spiralis-infected mice that comprised early 2-hour and late classical 24-hour responses. In vitro, Tric-F induced serotonin release by mast cells in the presence of T. spiralis antigen. Mast cells sensitized with Tric-F formed rosettes with antigen-coated sheep erythrocytes. It is suggested that Tric-F, an antigen-binding molecule that is T-cell-derived, mediates the early 2-hour component of delayed-type hypersensitivity and is involved in the initiation and regulation of T-cell-mediated intestinal inflammation during a T. spiralis infection in mice.

Serotonin and beta-thromboglobulin release reaction from platelet as triggered by interaction with polypeptide derivatives.

The effect of wettability of 14 polypeptide derivatives upon adhesion and activation of platelets was investigated with reference to release reactions from adhered platelets, using radioisotope labeling and radioimmunoassay method. The serotonin release was more significant from platelets adhered to polymer materials to which a large number of platelets are adhered. However, no clear relationship was found between adhesion of platelets and beta-thromboglobulin release from adhered platelets. Therefore, stimuli inducing serotonin release and beta-thromboglobulin release were considered to be from different origins. The trend in beta-thromboglobulin release was well correlated with the extent of morphological change of adhered platelets as observed by scanning electron microscope. It was suggested that the determination of released beta-thromboglobulin in association with the measurement of platelet adhesion could be useful for evaluation of blood compatibility of materials.

Demonstration of a specific C3a receptor on guinea pig platelets.

Guinea pig platelets reportedly contain receptors specific for the anaphylatoxin C3a based on both ligand-binding studies and functional responses. A portion of the human 125I-C3a that binds to guinea pig platelets is competitively displaced by excess unlabeled C3a; however, the majority of ligand uptake was nonspecific. Uptake of 125I-C3a by guinea pig platelets is maximal in 1 min, and stimulation of guinea pig platelets by thrombin, ADP, or the Ca2+ ionophore A23187 showed little influence on binding of the ligand. Scatchard analysis indicated that approximately 1200 binding sites for C3a exist per cell with an estimated Kd of 8 x 10(-10) M. Human C3a des Arg also binds to guinea pig platelets, but Scatchard analysis indicated that no specific binding occurred. Because the ligand-binding studies were complicated by high levels of nonspecific uptake, we attempted to chemically cross-link the C3a molecule to a specific component on the platelet surface. Cross-linkage of 125I-C3a to guinea pig platelets with bis(sulfosuccinimidyl)suberate revealed radioactive complexes at 105,000 and 115,000 m.w. on SDS-PAGE gels by autoradiographic analysis. In the presence of excess unlabeled C3a, complex formation was inhibited. No cross-linkage could be demonstrated between the inactive 125I-C3a des Arg and the putative C3a-R on guinea pig platelets. Human C3a, but not C3a des Arg induces serotonin release and aggregation of the guinea pig platelets. Human C3a was unable to induce either serotonin release or promote aggregation of human platelets. Uptake of human 125I-C3a by human platelets was not saturable, and Scatchard analysis was inconclusive. Attempts to cross-link 125I-C3a to components on the surface of human platelets also failed to reveal a ligand-receptor complex. Therefore, we conclude that guinea pig platelets have specific surface receptors to C3a and that human platelets appear devoid of receptors to the anaphylatoxin.

Cerastes cerastes (Egyptian Sand Viper) venom induced platelet aggregation as compared to other agonists

The Egyptian Sand Viper (Cerastes cerastes) crude venom and subfractions were, for the first time, shown to induce platelet aggregation with agonist activities present in two subfractions. The combined activities of the crude venom components behaved in a unique fashion as compared to the platelet agonists, ADP, collagen and thrombin. The action of the venom was inhibited by conditions that increased cAMP, partially required the formation of thromboxane A2 and was inhibited by the serine protease inhibitor PMSF while being only partially sensitive to leupeptin or soybean trypsin inhibitor. One of the fractionated venom agonists strongly induced serotonin release while the other venom agonist essentially did not. Further characterization of the Cerastes cerastes venom components should broaden our knowledge of the pathology of snake venoms, platelet aggregation and their potential therapeutic value.

Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation.

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.

Mutual relationship among cytosolic pH, Na + and Ca 2+ ions in the degranulation of rat leukemic basophils

Reagents which affect the cytosolic concentrations of protons and sodium ions markedly affect the degranulation process of mast cells. The proton-sodium exchanging ionophore, monensin, is found to cause noncytolytic dose dependent serotonin release from the rat leukemic basophils (line RBL-2H3). Its half maximal dose of ca. 2 μM leads to secretion of ca. 20% of these cells' serotonin content. Monensin induced serotonin secretion increases with external pH and decreases upon lowering external sodium ion concentrations, yet is independent on external calcium. Monitoring cytosolic pH and free Ca 2+ concentrations with BCECF and quin2, respectively, shows that a rise in pH i and [Ca 2+] i is caused by the ionophore. Amiloride, the blocker of cellular Na +/H + antiporter, is found to be an effective inhibitor of antigen or monensin induced serotonin release. However, it does not by itself cause secretion. In contrast, ouabain, which inhibits the cellular Na +/K + ATPase, does induce secretion. Cellular levels of pH, Na + and Ca 2+ ions are evidently linked and involve a manifold of activities. Though exchanging protons for sodium seems to be effective in causing mediator release, the present results do not provide sufficient support for proton/sodium ions having a second messenger role in the immunologically induced mediator release.

