Differentiation of the mechanisms inducing segmentation and asymetry of chromatids after treatment with 5-bromodeoxyuridine]

Rabbit anti-rat mast cell antibody is capable of liberating histamine from rat peritoneal mast cells in the presence of complement. The cytotoxicity of this complement-mediated histamine release mechanism is attested by a substantial reduction of cell ATP, release of ⁵¹Cr and ⁸⁶Rb and lytic ultrastructural changes. Inhibition of complement-dependent cytotoxic histamine release can be achieved by depressing mast cell ATP with 2,4-dinitrophenol (1 mM), antimycin A (0.2 μM) or potassium cyanide (1 mM). Restoration of cell ATP is accompanied by reversal of the inhibition of the cytotoxic histamine release. Ultrastructural analysis and ⁵¹Cr release studies reveal that in mast cells depleted of ATP, cytolysis occurs but perigranule membranes remain intact, thus preventing histamine release.     

Nature of the oxygen species generated by xanthine oxidase involved in secretory histamine release from mast cells

The present studies were carried out to characterize the nature of reactive oxygen species generated by the xanthine-xanthine oxidase system involved in the release of histamine by noncytotoxic and cytotoxic mechanisms. To distinguish secretory release from lytic release, mast cells were loaded with 51Cr and the release of 51Cr into the incubation medium was used as a measure of cell lysis. The secretory release of histamine was not inhibited by superoxide dismutase or catalase alone. However, together these agents inhibited the release. This suggests that the combination of superoxide and hydrogen peroxide can evoke secretory release. The lytic release of histamine, as monitored by concomitant release of 51Cr from mast cells at higher concentration of xanthine oxidase or longer periods of incubation, seems to be related to hydrogen peroxide production since catalase inhibited the cell lysis. Since it has been reported that exogenously added hydrogen peroxide at concentrations below 10 mM did not induce cell lysis, the lytic release, although hydrogen peroxide dependent, may not be due to its direct effect on the cell surface. The cell lysis observed in the xanthine-xanthine oxidase system seems to be brought about by a complex mechanism involving the interactions of hydrogen peroxide and superoxide with cellular components. These studies indicate that the beneficial effects of superoxide dismutase seen in biological systems may partly be due to inhibition of the secretory processes stimulated by superoxide.     

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the radionuclide Chromium 51 (⁵¹Cr) to label target cells. These targets are then exposed to effector cells and the release of ⁵¹Cr from target cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of ⁵¹Cr. As effector cells, we used an activated autoreactive clonal population of CD8⁺ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with ⁵¹Cr labeled target NIT cells for 16 hours, release of ⁵¹Cr was recorded to calculate specific lysis Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37°C, with 5% CO₂, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.     

Effects of L-leucine methyl ester (Leu-OMe) on mouse peritoneal mast cells: characterization of histamine release versus cytotoxicity.

L-Leucine methyl ester (Leu-OMe), a lysosomotropic compound, has been found to eliminate several lysosome-rich cellular subtypes and all natural killer cell function from peripheral blood mononuclear cells. In this report, the effect of Leu-OMe on mouse peritoneal mast cells is described. The L-Leu-OMe induced the release of histamine from mouse peritoneal mast cells in a dose-dependent manner (0.25 to 3 mM), while its D-stereoisomer had no effect. L-Leu-OMe displayed also a potent histamine release effect on purified mast cells, indicating a direct effect on mast cells. The monitoring of radioactive chromium release versus histamine release showed that both processes may be unrelated for Leu-OMe concentrations inferior to 1.5 mM. At higher doses, L-Leu-OMe, but not its D-stereoisomer, exerted a potent cytotoxic effect on mast cells. The secretory effect of Leu-OMe was temperature- and energy-dependent. Experiments performed in the absence of extracellular calcium and magnesium demonstrated that these divalent cations were not necessary for the Leu-OMe-induced histamine release, and their deprivation even involved a higher histamine release. The secretory characteristics of the Leu-OMe-induced histamine release appeared to be different from those of the IgE-induced ones. These results support the conclusion that exposure of mouse peritoneal mast cells to high doses of L-Leu-OMe results in killing of these cells, that are new targets of this lysosomotropic agent.     

