PubMed Health⌕ Search

Biomedical subjects

B D Gomperts

Publications and source records attributed to B D Gomperts.

At least 55 records · Page 3Linked to original sources
PubMed1988

Characterisation of the ATP4- receptor that mediates permeabilisation of rat mast cells.

ATP (as the tetrabasic acid, ATP4-) applied externally to rat mast cells causes the formation of lesions which permit influx and efflux of low molecular weight, normally impermeant aqueous solutes. To monitor membrane permeabilisation we have used two fluorescent dyes, ethidium which stains the nucleus, and TMA-DPH which stains the cytosolic surfaces of intracellular membranes following entry into the cells Permeabilisation by ATP is not affected by the metabolic status of the cells, and is maintained at temperatures as low as 8 degrees C. We have tested the ability of 30 structural analogues of ATP to effect mast cell permeabilisation. The analogues include those having substituents in the 2- and 8-positions of the purine ring, structural and optical isomers of the ribose sugar, and variations in the triphosphate chain. The pattern of selectivity displayed by the rat mast cell ATP4- receptor is distinct from those characteristic of the P1 purinoceptor for adenosine and the P2X and P2Y purinoceptors for adenine nucleotides.

Adenosine Triphosphate↗
PubMed1988

Stimulation of exocytotic degranulation by microinjection of the ras oncogene protein into rat mast cells.

To investigate the possible role of ras proteins in the secretory process, we have microinjected the proto oncogenic and oncogenic forms of the human H-ras protein into rat peritoneal mast cells. Mast cells are secretory cells which, upon appropriate stimulus, liberate histamine and other mediators of the acute inflammatory reaction by exocytotic degranulation. We report here that microinjection of the ras oncogene protein into mast cells induces exocytotic degranulation. In contrast, microinjection of similar amounts of the proto-oncogenic protein has little apparent effect on mast cells. Degranulation induced by injection of the ras oncogene protein occurs in the absence of an external stimulus and requires the presence of external calcium. The ultrastructural features of exocytotic degranulation in mast cells injected with the ras oncogene protein are similar to those seen when mast cells are activated by soluble ligands. Our results suggest that ras proteins may be involved, possibly as regulatory elements, in cellular functions that control exocytosis.

Animals↗
PubMed1987

Rat mast cells permeabilised with streptolysin O secrete histamine in response to Ca2+ at concentrations buffered in the micromolar range.

Rat peritoneal mast cells have been permeabilised by treatment with streptolysin O which generates membrane lesions of macromolecular dimensions. In the presence of Ca2+ buffered at concentrations in the micromolar range, the permeabilised mast cells release histamine, beta-N-acetylglucosaminidase and lactate dehydrogenase. Release of the two secretory components (but not lactate dehydrogenase) has an obligatory requirement for a nucleoside triphosphate and micromolar concentrations of Ca2+. Inosine triphosphate (ITP) supports the release reaction better than ATP does. It is concluded that the secretory materials are released from the cells by an exocytotic mechanism, while lactate dehydrogenase leaks from the cells through the toxin-generated lesions. By initially withholding and then supplying Ca2+ to the permeabilised cells, it is shown that the exocytotic secretory reaction can persist even when the cytosol is depleted of the bulk of soluble proteins. The streptolysin O treated mast cell preparation represents a simplified system with which to study the mechanism of exocytosis.

Acetylglucosaminidase↗
PubMed1987

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

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.

Animals↗
PubMed1987

Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilized 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.

Animals↗
PubMed1987

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.

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.

Animals↗
PubMed1987

Differential control of azurophilic and specific granule exocytosis in Sendai-virus-permeabilized rabbit neutrophils.

