Rat Mast Cell Granules Research Articles

Signal transduction pathways in mast cell granule-mediated endothelial cell activation.

BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen-activated protein kinase activation and nuclear factor-kappaB translocation.

INTRAVESICAL SODIUM HYALURONATE INHIBITS THE RAT URINARY MAST CELL MEDIATOR INCREASE TRIGGERED BY ACUTE IMMOBILIZATION STRESS

Mast cell activation and stress have been suggested as factors in the pathogenesis of interstitial cystitis, a painful disorder of the bladder that is diagnosed more frequently in women and characterized by increased urgency and frequency with absent infection. Intravesical sodium hyaluronate has been used to treat interstitial cystitis due to its possible replenishment of bladder glycosaminoglycans. We investigated the effect of sodium hyaluronate on the activation of bladder mast cell and release of proinflammatory mediators in the urine induced by acute immobilization stress in rats. Using anesthesia a catheter was inserted in the bladder of 170 gm. female Sprague-Dawley rats. After emptying post-void residual urine a solution of normal saline, 0.08% or 0.4% sodium hyaluronate was introduced for 30 minutes. Each rat was allowed to recover from anesthesia and stressed for 30 minutes by confining it in a clear acrylic plastic immobilizer, while urine was continuously collected. Urinary histamine, rat mast cell protease-I and interleukin (IL)-6 were then determined. At the end of the experiments each rat was sacrificed by CO2 asphyxiation, and the bladder was removed and fixed with 4% paraformaldehyde. Frozen sections were stained with acidified toluidine blue, and the mast cell number and degree of activation were determined by granule extrusion and reduced cellular staining. Mean bladder mast cell activation plus or minus standard deviation in 6 control rats was 30.4% +/- 3.7% but it increased to 76.2% +/- 6.1% in 6 stressed animals (p = 0.0001). Intravesical administration of 0.4% sodium hyaluronate for 30 minutes in 6 rats before stress reduced mean bladder mast cell activation by 69.7% to 23.1% +/- 6.1% compared with stressed controls (p = 0.0003). However, compared to itself before stress there was no significant difference, indicating complete inhibition in 6 rats. Intravesical 0.08% sodium hyaluronate had a weaker inhibitory effect in 6 rats, decreasing mean degranulation by 22.5% to 59.1% +/- 7.6% (p = 0.02). In 6 rats stress increased the total mean amount of urinary rat mast cell protease-I by 271% from 0.14 +/- 0.09 to 0.52 +/- 0.17 ng. (p = 0.008). Pretreatment with 0.4% sodium hyaluronate reduced mean rat mast cell protease-I 80.8% compared with stressed controls (p = 0.008) and prevented any increase in response to stress in the same group of 8 rats with a mean pre-stress and post-stress level of 0.09 +/- 0.04 and 0.1 +/- 0.04 ng., respectively (p = 0.8). Acute stress increased mean urinary histamine in 6 rats 40.2% from 137.3 +/- 29.7 before to 193.7 +/- 7.6 ng./ml. after stress (p = 0.004). Pretreatment with 0.4% sodium hyaluronate reduced mean histamine 7.1% compared with stressed controls but completely prevented any increase in the same group of 8 rats, in which it was 174.5 +/- 23.1 before and remained 179.4 +/- 9.9 ng./ml. after stress (p = 0.75). Acute stress in 7 rats also increased the mean amount of IL-6 released in the urine by 31.5% from 775.9 +/- 69.2 to 1,021.1 +/- 93.3 pg./ml. (p = 0.007). Pretreatment with 0.4% sodium hyaluronate in 9 rats reduced mean IL-6 17% compared with stressed controls but again prevented any increase from baseline, since the value was 898.6 +/- 299.3 before and 824.4 +/- 196.4 pg./ml. after stress (p = 0.03). Immobilization stress induces bladder mast cell activation and the secretion of proinflammatory mediators, which are inhibited by sodium hyaluronate. Intravesical sodium hyaluronate may be a useful therapeutic option for interstitial cystitis, especially in patients with bladder mastocytosis who have symptom exacerbation with stress.