Histamine H-1 receptors on rat peritoneal mast cells

In order to characterize the receptor subtype involved in histamine stimulation of increased cyclic AMP levels in rat mast cells with consequent impairment of anaphylactically induced mediator release, the binding of the H-1 receptor antagonist [³H] pyrilamine to mast cells was examined. Pyrilamine bound rapidly, in a saturable and reversible fashion, and with increased binding at 4°C as compared with 21°C and 37°C. [³H] Pyrilamine binding was displaced by H-1 antagonists (tripelnnamine > yrilamine ≧ iphenhydramine) > histamine > the H-2 antagonist, cimetidine. H-1 agonists displaced pyrilamine binding less efficiently than histamine but better than H-2 agonists. Rat mast cells have a single homogeneous population of low affinity (K_D = 222 ± 33 nM) H-1 receptors with a Bmax of 9.7 ± 2.3 pm/10⁶ mast cells and 5.4 ± 0.92 × 10⁶ binding sites per mast cell. Thus, the mast cell has an H-1 type histamine receptor which is probably involved in histamine-induced cyclic AMP increases.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

Mast Cell-Nerve Cell Interaction at Acupoint: Modeling Mechanotransduction Pathway Induced by Acupuncture

Mast cells are found abundant at sites of acupoints. Nerve cells share perivascular localization with mast cells. Acupuncture (mechanical stimuli) can activate mast cells to release adenosine triphosphate (ATP) which can activate nerve cells and modulates pain-processing pathways in response to acupuncture. In this paper, a mathematical model was constructed for describing intracellular Ca²⁺ signal and ATP release in a coupled mast cell and nerve cell system induced by mechanical stimuli. The results showed mechanical stimuli lead to a intracellular Ca²⁺ rise in the mast cell and ATP release, ATP diffuses in the extracellular space (ECS) and activates the nearby nerve cells, then induces electrical current in the nerve cell which spreads in the neural network. This study may facilitate our understanding of the mechanotransduction process induced by acupuncture and provide a methodology for quantitatively analyzing acupuncture treatment.     

The effect of Na+-K+ ATPase inhibition by ouabain on histamine release from human cutaneous mast cells

Background: There are controversial reports on the effect of sodium-potassium adenosine triphosphatase (Na⁺-K⁺ ATPase) inhibition on mast cell mediator release. Some of them have indicated that ouabain (strophanthin G), a specific Na⁺-K⁺ ATPase inhibitor, inhibited the release, whereas the others have shown that ouabain had no effect or even had a stimulatory effect on the mediator secretion. Most of these studies have utilized animal-derived mast cells. The aim of this study was to determine the effect of Na⁺-K⁺ ATPase inhibition on human skin mast cells. Methods: Unpurified and purified mast cells were obtained from newborn foreskins and stimulated by calcium ionophore A23187 (1 μM) for 30 min following a 1 hr incubation with various concentrations (10⁻⁴ to 10⁻⁸ M) of ouabain. Histamine release was assayed by enzyme-linked immunosorbent assay (ELISA). Results: The results indicated that ouabain had no significant effect on the non-immunologic histamine release from human skin mast cells, in vitro. Conclusions: Na⁺-K⁺ ATPase inhibition by ouabain had no significant effect on the non-immunologic histamine release from human cutaneous mast cells and suggested differences between human and animal mast cells.     

