Techniques and concepts in exocytosis: focus on mast cells.
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Adenosine Triphosphate↗
Biomedical subjects
B D Gomperts
Publications and source records attributed to B D Gomperts.
Modulation of the exocytotic reaction of permeabilised rat mast cells by ATP, other nucleotides and Mg2+.
In the terminal stages of exocytosis from permeabilised mast cells, ATP has a number of modulatory actions, although its presence (and by implication, phosphorylation) is not obligatory for secretion to occur. These effects include (1) the enhancement of the sensitivity to both of the essential effectors (Ca2+ and guanine nucleotide); (2) the maintenance of the responsiveness of permeabilised cells; (3) restoration of responsiveness to cells rendered refractory by previous permeabilisation, and (4) induction of delays in the onset of exocytosis from permeabilised cells. We define the modulatory reactions induced by ATP by characterising their specificity to other potential phosphorylating nucleotides and their requirement for Mg2+. GTP and AppNHp are without effect in any of the modulatory actions. ATP, ATP-gamma-S, ITP, XTP, CTP and UTP all appear to support an enhancement of the sensitivity to GTP-gamma-S when applied immediately at the time of permeabilisation. However, the non-adenine nucleoside triphosphates appear to mediate their effect by transphosphorylation to ADP, and therefore the active species appears to be ATP. Only ATP is capable of maintaining and restoring responsiveness (2 and 3 above). Only ATP and ATP-gamma-S induce onset delays and do so moreover in the absence (less than 10(-8) M) of Mg2+. We conclude that three of the modulatory effects (1, 2 and 3 above) which all express a requirement for Mg2+, and can be prevented by inhibitors of protein kinase C are likely to result from phosphorylation reactions. The induction of delays by ATP is unlikely to incur phosphorylation.
Adenosine Triphosphate↗
Mechanisms of granule enzyme secretion from permeabilized guinea pig eosinophils. Dependence on Ca2+ and guanine nucleotides.
The mechanisms of granule protein secretion have been studied in streptolysin-O-permeabilized guinea pig eosinophils. Secretion of the granule-associated enzyme N-acetyl-beta-D-glucosaminidase was dependent on both Ca2+ and a nonhydrolyzable GTP analogue, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting roles for both calcium and GTP binding proteins. Secretion was maximal by 7 min, and varied between 35 and 60% of the total enzyme activity. Other GTP analogues also elicited secretion, with rank order GTP-gamma-S greater than guanylyl-imidophosphate greater than guanylyl (beta-gamma-methylene-diphosphate). Unrelated nucleotide triphosphates showed little or no effect confirming the specificity of the G protein. Transmission electronmicroscopy confirmed that permeabilization alone did not result in loss of granules and that exocytosis was dependent on the addition of the effectors, Ca2+ and GTP-gamma-S. ATP enhanced the magnitude of the secretory response and also enhanced the effective affinities for both Ca2+ and GTP-gamma-S. In the presence of 10(-5) M GTP-gamma-S the ED50 (Ca2+) was pCa 5.57 +/- 0.04 (2.69 microM) in the absence of ATP and declined to pCa 6.16 +/- 0.03 (0.69 microM) in the presence of ATP (p less than 0.0001). Furthermore, ATP served to restore responsiveness in cells that had been rendered refractory by delaying stimulation after permeabilization. Pretreatment with PMA (an activator of PKC) inhibited the induction of a refractory state, whereas inhibition of PKC partially countered the ability of ATP to restore responsiveness, both observations pointing to a requirement for a specific component of the secretory mechanism to be in a phosphorylated state in order to condone the secretion process. These observations show that secretory mechanisms in eosinophils are similar to those in other myeloid cells, in particular neutrophils and mast cells, although the time course of secretion is more protracted.
Acetylglucosaminidase↗
Late events in regulated exocytosis.
To understand the intracellular mechanisms that control exocytosis it is necessary to have access to the cell interior. This is achieved by plasma membrane permeabilisation or by application of patch-pipettes. These conditions permit control over the cytosol composition and also allow leakage of soluble factors that may have roles in the exocytotic mechanism. Different permeabilisation methods allow different extents of leakage and therefore provide complementary data. The exocytotic machinery itself remains intact and can be activated by providing Ca2+ and/or a guanine nucleotide. In some cells there is evidence for the participation of two guanine nucleotide-binding proteins (GP and GE), as well as a Ca(2+)-binding protein. In others Ca2+ is the only requirement. In a number of cell types, ATP is not required for the late steps in the secretory pathway.
Adrenal Medulla↗
Secretory properties of eosinophils.
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Adenosine Triphosphate↗
Intracellular mechanisms regulating exocytotic secretion in mast cells.
