by Wan Sze Ping. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 172-185). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Publications --- p.vi / Abbreviations --- p.vii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Historical Background --- p.2 / Chapter 1.2. --- Origin and heterogeneity of mast cells --- p.2 / Chapter 1.3. --- Mast cell mediators --- p.4 / Chapter 1.3.1. --- Preformed mediators --- p.5 / Chapter 1.3.2. --- Newly synthesised mediators --- p.6 / Chapter 1.4. --- Mast cell activation --- p.8 / Chapter 1.4.1. --- Antigenic/Immunological pathway --- p.8 / Chapter 1.4.1.1. --- Aggregation of IgE Receptors (FcsRI) --- p.9 / Chapter 1.4.1.2. --- Biochemical events following FcsRI aggregation --- p.10 / Chapter 1.4.1.3. --- Phospholipase C activation --- p.11 / Chapter 1.4.1.4. --- Phospholipase A2 activation --- p.12 / Chapter 1.4.1.5. --- Adenylate cyclase activation --- p.14 / Chapter 1.4.1.6. --- Role of calcium --- p.15 / Chapter 1.5. --- Roles of chloride channels in mast cell activation --- p.18 / Chapter 1.6. --- Aims of the study --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.23 / Chapter 2.1. --- Materials --- p.24 / Chapter 2.1.1. --- Secretagogues --- p.24 / Chapter 2.1.2. --- Anti-allergic compounds --- p.24 / Chapter 2.1.3. --- Chloride channel blockers --- p.24 / Chapter 2.1.4. --- Materials for buffers --- p.25 / Chapter 2.1.5. --- Materials for rat sensitisation --- p.25 / Chapter 2.1.6. --- Materials for histamine assay --- p.26 / Chapter 2.1.7. --- Materials for calcium measurement --- p.26 / Chapter 2.1.8. --- Miscellaneous --- p.27 / Chapter 2.2. --- Buffers --- p.28 / Chapter 2.3. --- Stock solutions of drugs --- p.30 / Chapter 2.4. --- Source of mast cells --- p.31 / Chapter 2.4.1. --- Animals --- p.31 / Chapter 2.4.2. --- Sensitization of animals --- p.31 / Chapter 2.4.3. --- Isolation of rat peritoneal mast cells --- p.32 / Chapter 2.4.4. --- Purification of rat peritoneal mast cells --- p.32 / Chapter 2.4.5. --- Cell counting --- p.33 / Chapter 2.5. --- General protocol for histamine release --- p.35 / Chapter 2.6. --- Histamine assay --- p.35 / Chapter 2.7. --- Protocol for fura-2 fluorescence measurement of intracellular calcium --- p.39 / Chapter 2.7.1. --- Fura-2 cell loading --- p.39 / Chapter 2.7.2. --- Fura-2 fluorescence measurement by fluorescence microscope --- p.39 / Chapter 2.7.3. --- Calculation of intracellular calcium --- p.41 / Chapter 2.8. --- Protocol for 45Ca2+ influx measurement --- p.44 / Chapter 2.9. --- Measurement of 43Ca2+ influx --- p.45 / Chapter 2.10. --- Statistical analysis --- p.45 / Chapter Chapter 3 --- Effects of chloride depletion on mast cell activation --- p.46 / Chapter 3.1. --- Introduction --- p.47 / Chapter 3.1.1. --- Mechanism of non-immunological stimuli --- p.48 / Chapter 3.2. --- Materials and methods --- p.50 / Chapter 3.3. --- Results --- p.51 / Chapter 3.3.1. --- Effects of anti-IgE induced histamine release in chloride free buffers --- p.51 / Chapter 3.3.2. --- Effects of compound 48/80 induced histamine releasein chloride free buffers --- p.52 / Chapter 3.3.3. --- Effects of calcium ionophore A23187 induced histamine release in chloride free buffers --- p.53 / Chapter 3.3.4. --- Effects of ionomycin induced histamine release in chloride free buffers --- p.53 / Chapter 3.3.5. --- Effects of thapsigargin induced histamine releasein chloride free buffers --- p.53 / Chapter 3.3.6. --- Effects of Triton-X 100 induced histamine releasein chloride free buffers --- p.54 / Chapter 3.3.7. --- Effects of anti-IgE on the free intracellular calcium concentration of rat peritoneal mast cells in chloride free buffers --- p.54 / Chapter 3.3.8. --- Effects of compound 48/80 on the free intracellular calcium concentration of rat peritoneal mast cellsin chloride free buffers --- p.56 / Chapter 3.3.9. --- Effects of ionomycin on the free intracellular calcium concentration of rat peritoneal mast cellsin chloride free buffers --- p.58 / Chapter 3.3.10. --- Effects of thapsigargin on the free intracellular calcium concentration of rat peritoneal mast cellsin chloride free buffers --- p.59 / Chapter 3.4. --- Discussion --- p.86 / Chapter 3.5. --- Conclusion --- p.96 / Chapter Chapter 4 --- "DSCG and Chloride channel blockers (NPPB, DIDS, SITS, DPC, IAA-94 & Flufenamic acid)" --- p.97 / Chapter 4.1. --- Introduction --- p.98 / Chapter 4.1.1. --- Role of chloride channels --- p.98 / Chapter 4.1.1.1. --- Chloride channel classification --- p.99 / Chapter 4.1.1.2. --- Chloride channel blockers --- p.101 / Chapter 4.1.2. --- Aims of study --- p.106 / Chapter 4.2. --- Materials and methods --- p.107 / Chapter 4.3. --- Results --- p.108 / Chapter 4.3.1. --- Effects of DSCG and the test blockers on immunologically induced histamine release from rat peritoneal mast cells --- p.108 / Chapter 4.3.2. --- Effects of DSCG and the test blockers on compound48/80 induced histamine release from rat peritoneal mast cells --- p.110 / Chapter 4.3.3. --- Effects of DSCG and the test blockers on ionophore A23187 induced histamine release from rat peritoneal mast cells --- p.112 / Chapter 4.3.4. --- Effects of DSCG and the test blockers on thapsigargin induced histamine release from rat peritoneal mast cells --- p.113 / Chapter 4.3.5. --- Effects of the test blockers on Triton-X 100 induced histamine release from rat peritoneal mast cells --- p.114 / Chapter 4.3.6. --- Effects of DSCG and the test blockers on immunologically and non-immunologically induced 45Ca2+ uptake --- p.115 / Chapter 4.4. --- Discussion --- p.160 / Chapter 4.5. --- Conclusion --- p.167 / References --- p.172

by Catherine Mary (Kay) Roche. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 195-228). / Abstract / Acknowledgements / Publications / Abbreviations / Chapter Chapter 1 --- Introduction --- p.1-26 / Chapter 1.1 --- Historical Background / General information --- p.2 / Chapter 1.2 --- Mast Cell Mediators --- p.3 / Chapter 1.2.1 --- Pre-formed Mediators --- p.4 / Chapter 1.2.2 --- Newly Synthesised Lipid Mediators --- p.5 / Chapter 1.3 --- Mast Cell Activation --- p.7 / Chapter 1.3.1 --- The Antigenic Pathway of Mast Cell Activation --- p.7 / Chapter 1.3.2 --- The Non-Antigenic Pathway of Mast Cell Activation --- p.15 / Chapter 1.4 --- Phospholipase enzymes --- p.17 / Chapter 1.4.1 --- Phospholipase A2 --- p.18 / Chapter 1.4.2 --- The type I PLA2 --- p.20 / Chapter 1.4.3 --- The type II PLA2 --- p.22 / Chapter 1.5 --- Aims of the Study --- p.26 / Chapter Chapter 2 --- Materials and Materials --- p.27-49 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.2 --- Buffers --- p.30 / Chapter 2.3 --- Secretory stimuli --- p.31 / Chapter 2.4 --- Source of mast cells --- p.32 / Chapter 2.4.1 --- Sensitization of animals --- p.33 / Chapter 2.4.2 --- Isolation of rat peritoneal mast cells --- p.33 / Chapter 2.4.3 --- Purification of rat peritoneal mast cells --- p.34 / Chapter 2.4.4 --- Isolation of human lung mast cells --- p.34 / Chapter 2.4.5 --- Partial purification of human lung mast cells --- p.35 / Chapter 2.4.6 --- Isolation of guinea pig lung mast cells --- p.36 / Chapter 2.5 --- General procedure for studying histamine release from isolated mast cells --- p.36 / Chapter 2.5.1 --- Procedure for investigating the effect of sPLA2 on the spontaneous histamine release from isolated mast cells --- p.37 / Chapter 2.5.2 --- Procedure for investigating the effect of heat treated sPLA2-I on histamine release from isolated RPMC --- p.37 / Chapter 2.5.3 --- Procedure for investigating the effect of antimycin A on histamine release induced by sPLA2-I on isolated RPMC --- p.38 / Chapter 2.5.4 --- Procedure for investigating the effects of extracellular calcium on histamine release induced by sPLA2-I on isolated RPMC --- p.38 / Chapter 2.5.5 --- Procedure for investigating the effects of the sPLA2 enzymes on activated mast cells --- p.39 / Chapter 2.5.6 --- Procedure for investigating the effect of preincubation time on sPLA2 induced histamine release from immunologically activated RPMC --- p.39 / Chapter 2.5.7 --- Procedure for investigating the effects of the PLA2 inhibitors --- p.39 / Chapter 2.5.8 --- Procedure for investigating the effects of the cyclo- oxygenase and lipoxygenase inhibitors on sPLA2 --- p.40 / Chapter 2.6 --- Histamine Assay --- p.41 / Chapter 2.7 --- Measurement of prostaglandin D2 --- p.42 / Chapter 2.7.1 --- Prostaglandin D2 methoxime enzyme immunoassay kit --- p.42 / Chapter 2.7.2 --- The Prostaglandin D2 assay --- p.43 / Chapter 2.7.3 --- PGD2 release from purified rat peritoneal mast cells --- p.43 / Chapter 2.7.4 --- Derivatization of PGD2-MOX --- p.44 / Chapter 2.7.5 --- PGD2 assay procedure --- p.44 / Chapter 2.8 --- Measurement of intracellular calcium --- p.47 / Chapter 2.8.1 --- Fluorescent calcium indicator fura-2 --- p.47 / Chapter 2.8.2 --- Cell loading with the calcium indicator --- p.47 / Chapter 2.8.3 --- Measurement of fura-2 fluorescence --- p.47 / Chapter 2.9 --- Cell counts --- p.49 / Chapter 2.10 --- Data analysis --- p.49 / Chapter Chapter 3 --- Effects of sPLA2 on rat peritoneal mast cells --- p.50-157 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Methodology --- p.52 / Chapter Part 1 --- Effects of sPLA2 on unstimulated rat peritoneal mast cells --- p.52-86 / Chapter 3.3 --- Results --- p.52 / Chapter 3.3.1 --- Effects of sPLA2-I on the spontaneous histamine release from RPMC --- p.52 / Chapter 3.3.2 --- Time course of sPLA2-I induced histamine release --- p.53 / Chapter 3.3.3 --- Effect of heat treated sPLA2-I on histamine release --- p.53 / Chapter 3.3.4 --- Effects of antimycin A on sPLA2-I induced histamine release --- p.54 / Chapter 3.3.5 --- Effects of extracellular calcium on sPLA2-I induced histamine release --- p.54 / Chapter 3.3.6 --- Effects of PLA2 inhibitors on sPLA2-I induced histamine release --- p.54 / Chapter 3.3.7 --- Effects of sPLA2-I on PGD2 production --- p.56 / Chapter 3.3.8 --- Effects of sPLA2-II on the spontaneous histamine release from RPMC --- p.57 / Chapter 3.3.9 --- Effects of PLA2 inhibitors on sPLA2-II induced histamine release from purified RPMC --- p.57 / Chapter 3.3.10 --- Effects of sPLA2-II on PGD2 production --- p.57 / Chapter 3.4 --- Discussion --- p.75 / Chapter 3.4.1 --- Effects of sPLA2-I on RPMC --- p.75 / Chapter 3.4.2 --- Effects of sPLA2-II on RPMC --- p.83 / Chapter Part 2 --- Effects of sPLA2 on stimulated rat peritoneal mast cells --- p.87-130 / Chapter 3.5 --- Results --- p.87 / Chapter 3.5.1 --- Effects of sPLA2-I on histamine release from immunologically activated RPMC --- p.87 / Chapter 3.5.2 --- Effects of preincubation time on sPLA2-I enhanced histamine release from immunologically activated RPMC --- p.88 / Chapter 3.5.3 --- Effects of p-BPB on sPLA2-I enhanced histamine release from immunologically activated RPMC --- p.88 / Chapter 3.5.4 --- Effects of 12-epi-scalaradial on sPLA2-I enhanced histamine release from immunologically activated RPMC --- p.89 / Chapter 3.5.5 --- Effects of MAFP on sPLA2-I enhanced histamine release