Inhibition of compound 48--80 induced histamine liberation from mast cells by triton X-100]

Triton X-100 at concentrations preceding those which liberated histamine, produced dose-dependent inhibition of compound 48/80-induced histamine release from rat mast cells. Triton X-100 (0.00002 1/1) depleted ATP content in the mast cells and blocked compound 48/80-induced histamine release. The inhibition of compound 48/80-induced histamine release and depletion of the ATP content in the mast cells was reversed by glucose (10 mmole). It is concluded that inhibition by Triton X-100 of histamine release induced by compound 48/80 is dependent on inhibition of energy production.     

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity. I. General requirements of the reaction and temporal relationship between lethal hits and cytolysis.

The effect of various physical and chemical parameters on the cytotoxic reaction was studied in a ⁵¹Cr-release assay in order to analyze the mechanism by which human blood mononuclear cells (MC) damage antibody-sensitized target cells infected with herpes simplex virus. Centrifugation of the target cell-MC mixture consistently increased the velocity of the reaction. In addition, uncentrifuged target cell-MC cultures showed a sigmoidal kinetic curve of ⁵¹Cr release with an initial lag phase of at least 10 min, whereas ⁵¹Cr release in centrifuged cultures followed a linear pattern with time without an initial lag. These findings indicate that direct contact between target and effector cells is necessary for cytotoxicity to occur. The reaction as a whole was temperature dependent, proceeding well at 37 °C and not at all at 4 °C. Incubation of the MC at 46 °C for 10 min abolished their cytotoxic potential without affecting their viability; similar heating of the target cells did not affect their background isotope release or sensitivity to the lytic process. Heating target cell-MC mixtures at 46 °C for 10 min thus provided a tool by which the temporal relationship between the mounting of “lethal hits” and specific isotope release, or cell lysis, could be studied. Using this technique, we observed virtually simultaneous occurrence of lethal hits and cell lysis, measured at various intervals between 10 and 360 min postincubation. Likewise, we were unable to demonstrate a transient period of increased osmotic fragility in target cells after contact with MC but before actual cell lysis. Taken together, these findings imply either that cell lysis, as indicated by ⁵¹Cr release, results from a sudden nonosmotic injury to the target cell membrane or, alternatively, osmotic damage leading to ⁵¹Cr release occurs too rapidly to be detected by the methods employed in this study. These findings imply either a qualitative or a quantitative difference between antibody-dependent cellular cytotoxicity (ADCC) mediated by K cells and cytotoxicity mediated by sensitized T cells.The cytotoxic reaction was completely inhibited by 10 mM EDTA and did not occur in a Ca²⁺- and Mg²⁺-free medium. Neither Ca²⁺ nor Mg²⁺ alone produced as much cytotoxicity as the two cations in tandem; in addition, when added to the culture medium in suboptimal amounts, the two cations were either additive or synergistic. These observations suggest that both cations are necessary in ADCC and also that there may be separate Ca²⁺- and Mg²⁺-dependent events in the lytic pathway.     

Calcium requirement for the inhibition by theophylline of histamine release from mast cells

When added to Ca2+-free Hanks' solution, Ca2+ (0.1-2.5 mM) had no significant effect on antigen-induced histamine release from rat mast cells, but Sr2+ (1.0-3.0 mM) dose-dependently increased the release. Ba2+ (1.0 and 2.0 mM) also enhanced the release. Ca2+ and Ba2+ inhibited compound 40/80-induced histamine release, in a dose-dependent manner. In ordinary Hanks' medium, theophylline and 3-isobutyl-1-methylxanthine (IBMX) dose-dependently inhibited the antigen-induced histamine release but these drugs were ineffective in Ca2+-free medium. Theophylline (1.0 mM) also inhibited compound 48/80-induced histamine release in the presence but not absence of Ca2+. There was an optimal Ca2+ concentration for the theophylline effect. Sr2+ but not Ba2+ could substitute for Ca2+ in supporting the theophylline effect. Theophylline (1.0 mM) and IBMX (1.0 mM) increased mast cell cyclic AMP levels both in the presence and absence of Ca2+. These results suggest that Ca2+ is required in the interaction of theophylline and specific sites on mast cells or in the mast cell response to theophylline which probably does not involve the cyclic AMP increase and is linked to the inhibition of histamine release.     

