scholarly journals Changes in the state of actin during the exocytotic reaction of permeabilized rat mast cells.

1990 ◽  
Vol 111 (3) ◽  
pp. 919-927 ◽  
Author(s):  
A Koffer ◽  
P E Tatham ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Cortical Region ◽  
Filamentous Actin ◽  
Rhodamine Phalloidin ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Dependent Inhibition ◽  
Cytochalasin E ◽  
Actin Regulatory Proteins ◽  
Gamma S

The major part of mast cell actin is Triton-soluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, through filamentous actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be approximately 2.5 micrograms/10(6) cells (cytosolic concentration approximately 110 microM. After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, approximately 60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP-gamma-S together with Ca2+. In the presence of cytochalasin E the requirement for Ca2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by Ca2(+)-dependent actin regulatory proteins; their presence is indicated by a Ca2(+)-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca2+ and cause variable extents of Ca2(+)-independent secretion. Exposing the permeabilized cells to increasing concentrations of Ca2+ caused a progressive decrease in F-actin levels as measured by flow cytometry of RP-stained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP-gamma-S was not required for the Ca2(+)-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur.

scholarly journals Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilized rat mast cells.

1987 ◽  
Vol 105 (1) ◽  
pp. 191-197 ◽  
Author(s):  
T W Howell ◽  
S Cockcroft ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Rank Order ◽  
Regulatory Protein ◽  
Metabolic Inhibitors ◽  
Pyrimidine Nucleotides ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Gamma S ◽  
Sufficient Stimulus ◽  
Rat Mast Cells

Rat mast cells, pretreated with metabolic inhibitors and permeabilized by streptolysin-O, secrete histamine when provided with Ca2+ (buffered in the micromolar range) and nucleoside triphosphates. We have surveyed the ability of various exogenous nucleotides to support or inhibit secretion. The preferred rank order in support of secretion is ITP greater than XTP greater than GTP much greater than ATP. Pyrimidine nucleotides (UTP and CTP) are without effect. Nucleoside diphosphates included alongside Ca2+ plus ITP inhibit secretion in the order 2'-deoxyGDP greater than GDP greater than o-GDP greater than ADP approximately equal to 2'deoxyADP approximately equal to IDP. Secretion from the metabolically inhibited and permeabilized cells can also be induced by stable analogues of GTP (GTP-gamma-S greater than GppNHp greater than GppCH2p) which synergize with Ca2+ to trigger secretion in the absence of phosphorylating nucleotides. ATP enhances the effective affinity for Ca2+ and GTP analogues in the exocytotic process but does not alter the maximum extent of secretion. The results suggest that the presence of Ca2+ combined with activation of events controlled by a GTP regulatory protein provide a sufficient stimulus to exocytotic secretion from mast cells.

scholarly journals Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a rac/rhoGDI complex.

1996 ◽  
Vol 7 (3) ◽  
pp. 397-408 ◽  
Author(s):  
A J O'Sullivan ◽  
A M Brown ◽  
H N Freeman ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Peptide Sequencing ◽  
Bovine Brain ◽  
Nucleotide Exchange ◽  
Permeabilized Cells ◽  
Guanine Nucleotide Exchange ◽  
Exchange Inhibitor ◽  
Streptolysin O ◽  
Gamma S ◽  
Brain Cytosol

Mast cells permeabilized by treatment with streptolysin-O in the presence of Ca2+ and GTP-gamma-S can secrete almost 100% of their contained N-acetyl-beta-D-glucosaminidase. If these stimuli are provided to the permeabilized cells after a delay, the response is diminished and the ability of the cells to undergo secretion runs down progressively over a period of about 30 min. This is thought to be due to the loss of key proteins involved in the exocytotic mechanism. Using this effect as the basis of a biological assay, we have isolated a protein from bovine brain cytosol that retards the loss of responsiveness to stimulation by Ca2+ and GTP-gamma-S. Purification of this protein and peptide sequencing have enabled us to identify it as the small GTP-binding protein rac complexed to the guanine nucleotide exchange inhibitor rhoGDI. Both proteins are required to retard the loss of the secretory response, while purified rhoGDI applied alone accelerates the rundown.

scholarly journals Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin.