1. Rabbit neutrophils were permeabilized by treatment with Sendai virus. This was monitored by fluorescence measurement of the formation of the adduct of deoxyribonucleic acid (DNA) with ethidium bromide. 2. On addition of Ca2+, buffered (with EGTA) in the micromolar concentration range to the permeabilized cells, secretion of beta-glucuronidase (marker of azurophilic granules) and lysozyme (marker of specific granules) occurs. Lactate dehydrogenase (cytosol marker) is retained. Half-maximal secretion of beta-glucuronidase occurs at approximately pCa 6.3; lysozyme secretion occurs at approximately pCa 6.6. 3. Secretion is dependent on the provision of nucleoside triphosphates to the permeabilized cells. There is an absolute requirement for adenosine 5'-triphosphate (ATP) for the secretion of lysozyme, but beta-glucuronidase secretion can be partly supported by other nucleoside triphosphates in the order guanosine 5'-triphosphate (GTP) greater than uridine 5'-triphosphate (UTP) = xanthosine 5'-triphosphate (XTP) greater than cytidine 5'-triphosphate (CTP). 4. Secretion from both granules is complete within 10 min of adding Ca2+ to the permeabilized cells. There is a delay before commencement of beta-glucuronidase secretion of approximately half a minute; the secretion of lysozyme has no measurable delay.

Adenosine Triphosphate↗
PubMed1986

Potentiation and inhibition of secretion from neutrophils by phorbol ester.

We demonstrate that PMA exhibits multiple effects on fMetLeuPhe and ionophore-mediated secretion from rabbit and human neutrophils: Inhibition of receptor-mediated secretion appears to take place at an early stage in the stimulus-secretion coupling sequence since PMA inhibits the formation of phosphatidate. Protein phosphorylation is observed with both PMA and fMetLeuPhe and the extent of phosphorylation is additive in the presence of both stimuli. Another possible site of regulation by PMA lies distal to the rise in cytosolic Ca2+. This is indicated by the enhancement of inhibition by PMA of the A23187-induced secretory response at low and high external Ca2+ concentrations, respectively.

Animals↗
PubMed1986

Dual role for guanine nucleotides in stimulus-secretion coupling.

We have made mast cells and neutrophils permeable to gain access to the cytosol and thus to manipulate the composition of the cytosol. Secretion from both cell types can be triggered by elevation of cytosol Ca2+ to concentrations approaching 10-6 M; alternatively, secretion from mast cells, and of beta-glucuronidase (but not lysozyme) from neutrophils, can be triggered in the absence of Ca2+ by introducing stable analogs of GTP. We propose that GTP acts at two intracellular guanine nucleotide regulatory proteins (N proteins) in the stimulus-secretion sequence. By interaction with Np located on the inner face of the plasma membrane, it activates polyphosphoinositide phosphodiesterase to yield inositol phosphates and diacylglycerol. By interaction with Ne, situated distal to the site of action of Ca2+ and protein kinase C, it directly activates the exocytotic process without intervention of the products of polyphosphoinositide hydrolysis.

Acetylglucosaminidase↗
PubMed1985

Breakdown and synthesis of polyphosphoinositides in fMetLeuPhe-stimulated neutrophils.

The interconversions of the inositol-containing lipids (PI, PI-P and PI-P2) and their products (DG, inositol phosphates and PA) in human and rabbit neutrophils stimulated with fMetLeuPhe and PMA have been examined. PMA causes only the phosphorylation of PI to PI-P whereas fMetLeuPhe causes phosphorylation of both PI and PI-P yielding PI-P2 and the hydrolysis of all three lipids. While the predominant reaction is breakdown of PI to PA catalysed by phospholipase D, approx. 2% of PI is converted to polyphosphoinositides and then broken down by the phospholipase C route yielding inositol phosphates and DG. The latter reaction occurs without detectable lag and is a function of receptor occupancy. The amount of inositol trisphosphate thus formed would be sufficient to release Ca2+ from intracellular stores.

Animals↗
PubMed1983

Cytosol Mg2+ modulates Ca2+ ionophore induced secretion from rabbit neutrophils.