ヒト血小板のＰＭＡ（ｐｈｏｒｂｏｌ ｍｙｒｉｓｔａｔｅ ａｃｅｔａｔｅ）前処理による刺激応答の変化

The effects of tumor promoter (PMA, phorbol-12-myristate-13-acetate) on thrombin-stimulated human platelet responses and membrane phospholipid metabolism were investigated. Thrombin (0.1U/ml)-induced serotonin release was inhibited by pretreatment with PMA (50μg/ml) for 5min. This inhibition was associated with a suppression of polyphosphoinositide breakdown and increase of cytoplasmic free Ca2+ ([Ca2+] i). PMA was observed to increase the incorporation of [32P]phosphate into phosphatidylinositol 4, 5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP). This indicates that treatment with PMA suppresses the hydrolysis of PIP2 by phospholipase C, resulting in the insufficient supply of a Ca2+ releaser, IP3. A considerably high radioactivity in PIP would be due to the enhanced conversion of phosphatidylinositol (PI) to PIP via PI kinase rather than the inhibited PIP breakdown, since there was no indication of PIP hydrolysis during stimulation with thrombin alone. These observations provide evidence that protein kinase C subserves a negative feedback role in a receptor-mediated cell activation.

Leupeptin selectively inhibits human platelet responses induced by thrombin and trypsin; a role for proteolytic activation of phospholipase C

Thrombin and trypsin induce serotonin release and aggregation in human platelets. Both proteases induced activation of phospholipase C as reflected by formation of inositol phosphates and phospnorylation of the resultant 1,2-diacylglycerol to phosphatidic acid. Also, thrombin and trypsin activate protein kinase C and myosin light chain kinase as indicated, respectively, by phosphorylation of the 40,000 and 20,000 dalton proteins. Leupeptin, a known inhibitor of serine proteases, blocks all the observed responses of human platelets to trypsin and thrombin. Leupeptin does not inhibit serotonin release and aggregation induced by other platelet stimuli such as collagen, platelet-activating factor, ionophore A23187, and arachidonic acid. The implication of a proteolytic-mediated pathway in the transmembrane signalling involved in platelet activation is discussed.

The platelet activation induced by wheat germ agglutinin

In human platelets, wheat germ agglutinin (WGA) induced serotonin release without cell agglutination. WGA induced the phosphorylation of both 40-kDa and 20-kDa proteins in a parallel manner, and at least, the phosphorylation of 40-kDa protein was preceded by transient formation of endogenous diacylglycerol (DG) accompanied by a decrease in phosphatidylinositol (PI). Both phosphorylation of these two proteins and serotonin release were inhibited by prior treatment of platelets with dibutyryl cyclic AMP, W-7, or TMB-8. These results suggest that both phosphatidylinositol turnover and Ca 2+ mobilization play an essential role in WGA-induced platelet activation.

ヒト血小板におけるＣａ｀２＋´動態の再検討 細胞外Ｃａ｀２＋´の役割

The enhancement of cytoplasmic Ca2+ level upon stimulation is known to trigger the rapid response of secretory cells, such as platelets. The role of extracellular Ca2+ on activation of phospholipases A2 and C activities was studied.In the presence of 1mM EGTA, the level of platelet aggregation by thrombin was reduced to 20% of that in the presence of 1mM CaCl2. The EGTA-suppressed platelet restored aggregability by addition of extra Ca2+ concentration. Thrombin (0.3U/ml)-induced serotonin release assessed at 1min was independent of the concentrations of EGTA or CaCl2. The increased phosphatidylinositol (PI) turnover was observed as inferred by a decreased level of PI and increased 1, 2-diacyl glycerol and phosphatidic acid in [3H] arachidonic acid (AA)-labeled cells stimulated with thrombin (0.1 and 1U/ml). The increment of radioactivity in arachidonate and its metabolites was accounted for by the loss of [3H] phosphatidylcholine (PC) independent of the extracellular Ca2+ The degradation products of phospholipase A2, lysoPI and lysoPC were detected in stimulated cells with or without Ca2+ in the medium. Extracellular Ca2+ levels do not affect the degradation or resynthesis of phosphoinositides, PI, PI-4-monophosphate (DPI), and PI-4, 5-bisphosphate (TPI). These results indicate that the activation processes of phospholipases A2 and C in thrombinstimulated platelets are independent of extracellular Ca2+ level.

Increased biosynthesis of platelet-activating factor in activated human eosinophils.

