The dual effector system for exocytosis in mast cells: Obligatory requirement for both Ca2+ and GTP

1987 ◽  
Vol 7 (5) ◽  
pp. 369-381 ◽  
Author(s):  
B. D. Gomperts ◽  
S. Cockcroft ◽  
T. W. Howell ◽  
O. Nüsse ◽  
P. E. R. Tatham
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Cellular Response ◽  
Second Messengers ◽  
Phosphoglycerate Kinase ◽  
Secretory Process ◽  
Surface Receptors ◽  
Streptolysin O ◽  
Absolute Requirement ◽  
Intracellular Second Messengers

The secretory process is a coordinated cellular response, initiated by occupation of surface receptors and comprising an ordered sequence of biochemical steps subject to multiple controls. Conceptually we can divide the sequence into two main sections comprising early, receptor-mediated events leading to generation of intracellular second messengers, and later events leading to membrane fusion and exocytosis. With the discovery that occupation of Ca2+ mobilising receptors leads to activation of polyphosphoinositide phosphodiesterase (PPI-pde) through the mediation of a G-protein (Gp), all the early events can be ascribed to the plasma membrane. Investigation of the exocytotic stage of secretion has been simplified by the use of permeabilised cells in which the composition of the cytosol can be precisely controlled. We have used streptolysin-O, a bacterial cytolysin which generates protein-sized pores in the plasma membrane, to investigate the exocytotic mechanism of rat mast cells. We find that in addition to the activation of PPI-dpe, GTP also acts in concert with Ca2+ at, or close to, the exocytotic site. Exocytosis can occur after substantial depletion of cytosol lactate dehydrogenase and 3-phosphoglycerate kinase indicating that soluble cytosol proteins are unlikely to play any role. There is no absolute requirement for ATP or phosphorylating nucleotide in exocytosis though when present the effective affinities of the two obligatory effectors (i.e. Ca2+ and GTP) are substantially enhanced.

Soluble proteins as modulators of the exocytotic reaction of permeabilised rat mast cells

1989 ◽  
Vol 94 (3) ◽  
pp. 585-591
Author(s):  
A. Koffer ◽  
B.D. Gomperts
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Soluble Proteins ◽  
The Other ◽  
Guanine Nucleotide ◽  
Cytoplasmic Proteins ◽  
Streptolysin O ◽  
Two Factors ◽  
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.

Modulation of signalling initiated by phosphoinositidase-C-linked receptors

1993 ◽  
Vol 184 (1) ◽  
pp. 145-159
Author(s):  
R. J. Wojcikiewicz ◽  
S. R. Nahorski
Keyword(s):  
Cell Surface ◽  
Second Messengers ◽  
Receptor Internalization ◽  
Intensive Study ◽  
Receptor Stimulation ◽  
Surface Receptors ◽  
Regulatory Processes ◽  
Inositol 1,4,5 Trisphosphate ◽  
Cellular Responsiveness ◽  
Intracellular Second Messengers

An extensive group of cell surface receptors are coupled to phosphoinositidase C and thus to the production of the intracellular second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. While the mechanisms and consequences of phosphoinositidase C activation have been the target of intensive study for over a decade, information is scarce regarding the regulatory processes that modulate this system during receptor stimulation. This situation, however, is now beginning to change. Recent data indicate (a) that Ca2+, mobilized concurrently with activation of phosphoinositidase-C-linked receptors, is a feedback activator and amplifier of phosphoinositide hydrolysis, (b) that rapid desensitization, possibly associated with receptor phosphorylation, regulates phosphoinositidase-C-linked receptors, (c) that receptor internalization can mediate desensitization at later times and (d) that signalling can be regulated at additional sites downstream of phosphoinositidase C. These diverse regulatory events provide the means by which the breakdown of phosphoinositides and cellular responsiveness to their products are controlled during cell stimulation.

scholarly journals Arrest of membrane fusion events in mast cells by quick-freezing.