Roles of Superoxide Dismutase in Rat Mast Cell Granules

Background: It has been suggested that in the granules of rat mast cells there is some kind of superoxide dismutase (SOD), but details of this SOD in mast cells remain unclear. In the present study, we studied the mode of existence of SOD in mast cells and its releasing mechanism from the granules. In addition, we discussed the physiological role of SOD in allergic events. Methods: Purified rat mast cells were disrupted with a sonic disrupter and granules (sample I) were separated from supernatant (sample II) by centrifugation. The granules were treated with 1 mM Ca²⁺, and the supernatant (sample III) was separated from the pellet (sample IV). Sample III was applied to a heparin column and the eluate was used as sample V. SOD activity was measured in these samples. Results: SOD existed in mast cell granules as a heparin-binding and an inactive form. However, when granules were released and exposed to high Ca²⁺ concentration, SOD was discharged from heparin and shifted to the active form. The expression of macrophage inflammatory protein-1 alpha mRNA was enhanced when hydrogen peroxide (H₂O₂) or sample III with the xanthine-xanthine oxidase system were added to the culture media. Conclusions: These findings suggest that in stimulated rat mast cells, the released SOD may transform the generated superoxide anion into H₂O₂, and the generated H₂O₂ may enhance the expression of chemokine mRNA in the mast cells.

Mast Cell Granule Heparin Proteoglycan Induces Lacunae in Confluent Endothelial Cell Monolayers

The addition of rat mast cell granules to confluent bovine pulmonary artery endothelial cell monolayers resulted in the formation of numerous lacunae in the cultures. Several lines of evidence identified heparin proteoglycan as the component of the granule matrix responsible for the effect: presence of the activity in the proteoglycan fraction after chromatography of granule extracts, inhibition of granule activity by digestion with heparinase I, the failure of proteolysis of the proteoglycan fraction with proteinase K to significantly diminish its activity, and the failure of chymase and carboxypeptidase inhibitors to inhibit granule activity. The onset of hole formation was delayed for several hours after granule addition to the culture, and maximal hole formation occurred between 8 and 16 hours and was sustained as long as 24 hours. The lacunae formed by the separation of motile endothelial cells within the monolayer and was not attributable to cell contractile activity or cell loss. Time-lapse video recording showed that the holes were dynamic, individual holes expanding and regressing over a period of hours. Formation of lacunae occurred on gelatin and fibronectin surfaces alike. The presence of active chymase in the granules prevented the action of the proteoglycan. Heparin glycosaminoglycan as distinct from the proteoglycan did not similarly affect the endothelial monolayers but did block the action of granules added subsequently, indicating the likelihood of a heparin-reactive receptor or binding site.

Depletion of pre beta 1LpA1 and LpA4 particles by mast cell chymase reduces cholesterol efflux from macrophage foam cells induced by plasma.

Exposure of the LpA1-containing particles present in HDL3 and plasma to a minimal degree of proteolysis by the neutral protease chymase from exocytosed rat mast cell granules (granule remnants) leads to a reduction in the high-affinity component of cholesterol efflux from macrophage foam cells. In this study, we demonstrate for the first time, a role for mast cell chymase in the depletion of the lipid-poor minor components of HDL that are specifically involved in reverse cholesterol transport as initial acceptors of cellular cholesterol. Thus, addition of proteolytically active granule remnants or human skin chymase to cholesterol-loaded macrophages of mouse or human origin incubated with human apoA1, ie, a system in which prebeta1LpA1 is generated, resulted in a sharp reduction in the high-affinity cholesterol efflux promoted by apoA1. As determined by nondenaturing 2-dimensional polyacrylamide gradient gel electrophoresis, the granule remnants effectively depleted the prebeta1LpA1, but not the alphaLpA1, in HDL3 and in plasma during incubation at 37 degrees C for <1 hour. Incubation of plasma with granule remnants for 1 hour also led to near disappearance of the LpA4-1 and LpA4-2 particles, but did not affect the distribution of the apoA2-containing lipoproteins present in the plasma. We conclude that the reduced ability of granule remnant-treated HDL3 and granule remnant-treated plasma to induce cholesterol efflux from macrophage foam cells is caused by selective depletion by mast cell chymase of quantitatively minor A1- and A4-containing subpopulations of HDL. Because these particles, ie, prebeta1LpA1 and LpA4, are efficient acceptors of cholesterol from cell surfaces, their depletion by mast cells may block the initiation of reverse cholesterol transport in vivo and thereby favor foam cell formation in the arterial intima, the site of atherogenesis.

