Relationship between arachidonate generation and exocytosis in permeabilized mast cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Churcher Y
PROVIDER: S-EPMC1131109 | biostudies-other | 1990 Feb
REPOSITORIES: biostudies-other
Similar Datasets
Project description:We have measured the time course of secretion of hexosaminidase from rat mast cells permeabilized (in simple buffered NaCl solutions) in response to guanine nucleotides [GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S])] and Ca2+. In these experiments, ATP was excluded from the system (and the cells were pretreated with metabolic inhibitors). For cells permeabilized in the absence of Mg2+ but in the presence of Ca2+, secretion commences promptly in response to addition of GTP; when Mg2+ (2 mM) is provided, secretion commences after an extended delay, much higher concentrations of GTP are required, and the final extent of secretion is decreased. Ongoing secretion due to GTP and Ca2+ is abruptly terminated by addition of Mg2+ to cells initially stimulated in its absence. In contrast, although Mg2+ has no effect on the sensitivity to the non-hydrolysable analogue GTP[S], its absence does nevertheless cause delays in the onset of secretion triggered by the addition of GTP[S] to cells initially permeabilized in the presence of Ca2+ (micromolar range, again in the absence of ATP). However, exocytosis from cells triggered with Ca2+ after permeabilization in the presence of high concentrations of GTP[S] is instantaneous. The delays due to triggering by GTP[S] have GTP[S]-concentration-dependent and -independent components. The guanine-nucleotide-concentration-dependent component is expressed as an extended duration of delay as the concentration of GTP[S] is decreased, and may reflect the binding of GTP[S] to GE. The concentration-independent component is manifested as a limiting delay which cannot be further diminished by increasing the guanine nucleotide concentration. The duration of the limiting delay is sensitive to the identity of the stimulating nucleotide (GTP < GTP[S] < p[NH]ppG) and may reflect the time taken for an activating conformational change to occur after binding. Since both components of the delays are abolished by the presence of Mg2+, both the binding of guanine nucleotide and the activation of GE appear to be Mg(2+)-dependent. We therefore conclude that nucleotide binding, activation and the GTPase activity of GE are strongly dependent on Mg2+, in common with the same three processes in Gs and Gi.
| S-EPMC1132285 | biostudies-other
Project description:The relationship between Ca2(+)-dependent arachidonic acid release and exocytosis from digitonin-permeabilized bovine adrenal chromaffin cells was investigated. The phospholipase A2 inhibitors mepacrine, nordihydroguaiaretic acid and indomethacin had no effect on either arachidonic acid release or secretion. The phospholipase A2 activator melittin had no effect on secretion. The specific diacylglycerol lipase inhibitor RG80267 had no effect on secretion, but decreased basal arachidonic acid release to such an extent that the level of arachidonic acid in treated cells in response to 10 microM-Ca2+ was equivalent to that of control cells in the absence of Ca2+. Staurosporine, a protein kinase C inhibitor, was found to abolish Ca2(+)-dependent arachidonic acid release completely, but had only a slight inhibitory effect on Ca2(+)-dependent secretion. It is concluded that arachidonic acid is not essential for Ca2(+)-dependent exocytosis in adrenal chromaffin cells.
| S-EPMC1149600 | biostudies-other
Project description:The role of two G-proteins, Gp and Ge, in the stimulus-secretion pathway has been proposed on the basis of studies where GTP analogues have been introduced into permeabilized cell preparations. In this study, evidence is provided that two G-proteins are also involved when a receptor-directed agonist is used. Intact human neutrophils were made refractory to formylmethionyl-leucyl-phenylalanine (fMetLeuPhe) stimulation by metabolic inhibition and then permeabilized with streptolysin O to compare the intracellular requirements for exocytosis from specific and azurophilic granules and arachidonate release. In the presence of 1 microM-Ca2+ and 1 mM-MgATP, fMetLeuPhe or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induce secretion from both granule types as well as arachidonate release. Secretion and arachidonate release owing to fMetLeuPhe can occur in the absence of ATP, conditions under which G-protein-mediated activation of phospholipase C is suppressed. GTP[S]-induced secretion can also occur in the absence of MgATP, but GTP[S]-induced arachidonate release cannot. It is concluded that fMetLeuPhe, like GTP[S], stimulates secretion by interacting with another G-protein-mediated reaction apart from Gp. Evidence is provided that a possible target for the second G-protein-mediated reaction involved in fMetLeuPhe-induced secretion (but not GTP[S]-induced secretion) is phospholipase A2.