from immunologically activated RPMC --- p.89 / Chapter 3.5.6 --- Effects of sPLA2-I on PGD2 production from immunologically activated RPMC --- p.90 / Chapter 3.5.7 --- Effects of flurbiprofen and zileuton on sPLA2-I induced histamine release and enhanced histamine release from RPMC --- p.90 / Chapter 3.5.8 --- Effects of sPLA2-II on histamine release from immunologically activated RPMC --- p.91 / Chapter 3.5.9 --- Effects of preincubation time on sPLA2-II enhanced histamine release from immunologically activated RPMC --- p.92 / Chapter 3.5.10 --- Effects of p-BPB on sPLA2-II enhanced histamine release from immunologically activated RPMC --- p.92 / Chapter 3.5.11 --- Effects of 12-epi-scalaradial on sPLA2-II enhanced histamine release from immunologically activated RPMC --- p.92 / Chapter 3.5.12 --- Effects of MAFP on sPLA2-II enhanced histamine release from immunologically activated RPMC --- p.93 / Chapter 3.5.13 --- Effects of sPLA2-II on PGD2 production from immunologically activated RPMC --- p.93 / Chapter 3.5.14 --- Effects of flurbiprofen and zileuton on sPLA2-II induced histamine release and enhanced histamine release from RPMC --- p.93 / Chapter 3.6 --- Discussion --- p.124 / Chapter 3.6.1 --- Effects of sPLA2 on immunologically induced histamine release from activated RPMC --- p.124 / Chapter 3.6.2 --- Effects of sPLA2 on PGD2 production from immunologically activated RPMC --- p.128 / Chapter 3.6.3 --- Effects of flurbiprofen and zileuton on sPLA2 induced and enhanced histamine release from RPMC --- p.129 / Chapter Part 3 --- Investigation of the mode of action(s) of sPLA2 on RPMC --- p.131-157 / Chapter 3.7 --- Results --- p.131 / Chapter 3.7.1 --- Effects of sPLA2-I on non-immunologically activated RPMC --- p.131 / Chapter 3.7.2 --- Effects of sPLA2-II on non-immunologically activated RPMC --- p.131 / Chapter 3.7.3 --- Effects of sPLA2-I on [Ca2+]i in immunologically activated RPMC --- p.132 / Chapter 3.7.4 --- Effects of sPLA2-II on [Ca2+]i in immunologically activated RPMC --- p.133 / Chapter 3.8 --- Discussion --- p.145 / Chapter 3.9 --- Summary of the possible mechanism (s) of action of sPLA2 --- p.149 / Chapter 3.10 --- Conclusions --- p.156 / Chapter 3.11 --- Future studies --- p.157 / Chapter Chapter 4 --- Effects of sPLA2 on human and guinea pig lung mast cells --- p.158-194 / Chapter 4.1 --- Introduction --- p.159 / Chapter 4.2 --- Methodology --- p.162 / Chapter 4.3 --- Results --- p.163 / Chapter 4.3.1 --- Effects of sPLA2 on the spontaneous histamine release from HLMC --- p.163 / Chapter 4.3.2 --- Effects of sPLA2 on immunologically induced histamine release from HLMC --- p.163 / Chapter 4.3.3 --- Effects of sPLA2 on A23187 induced histamine release from HLMC --- p.164 / Chapter 4.3.4 --- Effects of p-BPB on the inhibitory activity of sPLA2-I --- p.165 / Chapter 4.3.5 --- Effects of sPLA2 on partially purified HLMC --- p.165 / Chapter 4.3.6 --- Effects of sPLA2 on the spontaneous histamine release from GPLMC --- p.166 / Chapter 4.3.7 --- Effects of sPLA2 on immunologically induced histamine release from GPLMC --- p.167 / Chapter 4.4 --- Discussion --- p.189 / Chapter 4.5 --- Conclusions --- p.194 / References --- p.195-228