The release of inflammatory mediators from mast cells occurs by a regulated exocytotic process. We have been able to study the intracellular events in this pathway by permeabilizing the plasma membrane of rat peritoneal mast cells and stimulating exocytosis by providing both Ca2+ and a guanine nucleotide. By this approach we have obtained evidence for the participation of at least two guanine nucleotide binding proteins in the control of exocytosis. We have also shown that ATP is unnecessary for the final events, but that it does have a number of modulatory functions, for instance in the control of the effective affinity of the proteins that bind Ca2+ and GTP. There is also evidence for a protein dephosphorylation in the later stages of the control pathway.
Adenosine Triphosphate↗
Rat mast cells degranulate in response to microinjection of guanine nucleotide.
Investigation of regulated exocytosis has frequently required the use of permeabilised cell preparations. This has provided evidence that Ca2(+)-binding and guanine nucleotide-binding proteins can mediate secretion. Since the manner and extent of membrane permeabilisation affect the requirements for Ca2+ and guanine nucleotide, we have introduced such effectors directly into intact, rat peritoneal mast cells by microinjection. During this brief procedure (approximately 1 s) a glass needle forms a seal with the plasma membrane. Following injection and withdrawal of the needle the membrane reseals without apparent loss of cell contents. Using fluorescent dye, we estimate that the volume injected is approximately 5 fl and that the dilution of injected solutes is approximately 100-fold. Injection of the nucleotides inosine triphosphate, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and guanosine 5'-[beta gamma-imido]triphosphate causes degranulation. The EC50 for GTP gamma S is approximately 10 microM (concentration in the needle) equivalent to an intracellular concentration of approximately 100 nM. However, the effect of GTP gamma S is dependent on the presence of Ca2+ in the external medium. This may be explained by a transitory influx of Ca2+ that occurs during impalement, since the seal between needle and membrane will not prevent the movement of small ions. Thus an increase in cytoplasmic Ca2+ appears to be necessary for secretion induced by GTP gamma S. Using metabolic inhibitors we have investigated the requirement for ATP. Under conditions where [ATP]i has fallen to 60 +/- 18 microM (S.E.M., n = 3) the mast cell agonist, compound 48/80, is unable to induce degranulation, yet injection of GTP gamma S still activates the cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
Intracellular application of guanosine-5'-O-(3-thiotriphosphate) induces exocytotic granule fusion in guinea pig eosinophils.
The mechanism of eosinophil secretion was studied in guinea pig eosinophils by measuring release of hexosaminidase from cell suspensions (greater than 98% pure) permeabilized with streptolysin-O and by whole-cell patch-clamp capacitance measurements. It is shown that release of eosinophil granule components occurs by an exocytotic mechanism in which individual granules fuse with the plasma membrane. Exocytosis can be induced by intracellular application of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting the involvement of a GTP-binding protein. The activation is modulated by the intracellular calcium concentration, with activation by GTP-gamma-S inducing transient elevations in the concentration of Ca2+. Thus, the nature and regulation of the release mechanism appear to be very similar to that of the mast cell and neutrophil.
Animals↗
Relationship between arachidonate generation and exocytosis in permeabilized mast cells.
Using rat mast cells permeabilized with streptolysin O we show that release of arachidonate generally occurs under similar but not identical conditions to those that cause exocytosis of beta-N-acetylglucosaminidase (hexosaminidase). Thus, hexosaminidase secretion and arachidonate release both require provision of Ca2+ together with a guanine nucleotide but exocytosis occurs at lower concentrations of both effectors. The kinetics of both processes are similar, with a delay in onset only when ATP is present. Arachidonate release occurs largely from a pool of arachidonyl phosphatidylcholine which appears to represent less than 1% of the total phosphatidylcholine of the cells. Despite the general similarity of the conditions causing exocytosis and arachidonate release, our results show that under some circumstances it is possible to obtain exocytosis without measurable release of arachidonate and that therefore phospholipase A2 activation is not an essential precursor of secretion.
Adenosine Triphosphate↗
Changes in the state of actin during the exocytotic reaction of permeabilized rat mast cells.
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.
Actin Cytoskeleton↗
ATP-dependent and ATP-independent pathways of exocytosis revealed by interchanging glutamate and chloride as the major anion in permeabilized 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.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Evidence for protein dephosphorylation as a permissive step in GTP-gamma-S-induced exocytosis from permeabilized mast cells.
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.
Adenosine Triphosphate↗
GE: a GTP-binding protein mediating exocytosis.
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Animals↗
The role and mechanism of the GTP-binding protein GE in the control of regulated exocytosis.
