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S Cockcroft

Publications and source records attributed to S Cockcroft.

At least 91 records · Page 5Linked to original sources
PubMed1987

Thyroid-stimulating hormone stimulates increases in inositol phosphates as well as cyclic AMP in the FRTL-5 rat thyroid cell line.

Studies were conducted to determine whether thyroid-stimulating hormone (TSH; thyrotropin), a hormone known to increase cytosol concentrations of cyclic AMP, also stimulates the formation of inositol phosphates in thyroid cells. TSH and noradrenaline both stimulated [3H]inositol phosphate formation in a concentration-dependent manner in the rat thyroid cell line, FRTL-5 cells, which had been prelabelled with [3H]inositol. The threshold concentration of TSH required to stimulate inositol phosphate formation was more than 20 munits/ml, which is approx. 10(3)-fold greater than that required for cyclic AMP accumulation and growth in these cells. We also demonstrate that membranes prepared from FRTL-5 cells possess a guanine nucleotide-activatable polyphosphoinositide phosphodiesterase, which suggests that activation of inositide metabolism in these cells may be coupled to receptors by the G-protein, Gp. Our findings suggest that two second-messenger systems exist to mediate the action of TSH in the thyroid.

Animals↗
PubMed1987

Fluoroaluminates mimic guanosine 5'-[gamma-thio]triphosphate in activating the polyphosphoinositide phosphodiesterase of hepatocyte membranes. Role for the guanine nucleotide regulatory protein Gp in signal transduction.

Fluoride and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) both activate the hepatocyte membrane polyphosphoinositide phosphodiesterase (PPI-pde) in a concentration-dependent manner. AlCl3 enhances the fluoride effect, supporting the concept that [A1F4]- is the active species. Analysis of the products of inositol lipid hydrolysis demonstrate that phosphatidylinositol bisphosphate is the major lipid to be hydrolysed. Guanosine 5'-[beta-thio]diphosphate (GDP beta S) is an inhibitor of activation of PPI-pde by both fluoride and GTP gamma S. These observations suggest that the guanine nucleotide regulatory protein (termed Gp) bears a structural resemblance to the well-characterized G-proteins of the adenylate cyclase system and the cyclic GMP phosphodiesterase system in phototransduction.

Aluminum↗
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↗
PubMed1987

Protein and diacylglycerol phosphorylation in the stimulus-secretion coupling of rat mast cells.

The pattern of endogenous protein phosphorylation during stimulation of rat peritoneal mast cells by two types of agonists has been compared. Compound 48/80, substance P and histone, which do not require the presence of external Ca2+ to trigger histamine release, induced a similar profile of phosphorylation comprising an increased phosphorylation of a 35,000 molecular weight (MW) protein and dephosphorylation of a 15,000 MW protein. The same profile was seen when the cells were stimulated with phorbol-12-myristate-13-acetate. The phorbol ester also induced histamine release, although less than that caused by the other secretagogues. The pattern of phosphorylation shared by both the phorbol ester and the basic secretagogues represented only part of that observed when the cells were stimulated in a Ca2+-free medium with anti-IgE. Under those conditions, two additional proteins of 68,000 and 56,000 MW became phosphorylated. The phosphorylation of these two proteins increased when anti-IgE was applied in the presence of Ca2+. In contrast, the extent of phosphorylation of the 35,000 MW protein was diminished. Both the basic secretagogues and anti-IgE, but not the phorbol ester, also enhanced the production of phosphatidic acid, indicating that diacylglycerol was generated. This process was independent of the presence of external Ca2+. It is suggested that protein kinase C activation is responsible for the phosphorylation observed with the basic secretagogues but not entirely with IgE-directed ligands.

Animals↗
PubMed1987

Inositol lipid metabolism in human T lymphocytes activated via the T3 complex.

Cloned human T lymphocytes and a mitogenic monoclonal antibody (UCHT1) that binds to the T3 antigen complex were used to study the role of inositol lipid hydrolysis in T-cell activation. Binding of the T3 molecular complex with anti-T3 antibody induced the generation of inositol trisphosphate after a lag of 1 min. While this is commensurate with the rise in cytosolic Ca2+ in these cells, examination of the inositol lipid revealed that phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate occurred before the generation of inositol trisphosphate. Thus, activation of an inositol lipid kinase appears to be one of the primary signals in cellular activation in human T lymphocytes.

Antigens, Surface↗
PubMed1986

The dependence on Ca2+ of the guanine-nucleotide-activated polyphosphoinositide phosphodiesterase in neutrophil plasma membranes.

The requirement for Ca2+ for the activation of polyphosphoinositide phosphodiesterase was studied with the guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). Levels of Ca2+ that pertain in unstimulated neutrophils (100 nM) are obligatory for the full expression of enzyme activity stimulated with GTP gamma S. Reduction of Ca2+ to 1 nM leads to inhibition. Increasing the level of Ca2+ from 100 nM to 1000 nM does not alter enzyme activity. Guanosine 5'-[beta-thio]diphosphate (GDP beta S) does not stimulate the phosphodiesterase but is an effective inhibitor of activation by GTP gamma S. Ca2+ in the millimolar range can also activate the phosphodiesterase alone and this is not inhibited by GDP beta S. It is also shown that Sr2+ in the millimolar range can stimulate enzyme activity similarly to Ca2+.

