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T R Hesketh

Publications and source records attributed to T R Hesketh.

At least 37 records · Page 2Linked to original sources
PubMed1986

c-fos and c-myc gene activation, ionic signals, and DNA synthesis in thymocytes.

Among the earliest responses to mitogens that have been detected in normal quiescent cells are ionic changes: we have described rapid increases in the cytosolic free Ca2+ concentration ([Ca]i) and in the intracellular pH (pHi) in mitogen-stimulated thymocytes and fibroblasts (Hesketh, T. R., Moore, J. P., Morris, J. D. H., Taylor, M. V., Rogers, J., Smith, G. A., and Metcalfe, J. C. (1985) Nature 313, 482-484). Here we investigate the relationship between these ionic signals and the subsequent expression of the c-fos and c-myc proto-oncogenes in murine thymocytes. We show that the plant lectin concanavalin A (ConA), the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the Ca2+-ionophore A23187 each causes a rapid increase in both c-fos and c-myc mRNAs. The activation of both genes is completely dependent on the extracellular Ca2+ concentration ([Ca]o) for A23187 and independent of [Ca]o for TPA. Activation of c-myc, but not c-fos, by ConA is partially dependent on [Ca]o. The pHi increases generated by ConA or TPA are not necessary for expression of mRNA from either gene in response to these mitogens. Exogenous 8-bromo-cyclic AMP (but not 8-bromo-cyclic GMP) inhibits the c-myc responses to ConA and TPA. The data also show that neither early c-fos nor c-myc expression is sufficient to commit the cells to DNA synthesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Free cytosolic Ca2+ measurements with fluorine labelled indicators using 19FNMR.

Characterisation by 19F NMR of fluorine-labelled indicators of cytosolic free Ca2+ concentration (by 5FBAPTA) and pH (by Fquene) is described, together with the techniques used to load the cell suspensions with the indicators for NMR spectroscopy. Useful features of the 19F NMR indicators include direct identification of the intracellular cation bound to the indicators, internal calibration of [Ca]i and pHi from the spectra, and simultaneous measurements of two or more indicators in the same cell suspension. Perturbations of cellular functions by 5FBAPTA and quin 2 are very similar, but vary widely in different cell systems. The [Ca]i and pHi responses of normal and transformed cells to mitogens and growth factors in serum are compared with data from similar experiments using fluorescence indicators. The only major discrepancy in [Ca]i measurements using the two independent assays was observed in Ehrlich ascites tumour cells. These cells have a high intracellular Zn2+ content which substantially quenches the quin 2 fluorescence, but does not affect [Ca]i measurements by 5FBAPTA. The Zn2+ present in the cells is detected as a separate response in the 5FBAPTA spectrum. The time course of the Ca signal in 2H3 cells stimulated by antigen to release histamine by exocytosis has been defined using 5FBAPTA and quin 2. Extension of the 19F NMR technique to [Ca] i and pHi measurements in perfused organs is illustrated in rat heart and responses to pharmacological agents are demonstrated. Developments in prospect to improve sensitivity and to measure [Na]i with a new family of indicators are outlined.

Aminoquinolines↗
PubMed1985

Early response pattern analysis of the mitogenic pathway in lymphocytes and fibroblasts.

The early biochemical responses stimulated by the action of mitogens and growth factors on mouse thymocytes and 3T3 fibroblasts are analysed as part of a systematic attempt to define the mitogenic pathways from G0 to S phase in these cells. Although the primary response to each mitogen can be distinguished by the pattern of secondary responses they initiate, there is substantial overlap in these responses. The aim is therefore to determine whether there is early convergence on a common mitogenic pathway, defined by a sequence of responses obligatory for progression from G0 to S phase for different mitogens and cell types. The 'dual-signal' hypothesis for the mitogenic stimulation of thymocytes is a simple version of a common mitogenic pathway. It proposes that the T-cell receptor initiates the pathway via the breakdown of phosphatidylinositol (4,5)-bisphosphate to generate a Ca signal (from the release of inositol (1,4,5)-trisphosphate) and to activate protein kinase C (from the release of diacylglycerol). The rationale for this hypothesis lies in the co-mitogenic action of the Ca2+-ionophore, A23187, and the phorbol ester, 12-o-tetradecanoyl phorbol 13-acetate, which is assumed to activate specifically protein kinase C. However, detailed analysis of the coupling between some of the early responses, including the Ca and pH signals, phosphatidylinositol (4,5)-bisphosphate metabolism, c-myc gene activation and general metabolic stimulation, indicates clearly that the hypothesis is inadequate to account for the initiation of the normal mitogenic pathway in thymocytes.

