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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Intracellular application of guanosine-5'-O-(3-thiotriphosphate) induces exocytotic granule fusion in guinea pig eosinophils. 213 56

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.
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PMID:Changes in the state of actin during the exocytotic reaction of permeabilized rat mast cells. 239 68

1. Stimulation of mast cells by externally applied secretagogues activated a slowly developing membrane current. With high external and low internal chloride (Cl-) concentrations, the current reversed at about -40 mV, but when external Cl- was made equal to internal Cl-, the reversal potential shifted to about 0 mV, demonstrating that the current carrier was Cl-. 2. In addition to external agonists, internally applied cyclic AMP and high concentrations of intracellular calcium [Ca2+]i could also activate the Cl- current. However, elevated [Ca2+]i produced only slow and incomplete activation. This suggests that the Cl- current is not directly Ca2+ activated. Also, activation of Cl- current by external agonists and by cyclic AMP was unimpaired when [Ca2+]i was clamped to low levels with internal ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA), indicating that elevated [Ca2+]i is not necessary for activation of the Cl- current. Although activation by cyclic AMP was faster than that produced by elevated [Ca2+]i, it still required tens of seconds; thus the effect of cyclic AMP was also likely to be indirect. 3. Internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) could also activate the Cl- current, suggesting the involvement of a G protein in the control of the current. 4. The variance associated with the Cl- current was small, and noise analysis gave a lower limit of about 1-2 pS for the single-channel conductance. The Cl- current was reduced by 4,4'-diisothiocyano-2,2'-stilbenedisulphonate (DIDS), and during DIDS blockade, the variance of the current increased. This suggests that DIDS enters and blocks the open channel. 5. Activation of the Cl- current would make the membrane potential negative following stimulation of a mast cell, thus providing a driving force for entry of external calcium via the stimulation-induced influx pathways described in the preceding paper (Matthews, Neher & Penner, 1989).
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PMID:Chloride conductance activated by external agonists and internal messengers in rat peritoneal mast cells. 255 69

Interaction of ligands with 'Ca2+-mobilizing' receptors is thought to result in the generation of two second messengers, inositol trisphosphate and diacylglycerol, from a common substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) (refs 1, 2), a component of plasma membranes. It is not known how the occupation of such receptors is translated into the activation of the catalytic unit polyphosphoinositide (PPI) phosphodiesterase, and then to cellular activation, but our recent experiments suggest that GTP regulatory proteins may be involved. In mast cells, non-hydrolysable analogues of GTP introduced and then trapped in the cytosol are able to substitute for external ligands in inducing exocytosis, a well-defined Ca2+-dependent process, suggesting that guanine nucleotide regulatory proteins may act by stimulating the catalytic activity of the PPI phosphodiesterase. We now provide evidence that mast cell secretion is inhibited by internalized neomycin, a compound known to interact with PPI. We also show that the PPI phosphodiesterase of human neutrophil plasma membranes can be activated simply by adding GTP analogues in the presence of concentrations of Ca2+ that pertain in unstimulated cells. These findings strongly support the idea that the coupling factor linking receptor and PPI phosphodiesterase is a guanine nucleotide binding protein analogous to those involved in the activation and inhibition of adenylate cyclase.
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PMID:Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase. 298 3

The patch-clamp technique was used to investigate the secretory responses of rat peritoneal mast cells at various intracellular calcium concentrations ([Ca2+]i). When Calcium was introduced into the cell with pipette-loaded dibromo-BAPTA, elevation of [Ca2+]i into the range 1-10 microM induced membrane capacitance increases indicative of exocytosis in a concentration-dependent manner. At higher concentrations a decrease of the response was observed. Cells that were exposed to micromolar [Ca2+]i underwent morphological alterations resulting in swelling, which is indicative of cytoskeletal alterations. The presence of dibromo-BAPTA (4 mM) strongly inhibited secretion induced by GTP-gamma-S, thus hampering the contribution of G-protein-mediated stimulation. Application of the Ca2+ ionophore ionomycin resulted in transient increases in [Ca2+]i which were parallelled by Ca2+-dependent secretion. Effective buffering of the cytosolic calcium level below 1 microM abolished the secretory response. Our results show that an increase in [Ca2+]i can trigger secretion, but only if it is high and sustained. During physiological stimulation, however, secretion proceeds at [Ca2+]i below 1 microM. It is, therefore, concluded that mast cell degranulation under physiological conditions is not simply a result of an increase in [Ca2+]i, but that other second messenger systems in conjunction with calcium act synergistically in order to ensure fast and efficient secretion.
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PMID:Secretory responses of rat peritoneal mast cells to high intracellular calcium. 312 72