Mediators released from the granules of antigen-activated mast cells contribute to allergies, inflammation and diseases such as asthma. One of the major models used to study mucosal mast cells is the RBL-2H3 mast cell line. While there has been considerable research on the initial signalling events following IgE receptor (Fc??RI) cross-linking, the movement of granules to sites of exocytosis is poorly understood. Understanding the mechanisms that control granule movement to and fusion with the plasma membrane could provide novel targets for improved asthma and allergy therapeutics. To this end, an isolated intact population of granules from the RBL-2H3 mast cell provides a powerful research tool and as such the primary aim of this work was to isolate intact granules from the RBL-2H3 mast cell. Using iso-osmotic Percoll gradients we have isolated an intact granule population from RBL-2H3 mast cells. This granule population contained three granule markers: ??- hexosaminidase, serotonin and chymase. Triton X-100 pre-lysis resulted in loss of granule markers from this main peak, indicating that the isolated granules are in fact intact. Further analysis of the granule population showed that it is free from bulk contamination with other organelles and plasma membrane. The granules were estimated to have a density of 1.055 ??? 1.092g/mL, significantly less dense than that of rat peritoneal mast cell granules (1.2g/mL; [1]). Using an intact versus lysed approach, granule-associated proteins and phosphoproteins, from unactivated RBL-2H3 cells, were determined. Nine unknown granule-associated proteins were found using silver staining of gradient fractions separated on a SDSPAGE gel. In addition, four unknown serine or threonine granule-associated phosphoproteins were found. Molecular weight comparison suggested overlap in some of the unknown proteins and phosphoproteins. Probing for protein kinase C (PKC) isoforms confirmed previous results suggesting that a small population of PKC?? localised to the granules [2], and extended these results to include a population of PKC??I. The serine/threonine phosphatase PP1 does not appear to be granule associated. However, there was a small loss of PP2A from the granules (upon lysis), suggesting that perhaps a subpopulation of PP2A is granule-associated. The main granule peak represents a secretion competent population as Fc??RI-mediated activation of the cells resulted in a significant loss of granule markers from this peak. At the peak rate of antigen-induced secretion a number of changes occur in the phosphorylation of granule-associated phosphoproteins. In addition to an increase in the phosphorylation of three of the phosphoproteins seen in resting mast cell granules, eight new proteins were seen. Whether these proteins are granule-associated is currently unknown. PKC?? was found to translocate away from the granules at the peak rate of secretion, perhaps representing an important control mechanism in granule exocytosis. None of the tested PKC isoforms were found to translocate to the granules, providing little clue as to the identity of the kinase that may be involved in these phosphorylation events. However, as PKC??I is granule-associated and does not translocate off the granules, it would suggest that this kinase might be important for some of the observed phosphorylations. Overall the studies in this thesis show for the first time a rapid gradient-based method for the isolation of intact granules from unactivated and activated RBL-2H3 mast cells. These granules were used to determine granule-associated proteins and phosphoproteins, as well as to investigate changes that occur during the secretory process. In addition, the results show that a number of proteins have increased serine/threonine phosphorylation at the peak rate of antigen-stimulated secretion. This implies that phosphorylation is likely to play a role in the control of granule exocytosis. The identity of these proteins deserves further investigation. Thus, isolated intact RBL- 2H3 mast cell granules provide a powerful research tool to further investigate the mechanism and control of granule exocytosis.