Animals↗
PubMed1984

Calcium signals and phospholipid methylation in eukaryotic cells.

Rat basophil leukaemic (2H3) cells, mast cells and mouse thymocytes respond to stimulation by specific ligands with an increase in the free cytosolic Ca2+ concentration. The time courses of these Ca signals and the biological responses have been compared with changes in phospholipid metabolism. Increased phosphoinositide metabolism coincides with the Ca signals and the responses in each cell system, whereas any increase in phospholipid methylation during the response is less than one molecule per receptor and at least 5-50-fold less than the increases reported previously. Furthermore, no significant changes were detected in the concentration of S-adenosylmethionine, the methyl-group donor in the synthesis of methylated phospholipids. The hypothesis that phospholipid methylation is obligatory for receptor-mediated Ca signals is not supported by these data and requires critical re-evaluation.

Animals↗
PubMed1984

The mechanism of the calcium signal and correlation with histamine release in 2H3 cells.

Rat basophil leukemic (2H3) cells ( Siraganian , R.P., McGivney , A., Barsumian , E. L., Crews, F. T., Hirata , F., and Axelrod , J. (1982) Fed. Proc. 41, 30-34) loaded with fluorescent Ca2+ indicator quin 2 ( Tsien , R. Y. (1980) Biochemistry 19, 2396-2404) showed a rapid increase in free cytosol calcium concentration [( Ca]i) when histamine release was induced. Intracellular quin 2 concentrations up to 7 mM did not affect release of histamine in response to antigen (aggregated ovalbumin) or concanavalin A with cells primed with antigen-specific monoclonal IgE, or in response to Ca2+ ionophores. The [Ca]i increased from approximately 105 nM to a maximum of approximately 1200 nM within 2 to 3 min after antigenic stimulation and then declined slowly over 30 min toward the level in unstimulated cells. Histamine release was most rapid as [Ca]i reached the maximum value and then decreased continuously with [Ca]i over the subsequent 30 min. Neither the Ca signal nor histamine release was observed when the Ca2+ concentration in the medium [( Ca]o) was less than 50 microM, but both responses were restored on readdition of Ca2+ to 1 mM. The maximal Ca signal was obtained when [Ca]o was approximately greater than 1 mM and was half-maximal at [Ca]o congruent to 0.4 mM. In marked contrast [Ca]i in unstimulated cells varied very little with [Ca]o from 0.1 to 1 mM. Maintenance of the Ca signal required the continuous presence of stimulating ligand, external Ca2+, and the maintenance of cellular ATP; metabolic inhibitors blocked or reversed the Ca signal. La+ ions also caused a rapid and reversible block of the Ca signal and histamine release. The data are interpreted in a model in which the Ca signal is generated by a La3+-sensitive signal influx pathway that is functionally independent of the normal Ca2+ influx pathway in unstimulated cells, and that allows a 10-fold or greater increase in rate of Ca2+ entry. The Ca signal is maintained dynamically by the balance between the increased Ca2+ influx and active Ca2+ efflux across the plasma membrane.

Aminoquinolines↗
PubMed1984

The calcium signal and phosphatidylinositol breakdown in 2H3 cells.

Phosphatidylinositol (PI) and its phosphorylated derivatives are rapidly broken down in 2H3 cells stimulated with antigen, with a time course which coincides with the generation of the Ca signal. Stimulated PI breakdown is absolutely dependent on Ca2+ in the medium with a concentration dependence similar to that of the Ca signal and histamine release described in the preceding paper. However, PI breakdown does not depend on the rise in free cytoplasmic Ca2+ concentration in stimulated cells over the range 100 nM to 1 microM. Thus, stimulation by the ionophore A23187 causes only a small increase in PI breakdown and the Ca signal stimulated by antigen can be selectively blocked with appropriate concentrations of Zn2+ (100 microM) or La3+ (10-100 microM) which have small or negligible effects on stimulated PI breakdown. Both PI breakdown and the Ca signal appear to depend on a common external Ca2+ site (or sites) with Km approximately equal to 0.4 mM, and the data are consistent with either independent activation of PI phosphodiesterase and the Ca signal after antigenic stimulation, or with PI breakdown as a component of the mechanism by which the Ca signal is generated.