Chymase in exocytosed rat mast cell granules effectively proteolyzes apolipoprotein AI-containing lipoproteins, so reducing the cholesterol efflux-inducing ability of serum and aortic intimal fluid.

Degranulated mast cells are present in human fatty streaks. Chymase in granules released from degranulated rat serosal mast cells, i.e., in granule remnants, proteolyzes human high density lipoprotein3 (HDL3), and so reduces its ability to induce cholesterol efflux from macrophage foam cells in vitro. In this study we found that remnant chymase, by proteolyzing human serum and human aortic intimal fluid, prevents these two physiologic fluids from effectively inducing cholesterol efflux from cultured macrophage foam cells. Inhibition was strongest when remnants were added to apolipoprotein AI (apoAI)-containing lipoproteins; the remnants had no effect on the weaker efflux produced by apoAI-deficient serum. Western blot analysis showed that granule remnants degrade apoAI in serum and in internal fluid. When released from remnants, chymase lost its ability to proteolyze HDL3 in the presence of serum. Thus, remnant chymase (but not isolated chymase) was able to resist the natural protease inhibitors present in serum and in intimal fluid. The results imply participation of exocytosed mast cell granules in foam cell formation in atherogenesis.

An histochemical approach to characterization of anionic constituents in mast cell secretory granules.

We used cationized colloidal gold in order to investigate the distribution of anionic sites in different secretory granules of rat and mouse mast cells. The localization of the anionic sites was performed by post-embedding labeling of thin sections of rat peritoneal cells or mouse skin tissue, fixed in Karnovsky's fixative and OsO4, and embedded in Araldite or LR white, respectively. In all cases anionic sites were demonstrated with a high density variation depending on cell type. In all mast cell secretory granules we have observed the highest density (ca. 500-900 gold particles/microns2), while in other peritoneal cell granules it was about 10 times less (ca. 40-80 gold particles/microns2). Pretreatment of the LR white sections with heparinase I and III resulted in a reduction of 97% and 72%, respectively, in the binding of the gold particles to the granules, indicating that the majority of the gold binding reactivity is due to heparin. Correlation of section profile area with labeling density revealed that the smaller granules were significantly more labeled when compared to the larger profiles. On the basis of these observations it seems that a post-translational change (mainly sulfation of heparin) of secretory content influences the granule anionic charge and thus may affect the intragranule buffer capacity.

Cytosolic calcium facilitates release of secretory products after exocytotic vesicle fusion

We monitored single vesicle exocytosis by simultaneous measurements of cell membrane capacitance as an indicator of fusion and amperometric detection of serotonin release. We show here that vesicle-plasma membrane fusion in rat mast cell granules is followed by a variable, exponentially distributed, delay before bulk release. This delay reflects the time required for the expansion of the exocytotic fusion pore, lasting, on average, 231 ms in resting cytosolic calcium, [Ca 2+] i (50 nM). In the presence of [Ca 2+] i in the low micromollar range, the lag between fusion and release was reduced to 123 ms. The characteristics of the amperometric signals were unchanged by [Ca 2+] i. These results show a novel site of regulation in the exocytotic process, the fusion pore, which may represent a different mechanism facilitating transmitter release.