| S-EPMC1150022 | biostudies-other
Project description:We have used a digitonin-permeabilized cell system to study the signal transduction pathways responsible for stimulus-secretion coupling in the rat peritoneal mast cell. Conditions were established for permeabilizing the mast cell plasma membrane without disrupting secretory vesicles. Exocytotic release of histamine from digitonin-permeabilized cells required a combination of micromolar concentrations of Ca2+ and the stable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but was independent of exogenous ATP. In the presence of 40 microM-GTP[S], exocytosis was half-maximal at 1.3 microM-Ca2+ and maximal at 10 microM-Ca2+; GTP[S] alone (100 microM) had no effect on histamine release in the absence of added Ca2+. In the presence of 10 microM free Ca2+, 5 microM-GTP[S] was required for half-maximal exocytosis. To examine the possible role of protein kinase C (PKC) in exocytosis, we utilized 12-O-tetradecanoylphorbol 13-acetate (TPA) to activate PKC and studied its effect on histamine release from permeabilized mast cells. Cells that had been incubated with TPA (25 nM for 5 min) exhibited increased sensitivity to both GTP[S] and Ca2+. The PKC inhibitor staurosporine blocked the effect of TPA without inhibiting normal exocytosis in response to the combination of GTP[S] and Ca2+. In addition, down-regulation of mast-cell PKC by long-term TPA treatment (25 nM for 20 h) blocked the ability of the cells to respond to TPA and inhibited exocytosis in response to Ca2+ and GTP[S] by 40-50%. These results suggest that the sensitivity of the exocytotic machinery of the mast cell can be altered by PKC-catalysed phosphorylation events, but that activation of PKC is not required for exocytosis to occur.
| S-EPMC1136896 | biostudies-other
Project description:Mast cells (MCs) are involved in host defenses against pathogens and inflammation. Stimulated MCs release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 13) proteins play essential roles in regulated exocytosis. Here, we found that MCs express Munc13-2 and -4, and we studied their roles using global and conditional knock-out (KO) mice. In a model of systemic anaphylaxis, we found no difference between WT and Munc13-2 KO mice, but global and MC-specific Munc13-4 KO mice developed less hypothermia. This protection correlated with lower plasma histamine levels and with histological evidence of defective MC degranulation but not with changes in MC development, distribution, numbers, or morphology. In vitro assays revealed that the defective response in Munc13-4-deficient MCs was limited to regulated exocytosis, leaving other MC secretory effector responses intact. Single cell capacitance measurements in MCs from mouse mutants differing in Munc13-4 expression levels in their MCs revealed that as levels of Munc13-4 decrease, the rate of exocytosis declines first, and then the total amount of exocytosis decreases. A requirement for Munc13-2 in MC exocytosis was revealed only in the absence of Munc13-4. Electrophysiology and EM studies uncovered that the number of multigranular compound events (i.e. granule-to-granule homotypic fusion) was severely reduced in the absence of Munc13-4. We conclude that although Munc13-2 plays a minor role, Munc13-4 is essential for regulated exocytosis in MCs, and that this MC effector response is required for a full anaphylactic response.
| S-EPMC5766930 | biostudies-literature
Project description:In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified "large-capacitance flickers" that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent "compound cavicapture," most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.
| S-EPMC3711296 | biostudies-literature
Project description:The effect of GTP analogues on catecholamine secretion and [3H]arachidonic acid release from digitonin-permeabilized adrenal chromaffin cells was examined. Several GTP analogues stimulated Ca2(+)-independent exocytosis, with the order of efficacy being XTP greater than ITP greater than guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The stimulatory effect of the GTP analogues appeared to be due to activation of a conventional GTP-binding protein, as it was inhibited by guanosine 5'-[beta-thio]diphosphate (GDP[S]). In contrast, Ca2(+)-dependent exocytosis was only partially inhibited by high doses of GDP[S]. GTP did not stimulate Ca2(+)-independent exocytosis, but instead was found to inhibit secretion caused by micromolar Ca2+. Arachidonic acid (100 microM) also stimulated Ca2(+)-independent catecholamine secretion. Determination of the effect of GTP analogues on release of free [3H]arachidonic acid into the medium showed that it was stimulated by GTP[S] but inhibited by GTP, p[NH]ppG, ITP and XTP. The inhibition of [3H]arachidonic acid release by XTP was not prevented by GDP[S]. These results demonstrate that activation of a GTP-binding protein by certain GTP analogues can induce Ca2(+)-independent secretion in adrenal chromaffin cells and that the effect of GTP analogues on Ca2(+)-independent secretion can be dissociated from generation of arachidonic acid.