1980 ◽  
Vol 86 (2) ◽  
pp. 666-674 ◽  
Author(s):  
D E Chandler ◽  
J E Heuser
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Membrane Fusion ◽  
Extracellular Space ◽  
Freeze Fracture ◽  
Secretory Process ◽  
Membrane Pores ◽  
Peritoneal Mast Cells ◽  
Quick Freezing ◽  
Rat Peritoneal Mast Cells

We have used quick-freezing and freeze-fracture to study early stages of exocytosis in rat peritoneal mast cells. Mast cells briefly stimulated with 48/80 (a synthetic polycation and well-known histamine-releasing agent) at 22 degrees C displayed single, narrow-necked pores (some as small as 0.05 micrometer in diameter) joining single granules with the plasma membrane. Pores that had become as large as 0.1 micrometer in diameter were clearly etchable and thus represented aqueous channels connecting the granule interior with the extracellular space. Granules exhibiting pores usually did not have wide areas of contact with the plasma membrane, and clearings of intramembrane particles, seen in chemically fixed mast cells undergoing exocytosis, were not present on either plasma or granule membranes. Fusion of interior granules later in the secretory process also appeared to involve pores; granules were often joined by one pore or a group of 2-4 pores. Also found were groups of extremely small, etchable pores on granule membranes that may represent the earliest aqueous communication between fusing granules.

Extracellular Calcium and cAMP: Second Messengers as “Third Messengers”?

Physiology ◽  
2007 ◽  
Vol 22 (5) ◽  
pp. 320-327 ◽  
Author(s):  
Aldebaran M. Hofer ◽  
Konstantinos Lefkimmiatis
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Second Messengers ◽  
Signaling Molecules ◽  
Extracellular Calcium ◽  
Cell Surface Receptors ◽  
Surface Receptors

Calcium and cyclic AMP are familiar second messengers that typically become elevated inside cells on activation of cell surface receptors. This article will explore emerging evidence that transport of these signaling molecules across the plasma membrane allows them to be recycled as “third messengers,” extending their ability to convey information in a domain outside the cell.

Practical considerations regarding the use of streptolysin-O as a permeabilising agent for cells in the investigation of exocytosis

1996 ◽  
Vol 16 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Karen Y. Larbi ◽  
Bastien D. Gomperts
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Experimental Manipulation ◽  
Cytolytic Activity ◽  
Guanine Nucleotide ◽  
Great Care ◽  
Streptolysin O ◽  
Commercial Source ◽  
Diagnostic Reagent ◽  
Substantial Extent

Streptolysin-O is widely used in cell biological investigations in order to make large (>12 nm) pores in the plasma membrane and so to render the cytosol directly accessible to experimental manipulation. We have compared the effect of streptolysin-O commercially formulated (Murex Diagnostics) as a diagnostic reagent in pathology with two pure reagents (a conventional purified protein, and a recombinant protein generated in E.coli) on exocytotic secretion from mast cells. For mast cells permeabilised by streptolysin obtained from the commercial source, exocytosis (of β-D-N-acetylglucosaminidase) is dependent on provision of both Ca2+ and a guanine nucleotide. In contrast, for cells permeabilised by either of the two pure proteins, a substantial extent of Ca2+-independent exocytosis can be elicited. When the Murex material is subject to dialysis or ultrafiltration, some secretion can be induced in the absence of Ca2+, indicating a modulatory function of the low mol wt additives of formulation, mainly phosphate and cysteine. However, Ca2+-independent exocytosis is still manifest when the pure proteins are reconstituted with ultrafiltrates from the Murex material. These observations indicate that reagents used to permeabilise cells should be characterised thoroughly and used with great care. Confirmation that the cytolytic activity of the Murex material derives from a cholesterol directed factor was demonstrated by inhibition of exocytosis when red blood cell derived (and hence cholesterol containing) sonicated liposomes were provided.