Ultrastructural immunogold localization of tumor necrosis factor-α to the cytoplasmic granules of rat peritoneal mast cells with rapid microwave fixation

Tumor necrosis factor-α (TNF-α) is a multifunctional, proinflammatory cytokine, which can be produced by mast cells and several other cell types. We used a newly developed microwave energy–assisted aldehyde fixation method to prepare purified rat peritoneal mast cells for the postembedding immunogold ultrastructural localization of TNF-α. These fixation methods were superior to chemical fixation alone in preserving both the ultrastructural morphology and immunoreactive TNF-α in rat mast cells. The percent of TNF-α–positive mast cells in samples prepared with microwave-assisted fixation in low (84%) and standard (81%) glutaraldehyde concentrations exceeded that for low (56%) and standard (15%) glutaraldehyde concentrations without the assistance of microwave energy. TNF-α was identified in the large storage granules of rat mast cells. The percent of positive granules in microwave-assisted standard (44%) and low (40%) glutaraldehyde samples was considerably higher than the percent of positive granules in standard (5%) and low (10%) glutaraldehyde-fixed samples without microwave assistance. This location of TNF-α in rat peritoneal mast cells suggests that this cytokine can use the regulated secretory route(s) for release from appropriately stimulated rat mast cells into the microenvironment. (J ALLERGY CLIN IMMUNOL 1994;94:531-6.)

Cytochemical Localization of Hyaluronan in Rat and Human Skin Mast Cell Granules

Rat and human skin were processed either by osmium tetroxide/microwave fixation followed by embedding in epoxy resin or by glutaraldehyde/microwave fixation and low-temperature embedding in Lowicryl K4M. Hyaluronan-binding proteins and link proteins (LP) were isolated from bovine nasal cartilage, coupled to 15-20-nm gold particles and employed as markers in a one-step post-embedding procedure for identifying hyaluronan (hyaluronic acid) at the ultrastructural level. Mast cell granules of both species were labeled. The specificity of the hyaluronan-binding probes was demonstrated by treatment of sections with testicular hyaluronidase, Streptomyces hyaluronidase, and chondroitinase ABC, and pre-incubation of probes with hyaluronan oligosaccharides. The results suggest that mast cell granules are a rich source of hyaluronan; this finding may account for the striking concurrence of hyaluronan accumulation with a mastocytotic condition in many tissues undergoing pathologic changes.

Electron microscopic cytochemical studies on sulfated glycosaminoglycans in rat mast cell granules.

Electron microscopic cytochemical studies have been made on sulfated glycosaminoglycans in mast cell granules from the rat peritoneal cavity by means of combined alcian blue (AB) pH 1.0-phosphotungstic acid (PTA) staining and enzyme (heparinase or chondroitinase ABC) digestion or chemical modification (nitrous acid) procedures. The results obtained revealed that the mast cell populations from the rat peritoneal cavity are grouped into at least three cell types on the basis of the molecular species of sulfated glycosaminoglycans contained in the cytoplasmic granules, the first cell type occupying the majority of the cell population with cytoplasmic granules containing primarily heparin, the second cell type being of an exceedingly small proportion with cytoplasmic granules containing primarily isomeric chondroitin sulfate (D and/or E), and the third cell type (intermediates between the first and second types) constituting a relatively small proportion with cytoplasmic granules containing two kinds of cytoplasmic granules containing heparin and isomeric chondroitin sulfate (D and/or E) respectively. In addition, unique substructures of cytoplasmic granules containing different sulfated glycosaminoglycans were disclosed such as tubular or thin thread-like elements, interstitial vesicles and surface spine-like figures.The present results are taken to represent the first electron microscopic cytochemical evidence that mast cell populations are heterogeneous in terms of molecular species of sulfated glycosaminoglycans contained in the cytoplasmic granules in mammals.

Supernormal Histamine Release and Normal Cytotoxic Activity of Beige (Chédiak-Higashi Syndrome) Rat Mast Cells with Giant Granules