Recent advances in the understanding of the terminal stages of the pathway of regulated secretion (exocytosis) have depended in large part on the use of permeabilized secretory cells in which the cytosol is directly accessible to manipulation from the outside. As well as Ca2+, a role for GTP, and hence a GTP-binding protein (GE), is plainly apparent in the exocytotic mechanism of some cells. In metabolically depleted and permeabilized mast cells, the combination of Ca2+ and a guanine nucleotide are a sufficient stimulus for the release of the total cell content of histamine and hexosaminidase. ATP is not required and so a phosphorylation reaction does not comprise a necessary step in the terminal pathway. Phosphorylating nucleotides, however, can modulate the exocytotic reaction in a manner that suggests that the reverse reaction, namely protein dephosphorylation, might be an enabling step. Support for this idea is derived from experiments in which the cells are conditioned to become responsive to Ca2+ alone; in these circumstances the dependence of secretion on the presence of a guanine nucleotide can be reimposed by okadaic acid, an inhibitor of protein phosphatases.
Adenosine Triphosphate↗
Exocytosis: the role of Ca2+, GTP and ATP as regulators and modulators in the rat mast cell model.
Cells and tissues release preformed materials contained in secretory granules into the extracellular environment by the mechanism of exocytosis which involves selective fusion of perigranular membranes with the plasma membrane. While the mechanism is believed to be shared between all cells that undergo exocytotic secretion, the molecular details of the interaction between the two fusing membranes are far from being understood. Recent advances have been based on the technique of membrane permeabilization which allows the composition of the cytosol to be precisely controlled. A role for a previously unsuspected GTP-binding protein (GE) in the regulation of the terminal steps of exocytosis has thereby been revealed. Among mammalian secretory cells, the mast cell has proved to be particularly amenable for investigations in this area.
Adenosine Triphosphate↗
ATP inhibits onset of exocytosis in permeabilised mast cells.
ATP is not required for exocytosis from permeabilised mast cells, and therefore there is no direct role for protein phosphorylation in the late stages of the activation pathway. We have measured the time-course of exocytosis from permeabilised cells triggered to release hexosaminidase following addition of Ca2+ to cells equilibrated for 2 min with GTP-gamma-S. If ATP is included at the time of permeabilization, then exocytosis commences after a delay, the duration of which depends on the square root of the product [Ca2+][GTP-gamma-S], and which may extend to beyond 3 min. When ATP is excluded then the maximal rate of exocytosis is established within 3 secs of completing the effector combination. These results suggest that the achievement of a new steady-state, induced by Ca2+ and GTP-gamma-S, and required for exocytosis is inhibited by ATP. From this we conclude that dephosphorylation of an unknown regulator protein may comprise a step in the exocytotic pathway.
Adenosine Triphosphate↗
Protein phosphorylation and the dependence on Ca2+ and GTP-gamma-S for exocytosis from permeabilised mast cells.
Exocytosis in permeabilised mast cells requires only that the concentrations of Ca2+ and GTP-gamma-S (the essential effectors) are elevated into the micromolar range of concentrations. These act through an unidentified Ca2(+)-binding protein and an uncharacterized G-protein (GE). There is no requirement for ATP in the final stages of the secretory pathway. However, mast cells permeabilised in the absence of ATP rapidly become refractory to stimulation due to a reduction in the affinity for the essential effectors. Here, we show that responsiveness may be restored by the addition of ATP. The characteristics of such ATP-dependent secretion have been examined. Preincubation (prior to permeabilization) of the cells with phorbol ester enhances affinity to Ca2+, and introduction of neomycin reduces Ca2+ affinity. AMG.C16, an ether-linked analogue of diglyceride, inhibits secretion in a manner which can be partially reversed by elevating the concentration of ATP. These observations indicate that while protein phosphorylation does not comprise a step in the triggering of exocytosis, a primed condition most likely involving a state of protein phosphorylation, and maintained by reactions catalysed by protein kinase C, is essential.
Adenosine Triphosphate↗
Soluble proteins as modulators of the exocytotic reaction of permeabilised rat mast cells.
This study addresses the question of the role of cytoplasmic proteins in exocytosis from permeabilised rat mast cells. We have used two different methods of cell permeabilisation (ATP4- and streptolysin O) to regulate the size of the plasma membrane lesions, and thus to dictate the rate and extent of efflux of the cytosolic proteins, and compared the secretory response of the two preparations. We report evidence for the existence of two factors present in the cytosol, which affect the exocytotic mechanism in opposing manners. One of these is required for the maintenance of cell responsiveness; it is retained for more than 120 min by ATP4- -permeabilised cells but lost within 60 min from cells permeabilised by streptolysin O. The other factor, which leaks immediately from cells treated from streptolysin O, but only gradually from cells treated with ATP4-, has the effect of suppressing the affinity for both Ca2+ and guanine nucleotide in the exocytotic reaction.
Adenosine Triphosphate↗