Animals↗
PubMed1984

Some mitogens cause rapid increases in free calcium in fibroblasts.

Quiescent 3T3 fibroblasts grown on microcarrier beads and loaded with the [Ca2+] indicator quin2 had a cytosolic free Ca2+ concentration ( [Ca2+]i) of 154 +/- 11 nM (SE; n = 32). Stimulation with the mitogens vasopressin, epidermal growth factor (EGF) or prostaglandin F2 alpha (PGF2 alpha) caused a very rapid increase in [Ca2+]i to a maximum of 200-500 nM after 60-90 s. [Ca2+]i declined thereafter to a level above that in quiescent cells which was maintained for at least 15 min. In contrast no immediate effects on [Ca2+]i were detected after the addition of the mitogens insulin or 12-O-tetradecanoylphorbol 13-acetate (TPA). These studies indicate that early changes in [Ca2+]i may be involved in the action on fibroblasts of some, but not all, mitogens.

Aminoquinolines↗
PubMed1984

Changes in free-calcium levels and pH in synaptosomes during transmitter release.

The free cytoplasmic Ca2+ concentration [( Ca2+]i) in rat brain synaptosomes estimated by the indicator quin 2 is 104 +/- 8 nM (S.D.) in artificial cerebrospinal fluid (1.2 mM Ca2+), but decreases at lower Ca2+ concentrations in the medium. The presence of quin 2 in the synaptosomes does not affect either the spontaneous release of transmitter (gamma-aminobutyric acid) or the release induced by K+ depolarisation. In quin 2-loaded synaptosomes, depolarisation by K+ causes an abrupt increase in [Ca]i (less than 2-fold) that is approximately proportional to the extent of depolarisation, whereas depolarisation by veratrine alkaloids produces a slow rise in [Ca]i. The increase in [Ca]i produced by K+ depolarisation does not occur in the absence of Ca2+ in the medium. The data are consistent with a direct correlation between [Cai] and transmitter release in functional synaptosomes. The pH in synaptosomes estimated by the indicator quene 1 is 7.04 +/- 0.07 and is stable in media containing 5 mM bicarbonate. The pH in synaptosomes was decreased by protoveratrine but not by K+ depolarisation.

Aminoquinolines↗
PubMed1984

cis-Unsaturated fatty acids uncouple mitochondria and stimulate glycolysis in intact lymphocytes.

In intact lymphocytes, linoleic acid acts as a typical mitochondrial uncoupler: it substantially decreases the mitochondrial membrane potential and the cellular ATP level, while stimulating oxygen consumption and glycolysis. Under these conditions the inhibition of cellular functions by linoleic acid cannot be attributed to its postulated effects on lipid domains in the plasma membrane.

Adenosine Triphosphate↗
PubMed1984

Stimulated release of histamine by a rat mast cell line is inhibited during mitosis.

Stimulated histamine release was depressed at least tenfold in mitotic 2H3 rat basophilic cells when compared with interphase cells even though both contained comparable amounts of histamine. Antigen stimulation of IgE-sensitized interphase cells initiated an influx of Ca2+ that preceded secretion of histamine and a similar Ca2+ influx occurred in stimulated mitotic cells. This strongly suggests that during mitosis there is a dramatic inhibition of one or more of the steps on the pathway leading from elevated intracellular Ca2+ to the fusion of secretory granules with the plasma membrane.

Animals↗
PubMed1983

Large effects of preparative techniques on lymphocyte cyclic AMP content.

When a cell suspension is formed by disruption of a pig lymph node into medium, large and transient increases in intracellular cyclic AMP occur. Similar effects are observed when pig lymphocytes are centrifuged and the cell pellets resuspended, or when the cells are subjected to rapid temperature changes. These observations define the conditions required to manipulate the cells while maintaining a stable cyclic AMP concentration. Under these conditions, neither concanavalin A nor ionophore A23187 at mitogenic concentrations have any early effect on cyclic AMP in pig lymphocytes, but small increases in cyclic AMP (less than 2-fold) were observed at supramitogenic concentrations of concanavalin A (50 microgram/ml) or A23187 (500nM). Mouse thymocytes show qualitatively similar but much smaller changes in cyclic AMP concentration in response to experimental manipulations, and no response to mitogenic or supramitogenic concentrations of concanavalin A below the cytotoxic value.

Animals↗
PubMed1983

Intracellular pH and free calcium changes in single cells using quene 1 and quin 2 probes and fluorescence microscopy.