1991 ◽  
Vol 113 (5) ◽  
pp. 1057-1067 ◽  
Author(s):  
M L Vitale ◽  
A Rodríguez Del Castillo ◽  
L Tchakarov ◽  
J M Trifaró
Keyword(s):  
Actin Filament ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Catecholamine Secretion ◽  
Cortical Region ◽  
Cortical Surface ◽  
Filamentous Actin ◽  
Cell Stimulation ◽  
Rhodamine Phalloidin ◽  
Filament Networks

Immunofluorescence and cytochemical studies have demonstrated that filamentous actin is mainly localized in the cortical surface of the chromaffin cell. It has been suggested that these actin filament networks act as a barrier to the secretory granules, impeding their contact with the plasma membrane. Stimulation of chromaffin cells produces a disassembly of actin filament networks, implying the removal of the barrier. The presence of gelsolin and scinderin, two Ca(2+)-dependent actin filament severing proteins, in the cortical surface of the chromaffin cells, suggests the possibility that cell stimulation brings about activation of one or more actin filament severing proteins with the consequent disruption of actin networks. Therefore, biochemical studies and fluorescence microscopy experiments with scinderin and gelsolin antibodies and rhodamine-phalloidin, a probe for filamentous actin, were performed in cultured chromaffin cells to study the distribution of scinderin, gelsolin, and filamentous actin during cell stimulation and to correlate the possible changes with catecholamine secretion. Here we report that during nicotinic stimulation or K(+)-evoked depolarization, subcortical scinderin but not gelsolin is redistributed and that this redistribution precedes catecholamine secretion. The rearrangement of scinderin in patches is mediated by nicotinic receptors. Cell stimulation produces similar patterns of distribution of scinderin and filamentous actin. However, after the removal of the stimulus, the recovery of scinderin cortical pattern of distribution is faster than F-actin reassembly, suggesting that scinderin is bound in the cortical region of the cell to a component other than F-actin. We also demonstrate that peripheral actin filament disassembly and subplasmalemmal scinderin redistribution are calcium-dependent events. Moreover, experiments with an antibody against dopamine-beta-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.

scholarly journals Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins.

1994 ◽  
Vol 126 (4) ◽  
pp. 1005-1015 ◽  
Author(s):  
J C Norman ◽  
L S Price ◽  
A J Ridley ◽  
A Hall ◽  
A Koffer
Keyword(s):  
Mast Cells ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Small Gtpases ◽  
Secretory Cells ◽  
Cortical Region ◽  
Permeabilized Cells ◽  
Gtp Binding Proteins ◽  
Gtp Binding

Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.

scholarly journals Evidence for protein dephosphorylation as a permissive step in GTP-gamma-S-induced exocytosis from permeabilized mast cells.

1990 ◽  
Vol 1 (7) ◽  
pp. 523-530 ◽  
Author(s):  
Y Churcher ◽  
K M Kramer ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Guanine Nucleotide ◽  
Kinase C ◽  
Streptolysin O ◽  
Effector System ◽  
Phosphorylation Reaction ◽  
Protein Dephosphorylation ◽  
Cellular Responsiveness ◽  
Gamma S ◽  
Micromolar Range

Mast cells permeabilized by streptolysin O secrete histamine and lysosomal enzymes in response to provision of a dual effector system comprising Ca2+ and a guanine nucleotide (e.g., GTP-gamma-S2) at concentrations in the micromolar range. These are both necessary and together they are sufficient. There is no requirement for adenosine triphosphate (ATP) and hence no obligatory phosphorylation reaction in the terminal stages of the exocytotic pathway. When exocytosis is induced by Ca2(+)-plus-GTP-gamma-S (i.e., no ATP) added at times after permeabilization (the permeabilization interval), cellular responsiveness declines so that there is no response to provision of the two effectors (both at 10(-5)M) if they are initially withheld and then added after 5 min. Here we show that this decline in responsiveness is characterized by a time-dependent reduction in the effective affinity for Ca2+. Affinity for Ca2+ and hence secretory competence can then be restored if ATP is added alongside the stimulus. Unlike cells stimulated to secrete at the time of permeabilization, exocytosis from cells that have undergone the cycle of permeabilization-induced refractoriness followed by ATP-induced restoration can be triggered by Ca2+ alone: after such conditioning there is no requirement for guanine nucleotide. In contrast, dependence on guanine nucleotide remains mandatory in cells that have been pretreated (i.e., before permeabilization) with okadaic acid (understood to be an inhibitor of protein phosphatases 1 and 2A) or phorbol myristate acetate (an activator of protein kinase C). These results indicate that obligatory dependence on guanine nucleotide is retained when the cells are treated under conditions conducive to maintained phosphorylation. It is concluded that the exocytotic mechanism of permeabilized mast cells is enabled by a dephosphorylation reaction and that the effector of the guanosine triphosphate (GTP)-binding protein (G epsilon) that mediates exocytosis is likely to be a protein phosphate.

scholarly journals Two G-proteins act in series to control stimulus-secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphodiesterase and exocytosis.