1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase catalyzes the conversion of biologically inactive lysophospholipid to bioactive platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) by an acetylation reaction. The activity of this enzyme in eosinophils isolated from patients with eosinophilia is stimulated (up to 4-fold) in a dose-, time-, and Ca2+/Mg2+-dependent manner after exposure to the eosinophil chemotactic factor of anaphylaxis (ECF-A), C5a, formyl-methionylleucylphenylalanine (fMLP), or ionophore A23187. The three naturally occurring chemotactic factors (ECF-A, C5a, and fMLP) cause a rapid and transient increase of enzyme activity, with a maximum at 1 or 3 min, whereas ionophore A23187 maintains an elevated level for up to 15 min. The activity of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase, an enzyme that catalyzes the breakdown of PAF to lyso-PAF, is not affected by C5a, fMLP, or ionophore A23187. The presence of PAF in eosinophils was established by demonstrating the lipid nature of the compound, the RF value being identical with that of synthetic 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine on thin layer chromatograms, and by its ability to induce serotonin release from rabbit platelets. Furthermore, ECF-A, C5a, fMLP, and ionophore A23187 all induce the secretion of PAF from eosinophils. These findings suggest that the generation and release of PAF could be a consequence of eosinophil chemotactic activation and may thus function in inflammatory and allergic reactions in which eosinophils participate.

Thrombin induced platelet reactions: Effect of substrates and inhibitors on binding of thrombin and serotonin release

Binding of 125I-thrombin to platelets and subsequent serotonin release were confirmed. The binding of unaltered thrombin to platelets was also measured by a new technique using a chromogenic substrate (S-2238). As compared to 125I-thrombin, this method gave similar constants for binding to the high affinity binding site, but lower for binding to the low affinity binding site. Furthermore, the results suggest that the platelets have two classes of independent binding sites. Substrates and inhibitors of thrombin inhibited thrombin induced serotonin release, suggesting that the release reaction depends on the proteolytic activity of thrombin. The serotonin release was more inhibited than the binding of thrombin, suggesting that the platelet binding site and the active site of thrombin are located in different parts of the thrombin molecule.

Protein phosphorylation and diglyceride production during serotonin release induced by epinephrine plus ADP in human platelets

Epinephrine (1 μM) plus ADP (5 μM) induced serotonin release from human platelets although neither epinephrine nor ADP alone brought about such a release. During this release reaction, the phosphorylation of 40K-dalton protein was induced to an extent similar to that induced by 0.5 unit/ml thrombin. The amount of diglyceride (DG) produced by epinephrine plus ADP, however, was much smaller than that produced by thrombin. The reaction velocities of these reactions induced by epinephrine plus ADP were slower than those induced by thrombin. Epinephrine or ADP alone hardly produced any DG and induced 40K-dalton protein phosphorylation only slightly. Indomethacin (1 μg/ml), a cyclooxygenase inhibitor, remarkably inhibited epinephrine plus ADP induced serotonin release, 40K-dalton protein phosphorylation and DG production although this agent had little effect on the same reactions induced by thrombin. These results suggest that prostaglandin endoperoxides or thromboxane A2 may be involved in serotonin release, 40K-dalton protein phosphorylation and DG production induced by epinephrine plus ADP.

Contrast media induced serotonin release in human blood.

Several contrast media were tested for their ability to release serotonin from whole blood. Renografin-60, Hypaque M-75, Vascoray and Conray were incubated with human blood at 37 degrees C for 60 min. Platelet-poor plasma was analysed for serotonin using a high performance liquid chromatographic technique. Seven non-ionic contrast media were similarly incubated and serotonin levels determined. Ionic contrast media induced a greater release of serotonin compared with non-ionic ones. The amount of serotonin was not proportional to iodine concentration in blood. These results suggest that hypertonic solutions of contrast media produce a physical injury to platelets causing them to release their granular contents.

Release of platelet constituents by monosodium urate crystals.

The release of human platelet constituents by the etiologic agent of gout, the monosodium urate crystal, is described here. In suspensions of washed platelets, response to urate crystals proceeded in two phases: A secretory phase involved the rapid active release of serotonin, ATP, and ADP with little loss of lactic dehydrogenase or beta-glucuronidase. A lytic phase involved the slower loss of all platelet constituents. Both phases were inhibited by iodoacetate plus dinitrophenol, suggesting an energy requirement. In ultrastructural studies, lysis of washed platelets which appeared to contain crystals was seen. Urate crystals were also shown to induce serotonin release and platelet lysis in citrated platelet-rich plasma. Since urate crystals are deposited at a variety of sites, urate crystal-platelet interaction in vivo is a possibility. Such interactions, leading to release of platelet constituents, might contribute to gouty inflammation or to enhanced atherogenesis.

Air-ionometry of hot, dry dessert winds(Sharaw) and treatment with air ions of weather-sensitive subjects.

Hot dry winds (Sharav) produce increased ionisation of the atmosphere, values for positive and negative ions going up from an average of 1, 000 per cm3 to 1, 500. There was almost always a slight preponderance of the positively-charged small ions. This increased air ionisation induces serotonin release in about one-quarter of the population with multiple complaints of a typical serotonin irritation syndrome. In 75% of 129 subjects suffering from serotonin ailments, the treatment with negative air ions (Ionotron) with an output of 3.5 × 105 ions/(cm3 · sec) at 1 m distance produced prophylactic and therapeutic relief when the patients were kept in a room of up to 4 × 4 m size. These results were controlled by serotonin and 5-HIAA urinalysis.