The beige rat is an animal model of the Chédiak-Higashi syndrome. Since mast cells can be easily purified from the peritoneal cavity of rats, we investigated the function of beige rat mast cells with giant granules by using quantitative methods. Beige and normal rat mast cells were sensitized with anti-dinitrophenol (DNP) IgE antibodies and stimulated by DNP conjugated with human serum albumin. The proportion of histamine released to total histamine was significantly greater in beige rat mast cells than in normal rat mast cells. Since the supernormal histamine release of beige rat mast cells was observed after treatment with 12-O-tetradecanoylphorbol 13-acetate, calcium ionophore A23187, substance P or compound 48/80, it appeared to be attributable to the enlargement in granules in beige rat mast cells. Spontaneous cytotoxic activity of mast cells was assayed by incubating purified mast cells with 51Cr-labelled WEHI-164 cells. Both beige and normal rat mast cells showed significant cytotoxic activity, but no significant difference was detectable between beige and normal rat mast cells. Even after IgE-mediated stimulation, no significant difference in cytotoxic activity was detectable between beige and normal rat mast cells either. Giant granules of beige rat mast cells did not appear to influence the cytotoxic activity of mast cells.

Magnesium and sulfur in mast cell and basophil granules of lower vertebrates in relation with histamine

Mast cell and basophil granules of the vertebrate contain heparin or related sulfated proteoglycans. Histamine is also present in mammalian mast cells and basophils. However, no histamine is detected in mast cell granules of the amphibian or fish, while it is shown in those of reptiles and birds A quantitative x-ray microanalysis of mast cell granules of fresh frozen dried ultrathin sections of the tongue of Wistar rats and tree frogs disclosed high concentrations of sulfur in rat mast cell granules and those of sulfur and magnesium in the tree frog granules. Their concentrations in tree frog mast cell granules were closely correlated (r=0.94).Fresh frozen dried ultrathin sections and fresh air-dried prints of the tree frog tongue and spleen and young red-eared turtle (ca. 6 g) spleen and heart blood were examined by a quantitative energy-dispersive x-ray microanalysis (X-650, Kevex-7000) for the element constituents of the granules of mast cells and basophils. The specimens were observed by transmission electron microscopy (TEM) (80-200 kV) and followed by scanning transmission electron microscopy (STEM) under an analytical electron microscope (X-650) at an acceleration voltage of 40 kV and a specimen current of 0.2 nA. A spot analysis was performed in a STEM mode for 100 s at a specimen current of 2 nA on the mast cell and basophil granules and other areas of the cells. Histamine was examined by the o-phthalaldehyde method.

ラット肥満細胞か粒におけるＨＮＫ‐１エピトープの存在 （続報） Ｍ６７６４抗体を用いての検討

Rat mast cell granules and the plasma membrane fractions were obtained by the homogenization of highly purified rat mast cells and their isolation in a Percoll or sucrose gradient respectively. Immuno-stainings of non-stimulated rat mast cells, compound 48/80 or calcium ionophore A 23187-stimulated rat mast cells, the granules and the plasma membrane fractions were performed with a monoclonal antibody, M 6764, which recognizes HNK-1 epitope. HNK-1 epitope was present in rat mast cells and the granules, but not in the plasma membrane fractions. The granules fixed with glutaraldehyde showed ring-like figures by immunostaining with M 6764. Rat mast cells and the granules treated with chloroform-methanol after fixation were also densely stained with M 6764. Western blot analysis of rat mas tcells and the granule homogenates with M 6764 antibody showed broad protein bands ranging from 100 to 250 KDa.These results showed the presence of HNK-1 epitope in rat mast cell granules. This HNK-1 epitope could be a glycoprotein.

In vivo internalization of rat mast cell granules by intraperitoneal transplanted Yoshida tumor cells and resident macrophages

In vivo internalization of rat mast cell granules by intraperitoneal transplanted Yoshida tumor cells and resident macrophages

Mapping of the rat mast cell granule proteinases RMCPI and II by enzyme‐linked immunosorbent assay and paired immunofluorescence