Differential Ca2+ mobilization and mast cell degranulation by FcεRI- and GPCR-mediated signaling

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca²⁺ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca²⁺ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca²⁺ levels, respectively. Histamine release and Ca²⁺ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca²⁺ increase. FcεRI-induced Ca²⁺ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca²⁺ release was both necessary and sufficient for mast cell degranulation, extracellular Ca²⁺ influx enhanced the process. Furthermore, cytosolic Ca²⁺ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca²⁺ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca²⁺ signaling in regulating histamine degranulation.     

Calcium requirement in compound 48/80 induced histamine release from rat mast cells in vitro

The effect of magnesium and EDTA on compound $\text{48}\text{80}$-induced histamine release and adenosine triphosphate (ATP) content of mast cells has been studied. Inhibition of histamine release after preincubation of the cells with or without EDTA in the absence of calcium and the reversal by calcium indicate that calcium is required for compound $\text{48}\text{80}$-induced histamine release. The presence of magnesium potentiate the inhibition caused by the lack of calcium. The inhibition of histamine release is not related to changes in cellular ATP content. The observations with EDTA suggest that calcium may be provided for the release process from intracellular sources.     

Ca2+-Mg2+-activated adenosine triphosphatase in plasma and granule membranes in non-secreting and secreting mast cells

An adenosine triphosphatase (ATP) activated by Ca²⁺ or Mg²⁺ is shown morphologically on the outer surface of non-secreting and secreting rat peritoneal mast cells. ATPase having the same properties is also seen on the external surface of the other peritoneal cells, i.e. macrophages, mononuclear cells and lymphocytes. When histamine release from the mast cells was induced by exposing them to antigen (anaphylactic reaction) or compound 48/80, ATPase activated by Ca²⁺ or Mg²⁺ could in addition be demonstrated in the granule membranes. Granule membrane ATPase is also shown in non-secreting mast cells after freezing and thawing. ATPase on the outer surface of the plasma membrane is seen in the secreting mast cells as in the non-secreting cells except in the areas where the plasma membrane fuses with the granule membrane. The role of ATPase in granule secretion process has been discussed.     

Cytopathic Action of Naegleria fowleri Amoebae on Rat Neuroblastoma Target Cells

The axenically cultured, weakly pathogenic Naegleria fowleri LEE and the highly pathogenic, mouse passaged N. fowleri LEEmp are cytopathic for B103 rat nerve cells in culture. Cytopathogenicity was measured by release of radiolabeled rubidium or radiolabeled chromium from B103 target cells. Cytopathogenicity was time-dependent for up to 18 h and dependent upon amoebae effector to nerve cell target ratios of less than 1:1. Release of⁵¹ Cr from B103 cells by either LEE or LEEmp amoebae was enhanced by addition of calcium or magnesium to medium free of these divalent cations but the ion-channel inhibitor, verapamil, or the ionophore A23187 and phorbol myristate acetate did not alter release of ⁵¹ Cr from B103 cells cocultured with the amoebae. Cycloheximide or actinomycin D impaired release of ⁵¹ Cr from B103 target cells injured by either LEE or LEEmp amoebae. Both strains of amoebae were fractionated by glass bead disruption and high speed centrifugation into membrane and soluble fractions. Each fraction was incubated with either ⁸⁶Rb or ⁵¹ Cr labeled nerve cells. The membrane fraction from LEEmp was more active than the soluble fraction in facilitating rubidium and chromium release. In contrast, the soluble fraction from LEE was more active than the membrane fraction in facilitating rubidium release from radiolabeled target cells. The sequential release of ⁸⁶Rb and ⁵¹ Cr from target cells rather than the simultaneous release of the two isotopes indicates that target cell death is due to the release of ions followed later by the release of large macromolecules. The results indicate that N. fowleri amoebae injure nerve cells by two alternate mechanisms, trogocytosis or contact-dependent lysis.     

