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 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 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 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.

Ca(2+)-dependent amylase secretion from SLO-permeabilized rat pancreatic acini requires diffusible cytosolic proteins

1995 ◽  
Vol 269 (5) ◽  
pp. G647-G652 ◽  
Author(s):  
P. J. Padfield ◽  
N. Panesar
Keyword(s):  
Maximal Response ◽  
Rapid Decline ◽  
Amylase Secretion ◽  
Alpha Toxin ◽  
Pancreatic Acini ◽  
Permeabilized Cells ◽  
Membrane Pores ◽  
Streptolysin O ◽  
The Brain ◽  
Brain Cytosol

Streptolysin O (SLO)-permeabilized pancreatic acini are now frequently used to study regulated exocytosis in the exocrine pancreas. In this paper we introduce alpha-toxin as a possible alternative permeabilization agent to SLO. Both alpha-toxin and SLO are bacterial cytolysins, but the membrane pores generated by SLO are approximately 5-10 times larger than those formed by alpha-toxin. The Ca2+ requirements for amylase secretion from both types of permeabilized acini were identical, maximal amylase secretion being obtained at 30 microM Ca2+ with an effective concentration of approximately 3-4 microM Ca2+ producing 50% of the maximal response. However, Ca(2+)-stimulated amylase secretion from the SLO-permeabilized acini stopped after 10-15 min, unlike secretion from the alpha-toxin-permeabilized cells, which continued for at least 50 min. The rapid cessation of secretion from the SLO-treated acini reflects the rapid decline in the responsiveness of the cells observed after permeabilization. This decline in Ca(2+)-dependent secretion appears to be due to the loss of cytosol, since addition of purified rat brain cytosol to nonresponsive SLO-permeabilized acini reconstituted regulated secretion. Because alpha-toxin-permeabilized acini maintained their responsiveness, the cytosolic factors lost from the SLO-permeabilized cells must be retained within the toxin-treated cells. The reconstitutive activity of the brain cytosol was nondialyzable but heat and trypsin sensitive, suggesting that the factors responsible are proteins. Of the cytosols screened (brain, liver, spleen, muscle, and lacrimal) only those prepared from brain or lacrimal gland reconstituted Ca(2+)-dependent amylase secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Effect of pertussis toxin and neomycin on G-protein-regulated polyphosphoinositide phosphodiesterase. A comparison between HL60 membranes and permeabilized HL60 cells

1988 ◽  
Vol 256 (2) ◽  
pp. 343-350 ◽  
Author(s):  
S Cockcroft ◽  
J Stutchfield
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dimethyl Sulphoxide ◽  
Hl60 Cell ◽  
Dependent Manner ◽  
Dibutyryl Cyclic Amp ◽  
Hl60 Cells ◽  
Permeabilized Cells ◽  
Streptolysin O

The promyelocytic HL60 cell can be differentiated with dimethyl sulphoxide or dibutyryl cyclic AMP leading to the appearance of fMetLeuPhe receptors on the cell surface. G-protein-stimulated polyphosphoinositide phosphodiesterase (PPI-pde) activity was assessed in membranes prepared from both differentiated and non-differentiated HL60 cells. Both the extent of the response and the rank order of potency of the GTP analogues to stimulate PPI-pde activation (guanosine 5′-[gamma-thio]triphosphate (GTP[S]) greater than guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5′-[beta gamma-methylene]triphosphate (p[CH2]ppG) remains unchanged after differentiation with dimethyl sulphoxide. In comparison, differentiation by dibutyryl cyclic AMP leads to diminution of PPI-pde activity when stimulated by GTP[S] or fluoride, but not by millimolar concentrations of Ca2+. GTP[S]-stimulated PPI-pde in membranes is sensitive to the presence of Ca2+ (pCa 8-5). Pertussis-toxin pretreatment of intact HL60 cells leads to inhibition of both the secretory response and the formation of inositol phosphates when stimulated by fMetLeuPhe. In contrast, pertussis-toxin pretreatment has no effect on either GTP[S]- or fluoride-stimulated PPI-pde. Neomycin in a concentration-dependent manner inhibits both GTP[S] plus Ca2+ (pCa 5)-stimulated secretion and PPI-pde activation in streptolysin-O-permeabilized cells. The extent of PPI-pde activation in membranes compared with streptolysin-O-permeabilized cells reveals that the membrane preparation does not possess all the components that make up the inositide signalling system.

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 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 Activated release of membrane-anchored TGF-alpha in the absence of cytosol

1993 ◽  
Vol 122 (1) ◽  
pp. 95-101 ◽  
Author(s):  
MW Bosenberg ◽  
A Pandiella ◽  
J Massagué
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Atp Hydrolysis ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Vesicular Traffic ◽  
Kinase C ◽  
Streptolysin O ◽  
Independent Protein

The ectodomain of proTGF-alpha, a membrane-anchored growth factor, is converted into soluble TGF-alpha by a regulated cellular proteolytic system that recognizes proTGF-alpha via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-alpha cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca(2+)-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-alpha cleavage is also stimulated by GTP gamma S through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA-sensitive Ca(2+)-independent protein kinase C. Activated proTGF-alpha cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-alpha that has reached the cell surface. These results indicate that proTGF-alpha is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol.

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.

scholarly journals Activation of protein kinase C is not an absolute requirement for amylase release from permeabilized rat pancreatic acini

1992 ◽  
Vol 285 (2) ◽  
pp. 597-601 ◽  
Author(s):  
A J O'Sullivan ◽  
J D Jamieson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Peptide Inhibitor ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Streptolysin O ◽  
Absolute Requirement ◽  
Messenger System

The effect of protein kinase C (PKC) on amylase discharge from streptolysin-O-permeabilized rat pancreatic acini was investigated. Addition of phorbol 12-myristate 13-acetate (PMA) to permeabilized cells potentiated Ca(2+)-stimulated release, but had no effect on discharge at non-stimulatory Ca2+ concentrations. PMA markedly shifted the Ca(2+)-concentration-dependence of amylase discharge to the left, by enhancing the time over which the permeabilized cells release. This effect was inhibited by both staurosporine and PKC-19-31-amide peptide inhibitor, indicating that the effect of PMA was due to its action on PKC. Staurosporine also partially inhibited amylase release at the optimal concentration of Ca2+; this effect was not replicated by the more specific PKC-19-31-amide peptide inhibitor and may be due to an effect on another second-messenger system. PKC appears to be an important modulator of release in pancreatic acini, but its activation is not an absolute requirement for Ca(2+)-dependent amylase discharge.

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