Photometric fluorescence microscopy has been used to measure intracellular pH (pHi) and free calcium concentrations [( Ca]i) in individual mouse thymocytes and 2H3 rat basophil leukaemic cells containing indicators for pH (quene 1) or calcium (quin 2). The pHi and [Ca]i measurements in individual 2H3 cells and mouse thymocytes and their responses to various stimuli were consistent with the corresponding data obtained from suspensions of these cells measured in a spectrofluorimeter. Photometric fluorescence microscopy of these indicators in individual cells provides a sensitive and fast method of following pHi and [Ca]i responses in individual cells.

Aminoquinolines↗
PubMed1983

Duration of the calcium signal in the mitogenic stimulation of thymocytes.

An increase in the free cytoplasmic Ca2+ concentration in thymocytes can be detected by the fluorescent indicator quin 2 within a few seconds of the addition of concanavalin A and the response is quantified from the increased proportion of quin 2 in the cells chelated by Ca2+ ('% Ca-quin 2'). The % Ca-quin 2 in untreated cells is 53 +/- 6%, increases to 64 +/- 7% immediately after the addition of concanavalin A and declines spontaneously over 24 h back to the level in untreated cells (53 +/- 6%). The increase in % Ca-quin 2 in response to concanavalin A is completely blocked when 50 mM-alpha-methyl D-mannoside is added before concanavalin A and completely reversed when the competing sugar is added immediately after the mitogen. Addition of alpha-methyl D-mannoside at increasing intervals after concanavalin A addition causes a progressively smaller decrease in % Ca-quin 2 and has a negligible effect after 24 h, when the % Ca-quin 2 is the same as that in untreated cells. The decline in the calcium signal defined by these experiments has a similar time course to cap formation by concanavalin A on the cells. It is concluded that the calcium signal lasts only while concanavalin A is bound to the cell surface and is terminated either by capping or by the addition of alpha-methyl D-mannoside.

Aminoquinolines↗
PubMed1983

Limits to the early increase in free cytoplasmic calcium concentration during the mitogenic stimulation of lymphocytes.

Three aspects of the calcium hypothesis we have proposed previously [Metcalfe, Pozzan, Smith & Hesketh (1980) Biochem. Soc. Symp. 45, 1-26] for the control of mitogenic stimulation of lymphocytes are examined in studies on the mitogenic action of the Ca2+ ionophore A23187 and its effect on cap formation. (1) Pig lymphocytes that were mitogenically stimulated by continuous incubation with 3H-labelled A23187 for 48 h contained between 3 and 15 amol of ionophore per cell. Lymphocytes exposed to 3H-labelled A23187 for 2h before washing the cells and resuspending them in ionophore-free medium were only stimulated mitogenically at 48h if the residual ionophore associated with the cells after washing was in the concentration range 3-15 amol per cell. When the cells were washed repeatedly after 2h incubation with ionophore to reduce the cell-associated ionophore below the critical concentration range, no mitogenic stimulation occurred as a result of short-term exposure to any ionophore concentration. Re-addition of ionophore to within the indicated range of cell-associated concentrations restored mitogenic stimulation at 48h. We conclude that large, short-term Ca2+ fluxes into the cells induced by the ionophore cannot generate a mitogenic signal that commits the cells to enter the cell cycle. (2) Further experiments with the ionophore showed that detectable mitogenic stimulation at 48h required a minimum of 3h exposure to optimal ionophore concentrations, and that maximal stimulation required at least 20h exposure. This is consistent with the view that a prolonged increase in the free cytoplasmic calcium concentration is required to stimulate the maximum proportion of the cells into the cell cycle. (3) Mouse splenic lymphocytes treated for short periods with very high ionophore concentrations (30 microM) in the presence of various external Ca2+ concentrations showed significant inhibition of cap formation of surface immunoglobulin receptors in the range 1-10 microM-Ca2+ in normal or depolarizing medium. We conclude that mitogens at optimal concentrations for the stimulation of lymphocytes do not cause any early increase in the free cytoplasmic Ca2+ concentration above 10 microM.

Animals↗
PubMed1983

The bivalent-cation dependence of phosphatidylinositol synthesis in a cell-free system from lymphocytes.