Nucleotides and divalent cations as effectors and modulators of exocytosis in permeabilized rat mast cells

1992 ◽  
Vol 336 (1276) ◽  
pp. 25-34 ◽  
Keyword(s):  
Mast Cells ◽  
Calcium Binding ◽  
Binding Protein ◽  
Divalent Cations ◽  
Guanine Nucleotides ◽  
Secretory Process ◽  
Activation Kinetics ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Kinetics Of

The idea that the universal trigger to exocytosis (the term inal step in the secretory process) is an elevation of the cytosol concentration of Ca 2+ , and that it is dependent on ATP, is no longer tenable. Working with streptolysin-O-permeabilized mast cells (and other myeloid cells) we have shown that non-hydrolysable analogues of GTP can stimulate exocytosis after depletion of Ca 2+ (i.e. at concentrations below 10 -9 m) and ATP. Such Ca 2+ - and ATP-independent exocytosis is strongly dependent on the presence of Mg 2+ , and the requirement for Mg 2+ declines as the concentration of Ca 2+ is brought up to 10 -7 m . We argue that Ca 2+ serves to regulate the binding of guanine nucleotides to G e, a GTP-binding protein that regulates exocytosis through its interaction with C E , a calcium-binding protein which serves as an intracellular pseudo-receptor. The onset of exocytosis, following provision of Ca 2+ and guanine nucleotides to the permeabilized cells, is preceded by delays which are sensitive to the order of provision of the two effectors (i.e. Ca 2+ and guanine nucleotides), the presence or absence of Mg 2+ , and the identity of the activating guanine nucleotide. In view of the similarity of these features with the activation kinetics of adenylyl cyclase, we argue that G E behaves as a member of the heterotrimeric class of signal transducing G-proteins such as G s .

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

2015 ◽  
Vol 57 ◽  
pp. 189-201 ◽  
Author(s):  
Jay Shankar ◽  
Cecile Boscher ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cancer Cell ◽  
Cellular Response ◽  
Spatial Organization ◽  
Essential Feature ◽  
Cell Signalling ◽  
Coated Pits ◽  
Signalling Molecules ◽  
Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

scholarly journals Ca2+ Microdomains, Calcineurin and the Regulation of Gene Transcription

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 875
Author(s):  
Gerald Thiel ◽  
Tobias Schmidt ◽  
Oliver G. Rössler
Keyword(s):  
Transcription Factors ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Gene Transcription ◽  
Intracellular Signaling ◽  
Second Messengers ◽  
Major Function ◽  
Surface Receptors ◽  
Dependent Protein

Ca2+ ions function as second messengers regulating many intracellular events, including neurotransmitter release, exocytosis, muscle contraction, metabolism and gene transcription. Cells of a multicellular organism express a variety of cell-surface receptors and channels that trigger an increase of the intracellular Ca2+ concentration upon stimulation. The elevated Ca2+ concentration is not uniformly distributed within the cytoplasm but is organized in subcellular microdomains with high and low concentrations of Ca2+ at different locations in the cell. Ca2+ ions are stored and released by intracellular organelles that change the concentration and distribution of Ca2+ ions. A major function of the rise in intracellular Ca2+ is the change of the genetic expression pattern of the cell via the activation of Ca2+-responsive transcription factors. It has been proposed that Ca2+-responsive transcription factors are differently affected by a rise in cytoplasmic versus nuclear Ca2+. Moreover, it has been suggested that the mode of entry determines whether an influx of Ca2+ leads to the stimulation of gene transcription. A rise in cytoplasmic Ca2+ induces an intracellular signaling cascade, involving the activation of the Ca2+/calmodulin-dependent protein phosphatase calcineurin and various protein kinases (protein kinase C, extracellular signal-regulated protein kinase, Ca2+/calmodulin-dependent protein kinases). In this review article, we discuss the concept of gene regulation via elevated Ca2+ concentration in the cytoplasm and the nucleus, the role of Ca2+ entry and the role of enzymes as signal transducers. We give particular emphasis to the regulation of gene transcription by calcineurin, linking protein dephosphorylation with Ca2+ signaling and gene expression.

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.

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