The guanine nucleotide-binding proteins (G proteins), which transduce hormonal and light signals across the plasma membrane, are heterotrimers composed of alpha, beta, and gamma subunits. Activation of G proteins by guanine nucleotides is accompanied by dissociation of the heterotrimer: G + alpha.beta.gamma in equilibrium alpha G + beta.gamma. Brain contains several G proteins of which the most abundant are alpha 39.beta.gamma and alpha 41.beta.gamma. We have used proteolysis by trypsin to study the functional domains of the alpha subunits. In the presence of guanosine 5'-(3-O-thio)triphosphate, trypsin removes a 2-kDa peptide from the amino terminus of these proteins (Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D., and Gilman, A. G. (1984) Science 226, 860-862; Winslow, J. W., Van Amsterdam, J. R., and Neer, E. J. (1986) J. Biol. Chem. 261, 7571-7579). Tryptic cleavage does not affect the GTPase activity of the truncated molecule nor the apparent Km for GTP. However, removal of the 2-kDa amino-terminal peptide prevents association of the alpha subunits with beta.gamma. Since the apparent substrate for pertussis toxin-catalyzed ADP-ribosylation is the alpha.beta.gamma heterotrimer, the trypsin-cleaved alpha subunit is not a substrate for the toxin. Digestion of the carboxyl terminus of alpha 39 with carboxypeptidase A prevents ADP-ribosylation by pertussis toxin but does not interfere with the formation of alpha 39.beta.gamma heterotrimers. We do not yet know whether the amino-terminal region of alpha 39 interacts with beta gamma directly or whether it is necessary to maintain a conformation of alpha 39 which is required for heterotrimer formation. Further studies are needed to define the nature of the contracts between alpha and beta gamma subunits since understanding the structural basis for their reversible interaction is fundamental to understanding their function.
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PMID:The amino terminus of G protein alpha subunits is required for interaction with beta gamma. 313 54

1. Mast cells, isolated from rat peritoneum, were studied under tight-seal, whole-cell recording conditions. Membrane conductance, membrane capacitance and the concentration of free intracellular Ca2+, [Ca2+]i, were measured simultaneously. 2. [Ca2+]i could be accurately buffered to values between 0 and 1.5 microM only if relatively high concentrations of calcium buffers (in the millimolar range) were added to the pipette filling solution against which the cytoplasm was dialysed. At lower buffer concentrations [Ca2+]i was markedly increased by hyperpolarizing the membrane. 3. When added to the pipette, guanosine-3-thio-triphosphate (GTP-gamma-S), a nonhydrolysable analogue of guanosine triphosphate, stimulated a 3.3-fold increase in membrane capacitance, which is indicative of mast cell degranulation (Fernandez, Neher & Gomperts, 1984). 4. In weakly buffered cells, GTP-gamma-S also induced a transient increase in [Ca2+]i which, usually, preceded degranulation. Calcium buffers at 1-5 mM concentration suppressed this transient. 5. High [Ca2+]i alone did not induce degranulation. However, it markedly accelerated GTP-gamma-S-induced degranulation. When [Ca2+]i was buffered to zero, an appreciable fraction of cells degranulated in response to GTP-gamma-S, but very slowly, and only after a long lag phase. 6. Transient increases in [Ca2+]i, evoked either by GTP-gamma-S, or by voltage changes, did not elicit capacitance changes during the lag phase, but accelerated the GTP-gamma-S-induced degranulation response at later times. 7. Internally applied inositol 1,4,5-trisphosphate (IP3) also induced transient increases in [Ca2+]i which did not lead to secretion in the absence of GTP-gamma-S. 8. It is concluded that an increase in [Ca2+]i is neither necessary nor sufficient for secretion from dialysed mast cells. [Ca2+]i, however, acts synergistically with other stimuli to promote secretion. It is the more efficient the more time the other stimulus had been allowed for priming the cell.
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PMID:The influence of intracellular calcium concentration on degranulation of dialysed mast cells from rat peritoneum. 313 23

