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J C Metcalfe

Publications and source records attributed to J C Metcalfe.

At least 109 records · Page 6Linked to original sources
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↗
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↗
PubMed1983

Intracellular calcium measurements by 19F NMR of fluorine-labeled chelators.

Symmetrically substituted difluoro derivatives of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (nFBAPTA) show large 19F NMR chemical shifts on chelating divalent cations. The complexes of Ca2+ with 4FBAPTA and 5FBAPTA show fast and slow exchange behavior, respectively, and the chemical shift or the areas of the resonances from the free and complexed forms can be used to determine the free Ca2+ concentration. The measurement of the free Ca2+ concentration by either ligand is unaffected by free Mg2+ concentrations less than 10 mM, by pH 6-8, or by contaminating divalent ions of high affinity (Zn2+, Fe2+, Mn2+). The tetraacetoxymethyl ester derivative of 5FBAPTA was used to load mouse thymocytes with 5FBAPTA to intracellular concentrations of 1 mM, and the 19F spectrum indicated a free intracellular Ca2+ concentration [( Ca]i) of 250 nM. The [Ca]i was increased to 350 nM by addition of succinylated concanavalin A at mitogenic concentrations, and the addition of A23187 saturated the intracellular chelator with Ca2+ from the external medium. The method provides a measurement of [Ca]i and other divalent cation concentrations with direct identification of the ionic species chelated.

Animals↗
PubMed1982

Kinetic evidence for a common mechanism of capping on lymphocytes.

1. Differences in the rates at which ligands cap various receptors on the same cells, and their sensitivity to various drugs, have been interpreted as evidence that there are distinct mechanisms for ;fast' and ;slow' cap formation. We have examined the factors which determine the rate of cap formation of three receptors on mouse splenic lymphocytes or thymocytes, and compared the effects of cytochalasin B or colchicine under conditions where the different receptors cap at similar rates. 2. When surface immunoglobulin, concanavalin A receptors, or theta antigen are induced to cap at their maximal rates by appropriate concentrations of one or more cross-linking ligands, the half-time for maximal capping of each receptor population is between 1.5 and 3.0min at 37 degrees C. Slower rates of cap formation are obtained by using non-optimal concentrations of the cross-linking ligands. 3. When the three receptors were induced to cap at similar rates (either maximal or slower), 10mum-cytochalasin B caused a similar decrease in the rate of cap formation for each receptor, without affecting the eventual extent of capping. At comparable capping rates on control cells, colchicine (10mum) increased the rate of cap formation for surface immunoglobulin and concanavalin A receptors to a similar extent, without affecting the eventual extent of cap formation. In contrast, colchicine had no detectable effect on the capping of theta antigen. 4. From these results, we conclude that there are no intrinsic differences in the rates at which different receptors can be induced to cap that can be used to diagnose differences in their mechanisms of cap formation. The observation that ligand concentration and the drugs acting on the cytoskeleton generally affect the rate but not the extent of cap formation accounts for the wide variation in reported effects of the drugs on cap formation measured at fixed times. The receptor-specific effect of colchicine on surface immunoglobulin and concanavalin A receptors, but not theta antigen, is not readily compatible with models of cap formation which depend on lipid or membrane flow.

Animals↗
PubMed1981

Cholesterol in sarcoplasmic reticulum and the physiological significance of membrane fluidity.

Vesicles of sarcoplasmic reticulum from rabbit muscle can be loaded with cholesterol to at least 20 mol% with respect to endogenous sarcoplasmic-reticulum phospholipid without effect on the ATPase activity at 32 degrees C. This applies both to sarcoplasmic-reticulum vesicles in which the ATPase activity is stably coupled to Ca2+ accumulation, and to sarcoplasmic-reticulum vesicles in which the sarcoplasmic-reticulum ATPase is activated severalfold by fully uncoupling the enzyme from net Ca2+ accumulation. Since the incorporation of cholesterol causes a large decrease in fluidity of sarcoplasmic-reticulum phospholipid bilayer, these results for sarcoplasmic reticulum raise the more general question of whether bilayer fluidity is important in modulating the function of membrane proteins under physiological conditions as is widely assumed, or whether the function of membrane proteins may be effectively buffered under normal operating conditions against changes in bilayer fluidity due to extraneous agents.