Calcium↗
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

Subcellular localisation of inositol lipid kinases in rat liver.

The subcellular distribution of the enzymes which phosphorylate phosphatidylinositol sequentially to form phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was investigated in rat liver. We demonstrate that whilst phosphatidylinositol kinase is present in Golgi, lysosomes and plasma membranes, the kinase that forms phosphatidylinositol 4,5-bisphosphate is localised predominantly at the plasma membrane. The role of the inositol lipid kinases in cell function is discussed.

1-Phosphatidylinositol 4-Kinase↗
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↗
PubMed1984

The fatty acid composition of phosphatidylinositol, phosphatidate and 1,2-diacylglycerol in stimulated human neutrophils.

The fatty acid composition of phosphatidylinositol (PtdIns), phosphatidate (PtdOH) and 1,2-diacylglycerol (DG) was determined in cytochalasin B-treated human neutrophils in the presence and in the absence of formylmethionyl-leucyl-phenylalanine. The compositions of PtdOH and DG in stimulated cells resemble closely that of PtdOH in control cells and are quite different from the composition of PtdIns. DG appears to be produced as a subsequent metabolite of PtdOH and not as an intermediate in the conversion of PtdIns into PtdOH. We conclude that PtdOH is produced directly from a small pool of newly synthesized PtdIns in stimulated neutrophils.

Diglycerides↗
PubMed1984

Ca2+-dependent conversion of phosphatidylinositol to phosphatidate in neutrophils stimulated with fMet-Leu-Phe or ionophore A23187.

Human and rabbit neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and A23187 show a loss of phosphatidylinositol and an increase in phosphatidate. In cells prelabelled with 32Pi it would be expected that the newly synthesised phosphatidate would have the same specific activity as cellular ATP, provided that the loss of phosphatidylinositol is by phospholipase C attack and the resultant diacyglycerol is phosphorylated by ATP. Instead, it is demonstrated that the specific activity of newly-formed phosphatidate is less than a tenth of the specific activity of ATP initially followed by a gradual increase. The time-course of mass and of [3H]glycerol-labelled phosphatidate formation (from cells pulse-labelled with [3H]glycerol) is similar to enzyme release but differs from the generation of 32P-labelled phosphatidate (from cells prelabelled with 32Pi). The source of the newly formed phosphatidate is most likely from phosphatidylinositol because: (a) The increase in phosphatidate is always accompanied by a loss of phosphatidylinositol with no changes in the other lipids. (b) Cells pulse-labelled with [3H]glycerol lose label from phosphatidylinositol only and this is accompanied by an increase in label in phosphatidate. (c) The specific activity of the newly synthesised phosphatidate is closest to the specific activity of phosphatidylinositol. One plausible explanation for these results is that phosphatidylinositol is directly converted to phosphatidate by phospholipase D action and the resulting phosphatidate accumulates radioactivity by exchange of its phosphate group with ATP. It is also shown that enzyme secretion and conversion of phosphatidylinositol to phosphatidate can depend on both intra- as well as extracellular Ca2+. Depletion of the intracellular pool of Ca2+ is essential to inhibit totally the enzyme secretion and the conversion of phosphatidylinositol to phosphatidate in agreement with our previous results on rabbit neutrophils (Cockcroft, S., et al. (1981) Biochem. J. 200, 501-508).

Adenosine Triphosphate↗
PubMed1984

The Ca2+-activated polyphosphoinositide phosphodiesterase of human and rabbit neutrophil membranes.

Addition of Ca2+ to a plasma-membrane fraction derived from human or rabbit neutrophils led to the specific breakdown of polyphosphoinositides. The degradation products were identified as diacylglycerol and inositol bis- and tris-phosphate, thus demonstrating the presence of a Ca2+-activated phospholipase C. The newly generated diacylglycerol resembled the polyphosphoinositides in its fatty acid composition, and in the presence of MgATP2- it was converted into phosphatidate. These results therefore demonstrate the presence in neutrophil plasma membranes not only of polyphosphoinositide phosphodiesterase but also of diacylglycerol kinase.

Animals↗
PubMed1983

The fatty acid composition of 1,2-diacylglycerol and polyphosphoinositides from human erythrocyte membranes.

The fatty acid compositions of 1,2-diacylglycerol and polyphosphoinositides have been determined in human erythrocyte membranes that have been incubated in the presence or in the absence of Ca2+. The results show that the diacylglycerol that is formed in Ca2+-treated membranes has a fatty acid composition closely similar to that of the inositides, consistent with previous indications that Ca2+ stimulates the activity of a polyphosphoinositide phosphodiesterase in the membranes. In contrast with some previous results, it appears that these plasma-membrane inositides and their derived diacylglycerols are rich in stearic acid and arachidonic acid.

Calcium↗