1987 ◽  
Vol 105 (6) ◽  
pp. 2745-2750 ◽  
Author(s):  
S Cockcroft ◽  
T W Howell ◽  
B D Gomperts
Keyword(s):  
Protein Kinase C ◽  
Mast Cells ◽  
Protein Kinase ◽  
G Proteins ◽  
Kinase C ◽  
Streptolysin O ◽  
In Series ◽  
Sparing Effect ◽  
Necessary And Sufficient ◽  
Gamma S

Provision of GTP (or other nucleotides capable of acting as ligands for activation of G-proteins) together with Ca2+ (at micromolar concentrations) is both necessary and sufficient to stimulate exocytotic secretion from mast cells permeabilized with streptolysin-O. GTP and its analogues, through their interactions with Gp, also activate polyphosphoinositide-phosphodiesterase (PPI-pde generating inositol 1,4,5-trisphosphate and diglyceride [DG]). We have used mast cells labeled with [3H]inositol to test whether the requirement for GTP in exocytosis is an expression of Gp activity through the generation of DG and consequent activation of protein kinase C, or whether GTP is required at a later stage in the stimulus secretion sequence. Neomycin (0.3 mM) inhibits activation of PPI-pde, but maximal secretion due to optimal concentrations of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) can still be evoked in its presence. When ATP is also provided the concentration requirement for GTP-gamma-S in support of exocytosis is reduced. This sparing effect of ATP is nullified when the PPI-pde reaction is inhibited by neomycin. We argue that the sparing effect of ATP occurs as a result of enhancement of DG production and through its action as a phosphoryl donor in the reactions catalyzed by protein kinase C.

scholarly journals Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: effects of ATP insulin, and GTP gamma S and localization of GLUT4 to clathrin lattices

1992 ◽  
Vol 117 (6) ◽  
pp. 1181-1196 ◽  
Author(s):  
LJ Robinson ◽  
S Pang ◽  
DS Harris ◽  
J Heuser ◽  
DE James
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Insulin Treatment ◽  
Protein S ◽  
Confocal Laser ◽  
Intact Cells ◽  
Intracellular Location ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Gamma S

Insulin stimulates the movement of two glucose transporter isoforms (GLUT1 and GLUT4) to the plasma membrane (PM) in adipocytes. To study this process we have prepared highly purified PM fragments by gently sonicating 3T3-L1 adipocytes grown on glass coverslips. Using confocal laser immunofluorescence microscopy we observed increased PM labeling for GLUT1 (2.3-fold) and GLUT4 (eightfold) after insulin treatment in intact cells. EM immunolabeling of PM fragments indicated that in the nonstimulated state GLUT4 was mainly localized to flat clathrin lattices. Whereas GLUT4 labeling of clathrin lattices was only slightly increased after insulin treatment, labeling of uncoated PM regions was markedly increased with insulin. These data suggest that GLUT4 recycles from the cell surface both in the presence and absence of insulin. In streptolysin-O permeabilized adipocytes, insulin, and GTP gamma S increased PM levels of GLUT4 to a similar extent as observed with insulin in intact cells. In the absence of an exogenous ATP source the magnitude of these effects was considerably reduced. Removal of ATP per se caused a significant increase in cell surface levels of GLUT4 suggesting that ATP may be required for intracellular sequestration of these transporters. When insulin and GTP gamma S were added together, in the presence of ATP, PM GLUT4 levels were similar to levels observed when either insulin or GTP gamma S was added individually. Addition of GTP gamma S was able to overcome this ATP dependence of insulin-stimulated GLUT4 movement. GTP gamma S had no effect on constitutive secretion of adipsin in permeabilized cells. In addition, there was no effect of insulin or GTP gamma S on GLUT4 movement to the PM in noninsulin sensitive streptolysin-O-permeabilized 3T3-L1 fibroblasts overexpressing GLUT4. We conclude that the insulin-stimulated movement of GLUT4 to the cell surface in adipocytes may require ATP early in the insulin signaling pathway and a GTP-binding protein(s) at a later step(s). We propose that the association of GLUT4 with clathrin lattices may be important in maintaining the exclusive intracellular location of this transporter in the absence of insulin.

scholarly journals ATP-induced pore formation in the plasma membrane of rat peritoneal mast cells.