The influence of extracellular Mg2+ on Ca2+ ionophore (A23187 and ionomycin) induced secretion and changes in the cytosol pH of rabbit neutrophils suspended in Ca2+-free buffer has been investigated. While extracellular Ca2+ is obligatory for ionomycin induced secretion, we have defined conditions under which A23187 can induce secretion in Ca2+-free media. The different behaviour of these two Ca2+ ionophores is discussed on the basis of their different counter cation specificities.

Animals↗
PubMed1983

Ionophore monensin induces Na+-dependent secretion from rabbit neutrophils. requirement for intracellular Ca2+ stores.

Rabbit (and human) neutrophils release the secretory enzyme beta-glucuronidase when treated with the ionophore monensin in the presence of Na+. Release of beta-glucuronidase occurs without loss of the cytosol enzyme lactate dehydrogenase and a number of other features of the release process lead us to conclude that a normal exocytotic mechanism is involved. These include sensitivity to metabolic inhibition, enhancement of release induced by cytochalasin B and a requirement for internal sources of Ca2+ when the cells are stimulated with monensin in the absence of extracellular Ca2+. The release process due to monensin differs from that due to receptor directed agonists such as fMet-Leu-Phe and the Ca2+ ionophores A23187 and ionomycin in respect of a prolonged time-course which extends over 20 min; nor do monensin-stimulated neutrophils generate the superoxide anion. The results are discussed in the light of reports which indicate a rôle for Na+ in the activation of neutrophils by other ligands.

Animals↗
PubMed1983

Detergent solubilisation of the rabbit neutrophil receptor for chemotactic formyl peptides.

Digitonin was found to be the only detergent (out of 24 tested) capable of solubilising the chemotactic formyl peptide receptor from rabbit neutrophil membranes in a form which retained its [3H]fMet-Leu-Phe binding activity. The solubilised material retained many of the characteristics of the membrane-bound receptor. [3H]fMet-Leu-Phe binding to the digitonin extract was measured at 4 degrees C using an equilibrium dialysis assay. Binding was saturable and of high affinity (Kd = 3.5 +/- 0.7 nM). The potencies of a series of synthetic peptides as inhibitors of [3H]fMet-Leu-Phe binding to the soluble receptor showed the same rank order as for inhibition of the membrane-bound receptor. In addition, binding to both preparations was sulphydryl dependent showing a parallel inhibition by p-chloromercuribenzene sulphonate which could be partially reversed by subsequent incubation with dithiothreitol.

Animals↗
PubMed1983

Rat mast cells permeabilized with Sendai virus secrete histamine in response to Ca2+ buffered in the micromolar range.

In the presence of low extracellular Ca2+, Sendai virus generates permeability lesions in the membrane of rat mast cells. This causes leakage of intracellular phosphorylated metabolites and lactate dehydrogenase; it also permits uptake of normally impermeant aqueous solutes such as the complex of Ca2+ with N-hydroxyethylethylenediaminetriacetic acid. We have used this system to buffer the concentration of cytosol Ca2+ in the micromolar range and thus to cause release of the contents of the secretory granules such as histamine and beta-N-acetylglucosaminidase. We argue that such release occurs by a normal exocytotic secretory mechanism for the following reasons. (1) While leakage of cytosol components is progressively inhibited by Ca2+ in the range 1-10 microM, release of histamine is dependent on Ca2+ in this range. Higher concentrations of Ca2+ are inhibitory to both permeabilization and histamine release. (2) While leakage of phosphorylated metabolites is enhanced in metabolically inhibited cells and the leakage of lactate dehydrogenase is insensitive to metabolic inhibition, the release of histamine and beta-N-acetylglucosaminidase is strictly dependent on an intact metabolic process.

Animals↗
PubMed1982

Plasma-membrane location of phosphatidylinositol hydrolysis in rabbit neutrophils stimulated with formylmethionyl-leucylphenylalanine.