Ca2+ influx,phosphoinositide hydrolysis,and histamine release induced by lysophosphatidylserine in mast cells

We have previously demonstrated that snake venom phospholipases A₂ (PLA₂s) and mammalian PLA₂s induced inflammatory processes. This effect was correlated with the activity of the enzymes and the release of lipid mediators. We have now determined the role of lysophosphatidylserine (LysoPS) as an inflammatory lipid mediator. Thus, we have studied the possibility that intracellular calcium concentration, phosphoinositide hydrolysis, and the subsequent histamine release in mast cells is due to the action of lysophosphatidylserine. Lysophosphatidylserine-stimulated release of histamine was significantly higher than release by other lysophospholipids. The contribution of increased phospholipase C activity and the intracellular Ca²⁺ influx were therefore examined. LysoPS increased mast cell calcium concentration, and this increment was associated with phospholipase C activation and release of inositol phosphates. The increase in intracellular calucium and histamine degranulation induced by LysoPS were inhibited by apomorphine. Pretreatment of mast cells with pertussis toxin decreased the secretagogic effect of LysoPS and compound 48/80 without modifying the effect of the ionophore A23187. These results suggest that pertussis toxinsensitive G-protein might be involved in the mast cell degranulation produced by lysophosphatidylserine and allow the increase in phospholipase C activity, thus enhancing intracellular calcium concentration, which then induces exocytosis of histamine. © 1995 Wiley-Liss Inc.     

Inhibitory effect of diethylstilbestrol on histamine release by rat mast cells and its relation to the cellular ATP content

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.     

Carnosine attenuates mast cell degranulation and histamine release induced by oxygen-glucose deprivation

Carnosine (beta-alanyl-histidine) is a naturally occurring dipeptide that has been characterized as a putative hydrophilic antioxidant. The protective function of carnosine has been demonstrated in neuronal cells under ischemic injury. The purpose of this study was to investigate the effects of carnosine on oxygen-glucose deprivation (OGD)-induced degranulation and histamine release from mast cells. Cultured mast cells were exposed to OGD for 4 h, and then the degranulation was observed immediately by microscopy. Histamine release was analyzed by high-performance liquid chromatography (HPLC). OGD caused degranulation of mast cells, and increased histamine and lactate dehydrogenase (LDH) release. Carnosine (at a concentration of 5 mM) alone did not produce any appreciable effect on degranulation, histamine, and LDH release from mast cells under normal condition, but significantly inhibited the degranulation, histamine, and LDH release of mast cells induced by OGD. These results indicate that carnosine can protect mast cells from degranulation and histamine release and it may be an endogenous mast cell stabilizer in the pathological processes induced by ischemia.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using H NMR

¹H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events folowing exocytosis, activate glycolysis.     

Does intracellular histamine mediate mast cell histamine release?

Previously, we demonstrated that through binding a novel intracellular receptor of microM affinity (HIC), histamine mediates, and the HIC antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine. HCl (DPPE) inhibits, platelet aggregation and serotonin granule secretion; the latter response is dependent upon the same processes that mediate histamine release from mast cell granules. We now show that, as for platelet serotonin release, DPPE blocks concanavalin A-stimulated mast cell histamine release with a potency (IC50 = 30 microM) greater than the H1-antagonist, pyrilamine (IC50 = 150 microM) or the H2-antagonist cimetidine (IC50 = 5 mM), correlating with rank order of potency to inhibit 3H-histamine binding in rat brain membranes and liver microsomes. We postulate that histamine release from mast cells is mediated at HIC by second messenger intracellular histamine. However, unlike platelets, mast cells do not appear to rely on newly synthesized histamine. Rather, as for calcium, histamine may be mobilized from bound stores to mediate histamine secretion.