| S-EPMC1131608 | biostudies-other
Project description:Rab44 is a large Rab GTPase containing a Rab GTPase domain and some additional N-terminal domains. We recently used Rab44-deficient mice to demonstrate that Rab44 regulates granule exocytosis in mast cells and IgE-mediated anaphylaxis. In mouse mast cells, Rab44 is expressed as two isoforms, namely, the long and short forms; however, the characteristics of these two isoforms remain unknown. Here, we investigated secretion and localization of the human long Rab44 isoform and the two mouse isoforms and their mutants expressed in rat basophilic leukemia (RBL)-2H3 cells. Expression of the human long isoform and both mouse isoforms caused an increase in β-hexosaminidase secretion. Confocal and quantitative analyses showed that both human and mouse long isoforms localized mainly to lysosomes while the mouse short isoform localized mainly to the ER. Live imaging with LysoTracker indicated that the size and number of LysoTracker-positive vesicles were altered by the various mutants. Ionomycin treatment partially altered localization of both long isoforms to the plasma membrane and cytosol, whereas it had little effect on colocalization of the short isoform with lysosomes. Mechanistically, both human and mouse Rab44 proteins interacted with vesicle-associated membrane protein 8 (VAMP8), a v-SNARE protein. Therefore, Rab44 isoforms similarly promote lysosomal exocytosis, but exhibit differential localization in mast cells.
| S-EPMC8016136 | biostudies-literature
Project description:In the neonatal mouse ovary, clusters of oocytes called nests break into smaller cysts and subsequently form individual follicles. During this period, we found numerous mast cells in the ovary of MRL/MpJ mice and investigated their appearance and morphology with follicular development. The ovarian mast cells, which were already present at postnatal day 0, tended to localize adjacent to the surface epithelium. Among 11 different mouse strains, MRL/MpJ mice possessed the greatest number of ovarian mast cells. Ovarian mast cells were also found in DBA/1, BALB/c, NZW, and DBA/2 mice but rarely in C57BL/6, NZB, AKR, C3H/He, CBA, and ICR mice. The ovarian mast cells expressed connective tissue mast cell markers, although mast cells around the surface epithelium also expressed a mucosal mast cell marker in MRL/MpJ mice. Some ovarian mast cells migrated into the oocyte nests and directly contacted the compressed and degenerated oocytes. In MRL/MpJ mice, the number of oocytes in the nest was significantly lower than in the other strains, and the number of oocytes showed a positive correlation with the number of ovarian mast cells. The gene expression of a mast cell marker also correlated with the expression of an oocyte nest marker, suggesting a link between the appearance of ovarian ? 4mast cells and early follicular development. Furthermore, the expression of follicle developmental markers was significantly higher in MRL/MpJ mice than in C57BL/6 mice. These results indicate that the appearance of ovarian mast cells is a unique phenotype of neonatal MRL/MpJ mice, and that ovarian mast cells participate in early follicular development, especially nest breakdown.
| S-EPMC3790711 | biostudies-literature
Project description:Mast cells degranulate and release the contents of intracellular secretory granules in response to the cross-linking of FcepsilonRI by multivalent antigens. These granules contain a variety of biologically active inflammatory mediators; however, it is not clear whether these granules are homogenous or whether there is heterogeneity within the secretory granule population in mast cells. By using genetically altered mice lacking specific vesicle-associated SNARE membrane fusion proteins, we found that VAMP-8-deficient mast cells exhibited defects in FcepsilonRI-regulated exocytosis, whereas synaptobrevin 2- or VAMP-3-deficient mast cells did not. Surprisingly, the defect in secretion in VAMP-8-deficient mice was limited to the subpopulation of mast cell secretory granules containing serotonin and cathepsin D, whereas regulated exocytosis of secretory granules containing histamine and TNF-alpha was normal. Confocal microscopy confirmed that serotonin and histamine were present in distinct intracellular granules and that most serotonin-containing granules were VAMP-8-positive. Thus, this study demonstrates that mast cells do indeed possess distinct subsets of secretory granules and that these subsets use different SNARE isoforms for exocytosis.
| S-EPMC2268179 | biostudies-literature