Rabbit peritoneal neutrophils, disrupted by sonication, were separated into three subcellular fractions by sucrose-step-gradient centrifugation and these were analysed with respect to biochemical markers. They comprised a high-speed supernatant containing the cytosol, a light particulate fraction enriched in Golgi and plasma membranes and a heavy particulate fraction enriched in granules and nuclei. The light particulate fraction was further separated into its components, which were identified as Golgi membranes (galactosyltransferase activity) and plasma membranes ((radioactivity derived from labelling intact cells with [125I]di-iodosulphanilic acid diazonium salt and [3H]formylmethionyl-leucylphenylalanine ([3H]fMet-Leu-Phe) binding)). In cells prelabelled with [3H]glycerol, the hydrolysis of phosphatidylinositol due to cell stimulation with fMet-Leu-Phe (10 nM) was shown to occur in the light particulate fraction. The [32P]Pi-labelling of phosphatidate, which is an early consequence of phosphatidylinositol hydrolysis, also occurred in this fraction. Analytical sucrose-gradient centrifugation of the light particulate fraction showed that the stimulated increment in [32P]phosphatidate (and thus by implication the initial phosphatidylinositol breakdown) was localized in the plasma membrane.

Animals↗
PubMed1981

The dependence on Ca2+ of phosphatidylinositol breakdown and enzyme secretion in rabbit neutrophils stimulated by formylmethionyl-leucylphenylalanine or ionomycin.

1. We have measured the breakdown of [3H]phosphatidylinositol in rabbit neutrophils prelabelled with [3H]glycerol by a pulse-chase procedure. With a view to defining a possible causal relationship between phosphatidylinositol breakdown and enzyme secretion in these cells, we have compared the characteristics of both these processes induced by either the receptor-directed agonist formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) or the Ca2+-ionophore ionomycin. 2. The dependence on fMet-Leu-Phe concentration of phosphatidylinositol breakdown and secretion is identical (half-maximal at 0.3 nM). This is 30-fold less than that required for half-maximal occupation of receptors. 3. Both secretion and breakdown of phosphatidylinositol due to fMet-Leu-Phe are modulated by extracellular Ca2+. The sensitivity to Ca2+ of both processes is enhanced by pretreatment to deplete cell Ca2+. The concentration of Ca2+ required to cause half-maximal effects of both processes in Ca2+-depleted cells on stimulation with 1nM-fMet-Leu-Phe is 100 microM. Ionomycin-stimulated secretion and breakdown of phosphatidylinositol are completely dependent on extracellular Ca2+ over similar concentration ranges. 4. Both secretion and phosphatidylinositol breakdown due to fMet-Leu-Phe approach completion by 10s. With ionomycin these processes are slower, terminating by 2 min. 5. In the presence of [32P]Pi, labelling of [32P]phosphatidic acid reaches a maximum 15 min after stimulation with either fMet-Leu-Phe or ionomycin. This precedes the labelling of [32P]phosphatidylinositol and shows the expected precursor-product relationship. 6. We conclude from these results that in rabbit neutrophils a rise in cytosol [Ca2+] is both sufficient and necessary to cause secretion and phosphatidylinositol breakdown. In cells depleted of Ca2+, the occupation of receptors by fMet-Leu-Phe is without effect on these two processes.

Animals↗
PubMed1981

A synthetic ionophore for Ca2+: studies with model and biological systems.

We show that the relatively simple beta-diketone, 1.1.1.2.2.3.3-heptafluoro-7,7-dimethyloctane-4,6-dione, known as "fod", will solvate Ca2+ ions in an organic phase. In consequence it will transport Ca2+ across phospholipid bilayers. It acts as predicted for a Ca2+-transporting ionophore in two biological systems: it will stimulate Ca2+-dependent K+ efflux from human red blood cells, and Ca2+-dependent histamine secretion from rat peritoneal mast cells.

Animals↗