An unusual type of posttranslational modification has been observed in a rat brain in vitro system. It consists in leucine addition to a preformed protein in such a way that the added leucine is not located at either the NH2 or the COOH terminus of the acceptor protein. The incorporation reaction requires ATP, ATP-generating components and tRNA. It is inhibited by aurintricarboxylic acid but does not require the presence of ribosomes or GTP. The incorporated leucine has a free NH2 group, and it is not released by leucine aminopeptidase or carboxypeptidase A. It is linked to the acceptor protein through a bond that is too alkali labile and too hydroxylamine labile to be a peptide bond. The simplest interpretation of the results consists in proposing that an ester bond is formed between the leucine and the side chain of a serine, threonine, or tyrosine in the acceptor protein.
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PMID:Transfer ribonucleic acid dependent but ribosome-independent leucine incorporation into rat brain protein. 717 78

Rab proteins are ras-like low molecular mass GTP-binding proteins, which are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. We have previously shown that synthetic peptides, corresponding to the effector domain of Rab3 proteins, stimulate a complete exocytotic response in mast cells. We have used a PCR-cloning strategy to investigate the presence of mRNA encoding Rab3 in mast cells. RNA based PCR was then performed on mast cell RNA using degenerate oligonucleotide primers based on two conserved sequences among Rab3 proteins. However, no PCR products were obtained, even for proteins known to be expressed in high copy numbers in mast cells (beta-actin and Fc receptor). We have found that the problem resides in the presence of mast cell secretory granule derived heparin, that copurifies with the RNA; heparin has been shown to inhibit the activity of reverse transcriptase and Taq polymerase in PCR. After treating the RNA (obtained from about 500 mast cells) with heparinase, several PCR products of varying size were obtained using primers specific for Rab3 proteins. These products were cloned and sequenced. We have found clones containing sequences that had a 100% homology at the deduced amino acid level to a portion of Rab3B and Rab3D (amino acids 16 to 83).
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PMID:RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells. 750 66

Stimulation of the mast cell line, RBL-2H3, with antigen via the tetrameric (alpha beta gamma 2) immunoglobulin E receptor (Fc epsilon R1) leads to the activation of cytosolic phospholipase A2 and the release of arachidonic acid. This pathway is dependent on the activation of the mitogen-activated protein (MAP) kinase. In this paper, we show that the MAP kinase/cytosolic phospholipase A2 pathway is linked to Fc epsilon R1 via the cytosolic tyrosine kinase, Syk, and that the GDP/GTP exchange factor, Vav, might be one candidate for accomplishing this link. Cross-linking of transmembrane chimeras containing the Fc epsilon R1 gamma motif, which is known to activate Syk, results in the tyrosine phosphorylation of Vav, activation of MAP kinase, and release of arachidonic acid. Cross-linking of chimeras containing the Fc epsilon R1 beta motif does not cause these events. Furthermore, stimulation of these events by antigen is enhanced by transient overexpression of a wild-type form of Syk and blocked by overexpression of a dominant negative form of Syk. By contrast, stimulation via the transfected, G protein-coupled, muscarinic m1 receptor is not influenced by either form of Syk and does not result in tyrosine phosphorylation of Vav. These data establish unequivocally that the two types of receptor are independently linked to the two types of receptor are independently linked to the MAP kinase/cytosolic phospholipase A2 pathway and demonstrate the existence of the Fc epsilon R1-Syk-MAP kinase pathway.
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PMID:A requirement for Syk in the activation of the microtubule-associated protein kinase/phospholipase A2 pathway by Fc epsilon R1 is not shared by a G protein-coupled receptor. 753 41


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