The distribution of the rat mast cell granule proteinases, rat mast cell proteinase I and II (RMCPI and II respectively) has been determined in rat tissues with the aid of highly sensitive and specific enzyme-linked immunosorbent assays (ELISA) and paired immunofluorescence. The major source of RMCPII is the gastrointestinal tract, although low concentrations were also detected in non-mucosal sites including thymus, mesenteric lymph nodes, liver, bone marrow, heart, kidney and spleen. Cellular localization by paired immunofluorescence showed that most cells contained either RMCPI or RMCPII, although a minor subpopulation in which individual cells contained both proteinases was also identified in a few tissues. RMCPII-containing cells predominated at mucosal surfaces but were also found in non-mucosal tissues. Individual cells expressing both RMCPI and II were present in lung, liver mesenteric lymph node and submucosa of stomach and were occasionally represented amongst serosal cells from the peritoneal cavity. Connective tissue mast cells of skin and tongue were identified as major sources of RMCPI, although this proteinase was widely distributed in all tissues examined. The present study demonstrates the heterogeneity of mast cell proteinase phenotypes in the rat and emphasises the difficulties in determining mast cell subtypes on tissue location alone.

Polyamines stimulate the phosphorylation of phosphatidylinositol in rat mast cell granules

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of polyamines, spermine and spermidine, to the granules caused an increase of DPI synthesis, which can be catalyzed by PI kinase. This effect of polyamines in the DPI synthesis was in a dose-dependent manner and maximal effects were observed at 1 mM spermine and 10 mM spermidine, respectively.

ラット肥満細胞か粒におけるＨＮＫ‐１エピトープの存在 抗ｍｙｅｌｉｎ‐ａｓｓｏｃｉａｔｅｄ ｇｌｙｃｏｐｒｏｔｅｉｎ（ＭＡＧ）抗体を用いての検討

Rat mast cell granules and plasma membrane fractions were obtained from highly purified intact cells by homogenization followed by Percoll and sucrose density gradient centrifugation respectively and immunostained with two types of mouse monoclonal anti-MAG antibodies: IgG antibody recognizing a polypeptide moiety of intact MAG (IgG-P) and IgM antibody recognizing a carbohydrate moiety (IgM-C) . Both intact cells and granule fraction, but not plasma membrane fraction were well stained with IgM-C.Granule fraction fixed with glutaraldehyde showed a ring-like pattern by immunostaining with IgM-C. On the contrary, neither intact cells nor granules and plasma membrane fractions were reacted with IgG-P. Both intact cells and granule fraction treated with chloroform-methanol were also densely stained with IgM-C., indicating that IgM-C positive epitope is present in the granule fraction of the mast cell and shares with a carbohydrate moiety of MAG.

Rat Mast Cell Granules Reacting with a Mouse Monoclonal Antibody M6764 which Recognizes the Same Epitope of a Mouse Monoclonal Antibody HNK-1

Rat mast cell granules and plasma membrane fractions were obtained by homogenization of highly purified rat mast cells and isolation in a Percoll gradient and a sucrose gradient, respectively. Immunostaining of rat mast cells, granules and plasma membrane fractions was performed with mouse monoclonal antibody M6764 which was produced against the crude membrane fractions of the neural tubes. Rat mast cells and granules were immunostained with the monoclonal antibody, but not the plasma membrane fractions. The granules fixed with glutaraldehyde-paraformaldehyde showed ring-like forms. Chloroform-methanol treatment did not effect the staining of rat mast cells and granules with the monoclonal antibody. Western blotting analysis of rat mast cells and granules with the monoclonal antibody showed broad protein bands ranging from 100 to 250 kD.

Inhibitory effect of adenine nucleotides and anti-allergic drugs on phosphorylation of phosphatidylinositol in rat mast cell granules.

Rat mast cell granules were obtained by sonication of highly purified rat mast cells and isolated in a Percoll gradient. Phosphorylation of endogenous phosphatidylinositol in rat mast cell granules, which is catalyzed by phosphatidylinositol kinase in the granules, was assayed by measuring the incorporation of 32P from [gamma 32P]ATP into phosphatidylinositol 4-phosphate. Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. Phosphatidylinositol 4-phosphate areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The phosphorylation reaction was inhibited by 50-500 microM adenosine, ADP and 500 microM AMP in a concentration-dependent manner. Among several anti-allergic drugs investigated. 100-1000 microM theophylline and 10-100 microM azelastine inhibited the phosphorylation reaction, but disodium cromoglycate and ketotifen had little effect.