1990 ◽  
Vol 95 (3) ◽  
pp. 459-476 ◽  
Author(s):  
P E Tatham ◽  
M Lindau
Keyword(s):  
Mast Cells ◽  
Single Channel ◽  
Noise Analysis ◽  
Hill Coefficient ◽  
Patch Clamp Technique ◽  
Permeabilized Cells ◽  
Cation Selectivity ◽  
Peritoneal Mast Cells ◽  
Rat Peritoneal Mast Cells ◽  
Gamma S

We have investigated the ATP-induced permeabilization of rat peritoneal mast cells using three different techniques: (a) by measuring uptake of fluorescent membrane and DNA marker dyes, (b) by voltage-clamp measurements using the patch-clamp technique, and (c) by measurements of exocytosis in response to entry of Ca2+ and GTP gamma S into permeabilized cells. In the absence of divalent cations cells become highly permeable at ATP concentrations as low as 3 microM. In normal saline containing 1 mM MgCl2 and 2 mM CaCl2, dye uptake and electric conductance are detectable at 100 microM ATP corresponding to 4 microM ATP4-. The permeabilization is half-maximal at an ATP4- concentration of 5-20 microM with a Hill coefficient near 2. The ATP-induced whole-cell conductance at saturating ATP concentrations was 35-70 nS, exhibiting only weak cation selectivity. The activation is very fast with a time constant less than or equal to 65 ms. Pores which are large enough to allow for permeation of substances of 300-900 D are expected to have a unit conductance of approximately 200-400 pS. However, in whole cells as well as outside-out patches, discrete openings and closings of channels could not be observed at a resolution of approximately 40 pS and the single-channel conductance obtained from noise analysis is approximately 2-10 pS. Entry of Ca2+ into cells permeabilized with ATP stimulates exocytosis at low but not at high ATP concentrations indicating loss of an essential intracellular component or components at a high degree of permeabilization. This inactivation is removed when GTP gamma S is provided in the medium and this leads to enhanced exocytosis. The enhancement only occurs at high ATP concentrations. These results strongly suggest that the ATP-induced pores are of variable size and can increase or decrease by very small units.

scholarly journals Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin O

1988 ◽  
Vol 250 (2) ◽  
pp. 375-382 ◽  
Author(s):  
J Stutchfield ◽  
S Cockcroft
Keyword(s):  
Rank Order ◽  
Guanine Nucleotides ◽  
Hl60 Cell ◽  
Maximal Response ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Streptolysin O ◽  
Competitive Inhibitors ◽  
In Series ◽  
Gamma S

The non-differentiated HL60 cell can be stimulated to secrete when Ca2+ and guanosine 5′-[gamma-thio]-triphosphate (GTP gamma S) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentration-dependence on Ca2+ between pCa 8 and pCa 5. The half-maximal requirements for Ca2+ for PPI-pde activation and secretion are pCa 6.4 +/- 0.1 and pCa 6.2 +/- 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTP gamma S greater than guanosine 5′-[beta gamma-imido]-triphosphate greater than guanosine 5′-[beta gamma-methylene]triphosphate greater than XTP approximately equal to ITP, but the maximal response achieved by each compound compared with GTP gamma S is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTP gamma S-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTP gamma S in stimulating secretion, suggesting that the effect of GTP gamma S cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2+ and GTP gamma S requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (GP) and the other for activating exocytosis (GE), acting in series.

scholarly journals ATP-dependent and ATP-independent pathways of exocytosis revealed by interchanging glutamate and chloride as the major anion in permeabilized mast cells.

1990 ◽  
Vol 1 (4) ◽  
pp. 337-346 ◽  
Author(s):  
Y Churcher ◽  
B D Gomperts
Keyword(s):  
Mast Cells ◽  
Electrolyte Solutions ◽  
Secretory Cells ◽  
Guanine Nucleotide ◽  
Mixed Electrolyte ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Effector System ◽  
Gamma S ◽  
Rat Mast Cells

Most investigations of the mechanism of regulated exocytosis have involved the use of secretory cells permeabilized in glutamate-based electrolyte solutions. In our previous work we have used NaCl-based electrolyte solutions. For secretion to occur from rat mast cells under these latter conditions, a dual effector system comprising Ca2+ and a guanine nucleotide are required; together they are sufficient. Here we compare the secretion from mast cells permeabilized in solutions of different electrolytes. Replacement of Na+ by K+ had little effect. Replacement of Cl- by Br-, SO4-, gluconate, isethionate, acetate, tartrate, succinate, etc. affected the maximal extent of secretion elicited by the dual effectors Ca2+ and guanosine-5'-O-(3-thiotriphosphate) (Ca2(+)-plus-GTP-gamma-S) but had little influence on the effective affinity for Ca2+. The dicarboxylic amino acids (L- and D-glutamate, and L-aspartate) permitted exocytosis to be elicited by Ca2+ or GTP-gamma-S alone. Secretion stimulated by GTP-gamma-S is strongly inhibited by Cl- (50% inhibition by 20 mM Cl-), whereas the extent of Ca2(+)-induced secretion is proportional to the concentration of glutamate in mixed electrolyte buffers. Unlike dual-effector stimulation, secretion due to the single effectors requires adenosine triphosphate (ATP) and is prevented by inhibitors of protein kinase C. These results point to the existence of two parallel pathways for control of exocytosis in permeabilized cells, one ATP dependent, the other ATP independent.

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