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 84-91). / ix, 91 leaves, bound ill. 29 cm

Mediators released from the granules of antigen-activated mast cells contribute to allergies, inflammation and diseases such as asthma. One of the major models used to study mucosal mast cells is the RBL-2H3 mast cell line. While there has been considerable research on the initial signalling events following IgE receptor (Fc??RI) cross-linking, the movement of granules to sites of exocytosis is poorly understood. Understanding the mechanisms that control granule movement to and fusion with the plasma membrane could provide novel targets for improved asthma and allergy therapeutics. To this end, an isolated intact population of granules from the RBL-2H3 mast cell provides a powerful research tool and as such the primary aim of this work was to isolate intact granules from the RBL-2H3 mast cell. Using iso-osmotic Percoll gradients we have isolated an intact granule population from RBL-2H3 mast cells. This granule population contained three granule markers: ??- hexosaminidase, serotonin and chymase. Triton X-100 pre-lysis resulted in loss of granule markers from this main peak, indicating that the isolated granules are in fact intact. Further analysis of the granule population showed that it is free from bulk contamination with other organelles and plasma membrane. The granules were estimated to have a density of 1.055 ??? 1.092g/mL, significantly less dense than that of rat peritoneal mast cell granules (1.2g/mL; [1]). Using an intact versus lysed approach, granule-associated proteins and phosphoproteins, from unactivated RBL-2H3 cells, were determined. Nine unknown granule-associated proteins were found using silver staining of gradient fractions separated on a SDSPAGE gel. In addition, four unknown serine or threonine granule-associated phosphoproteins were found. Molecular weight comparison suggested overlap in some of the unknown proteins and phosphoproteins. Probing for protein kinase C (PKC) isoforms confirmed previous results suggesting that a small population of PKC?? localised to the granules [2], and extended these results to include a population of PKC??I. The serine/threonine phosphatase PP1 does not appear to be granule associated. However, there was a small loss of PP2A from the granules (upon lysis), suggesting that perhaps a subpopulation of PP2A is granule-associated. The main granule peak represents a secretion competent population as Fc??RI-mediated activation of the cells resulted in a significant loss of granule markers from this peak. At the peak rate of antigen-induced secretion a number of changes occur in the phosphorylation of granule-associated phosphoproteins. In addition to an increase in the phosphorylation of three of the phosphoproteins seen in resting mast cell granules, eight new proteins were seen. Whether these proteins are granule-associated is currently unknown. PKC?? was found to translocate away from the granules at the peak rate of secretion, perhaps representing an important control mechanism in granule exocytosis. None of the tested PKC isoforms were found to translocate to the granules, providing little clue as to the identity of the kinase that may be involved in these phosphorylation events. However, as PKC??I is granule-associated and does not translocate off the granules, it would suggest that this kinase might be important for some of the observed phosphorylations. Overall the studies in this thesis show for the first time a rapid gradient-based method for the isolation of intact granules from unactivated and activated RBL-2H3 mast cells. These granules were used to determine granule-associated proteins and phosphoproteins, as well as to investigate changes that occur during the secretory process. In addition, the results show that a number of proteins have increased serine/threonine phosphorylation at the peak rate of antigen-stimulated secretion. This implies that phosphorylation is likely to play a role in the control of granule exocytosis. The identity of these proteins deserves further investigation. Thus, isolated intact RBL- 2H3 mast cell granules provide a powerful research tool to further investigate the mechanism and control of granule exocytosis.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Thesis (doctoral)--Ume̊a Universitet, 1982. / Extra t.p. with thesis statement inserted. eContent provider-neutral record in process. Description based on print version record. Includes bibliographies.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: Regulation of compound exocytosis. Includes bibliographical references (p. 191-243). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. Description based on print version record. Includes bibliographical references.