The bivalent-cation requirements of two enzymes involved in phosphatidylinositol synthesis were defined for pig lymphocyte membranes using a citric acid buffer. CTP:phosphatidic acid cytidylyltransferase (EC 2.7.7.41) is activated by free Mn2+ concentrations above 20nM and by free Mg2+ concentrations above 10 microM. When activated by Mg2+, the enzyme is weakly inhibited by Ca2+ (Ki greater than 250 microM), but Ca2+ has no effect when Mn2+ is used to stimulate CDP-diacylglycerol synthesis. The synthesis of phosphatidylinositol from phosphatidic acid is also stimulated by Mn2+ and Mg2+ concentrations similar to those above and is inhibited by free Ca2+ concentrations above 500nM, probably by its action on CDP-diacylglycerol:inositol 3-phosphatidyltransferase (EC 2.7.8.11). Taken together, these studies suggest that under physiological conditions phosphatidylinositol synthesis is activated by Mg2+ and it is possible that it is further regulated by the free concentrations of Ca2+ and/or Mn2+.

Animals↗
PubMed1983

Intracellular pH of stimulated thymocytes measured with a new fluorescent indicator.

A new fluorescent intracellular pH indicator is described ("quene 1") which is related to the tetracarboxylate Ca2+ indicator based on the quinoline fluorophor ("quin 2"). Quene 1 has excitation and emission maxima at 390 and 530 nm, respectively, and shows a 30-fold increase in fluorescence between pH 5 and 9 with a pK alpha of 7.3. The fluorescence is insensitive to Ca2+ and Mg2+ at free concentrations up to 10(-4) M and to the proportions of Na+ and K+ at total concentrations of Na+ and K+ from 100 to 200 mM. The indicator is loaded into thymocytes using the tetraacetoxymethyl ester derivative which is hydrolyzed in the cells to give the tetracarboxylate anion. Intracellular pH can be measured at intracellular quene 1 concentrations of approximately 0.1 mM and quene 1 does not perturb glycolysis or the ATP level in resting cells at concentrations up to 0.8 mM. The intracellular pH of mouse thymocytes indicated by quene 1 is 7.15 +/- 0.04 and it is insensitive to the concentration of Ca2+ or Mg2+ in the extracellular medium. The intracellular pH decreased when the pH of the medium was lowered by addition of HCl, but was insensitive to NaOH at extracellular pH values up to 8.0. Rapid transient changes in intracellular pH are induced by NH4Cl, NaCO2CH3, or HCO3-/CO2. The thymocytes showed no early changes (within 30 min) in intracellular pH in response to mitogenic concentrations of lectins or 4 beta-phorbol-12-myristate-13-acetate.

Aminoquinolines↗
PubMed1983

Free cytoplasmic calcium concentration and the mitogenic stimulation of lymphocytes.

The effects of the lectins concanavalin A, succinylated concanavalin A, and wheat germ agglutinin on the free cytoplasmic Ca2+ concentration in mouse thymocytes were measured using the fluorescent Ca2+ indicator "quin 2" (Tsien, R. Y. (1980) Biochemistry 19, 2396-2404) and compared with the metabolic and mitogenic effects of the lectins on the cells. Within 1 min of adding each ligand, there is a dose-dependent increase in the free cytoplasmic Ca2+ concentration reported by quin 2. This response is selective for Ca2+, but it does not coincide closely with the subsequent mitogenic stimulation at 48 h by concanavalin A or succinyl concanavalin A. The nonmitogenic lectin wheat germ agglutinin also causes an increase in free cytoplasmic Ca2+ concentration and early metabolic stimulation of the cells, but stimulation is self-aborted before DNA synthesis occurs. At the intracellular concentrations of quin 2 required for measurement of the free Ca2+ concentration, the chelator causes early metabolic stimulation of the cells very similar to that produced by concanavalin A and the mitogenic Ca2+ ionophore A23187. Thus, phosphatidylinositol metabolism and lactate production are stimulated in mouse thymocytes and pig lymphocytes within 1 h of loading with quin 2 and significant increases in RNA synthesis occur after 8 h. Quin 2 causes mitogenic stimulation of pig lymphocytes measured as increased [3H]thymidine uptake at 48 h, that is variable but substantial in most experiments (up to 100% of the stimulation by A23187). The chelator itself has no significant activity as a Ca2+ ionophore, but the apparent free Ca2+ concentration in the cells increases both with the concentration of intracellular quin 2 and with the extracellular Ca2+ concentration. These data leave open the possibility that quin 2 itself affects the concentration of free Ca2+ or other cations in the cells.

Aminoquinolines↗