Adenosine Triphosphatases↗
PubMed1981

The effect of bilayer thickness on the activity of (Na+ + K+)-ATPase.

The activities of purified (Na+ + K+)-ATPase supported by a series of phosphatidylcholines with monounsaturated (cis-9) fatty acyl chains (di(n : 1) phosphatidylcholine) varying in length from n = 12 to n = 23 were determined by the lipid titration technique. The ATPase activity at 20 degrees C decreased from 2.9 to 0.1 mumol/min per mg protein as n was decreased from 16 to 12 and decreased from 2.9 to 1.0 mumol/min per mg protein as n was increased from 20 to 23. In further experiments, the di(n : 1) phosphatidylcholine-ATPase complexes were treated with increasing proportions of n-decane, which has been shown previously to increase the thickness of black lipid membranes. n-Decane caused a large increase (greater than 20-fold) in activity of the short-chain complexes (n = 12,13); for n = 14--18, the ATPase activity first increased and subsequently decreased as the proportion of decane was increased, and for n = 20 or 23 decane caused a progressive decrease in activity with increasing concentration. These effects confirm qualitatively that a major factor determining the activity in each bilayer is its thickness. This behaviour closely parallels that of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum [1] and suggests that a major class of trans-membrane transport proteins may have a similar dependence on bilayer thickness.

Alkanes↗
PubMed1980

Cap formation by various ligands on lymphocytes shows the same dependence on high cellular ATP levels.

The effects of inhibitors of mitochondrial ATP synthesis and the calcium ionophore, A23187, on the capping of surface immunoglobulin, concanavalin A receptors and theta antigen on mouse spleen or thymus cells have been examined. (i) For all of these capping ligands and inhibitors, the cellular ATP level must be above 80% of the normal level in resting lymphocytes for 90% of maximal cap formation to occur. Below 50% of the normal ATP level, less than 10% of maximal capping occurs. There is, therefore, a common dependence for all three capping systems on the cellular ATP level, irrespective of the metabolic inhibitor used. (ii) Inhibition of cap formation by A23187 follows the same profile for ATP dependence as the mitochondrial inhibitors, but in contrast to those inhibitors, A23187 requires extracellular calcium to decrease the ATP level and inhibit capping. Other agents can affect cap formation without reducing the ATP level. For example, concanavalin A inhibits its own cap formation and cytochalasin B reduces the rate of cap formation at concentrations which do not alter the cellular ATP level. (iii) From these and other data we conclude that there are cellular functions essential for cap formation, other than the maintenance of ionic gradients, that require a high concentration of cellular ATP. The possibility that high levels of ATP are required for the function of the cytoskeleton in lymphocytes is discussed.

Adenosine Triphosphate↗
PubMed1980

Triggering of lymphocyte capping appears not to require changes in potential or ion fluxes across the plasma membrane.

Capping induced by anti-Ig antibody on mouse spleen lymphocytes was found to proceed normally over a wide range of membrane potentials from approx. 0 to -65 mV, as estimated with fluorescent probes. The potential was manipulated by ionic substitution in the medium and/or application of gramicidin. Various agents which inhibit capping had differing effects on the membrane potential, some producing no measurable change, others depolarising the cells. In particular valinomycin (10-7 M) was found to inhibit capping in cells both slightly hyperpolarised from the normal resting potential, and fully depolarised. Valinomycin was found to deplete the lymphocytes markedly of ATP and this effect was sufficient to account for the inhibition of capping. Capping occurred in a simplified (sucrose) medium lacking Na+, K+ and Ca2+, suggesting that fluxes across the plasma membrane of these ions are not required. It is concluded that after ligand binding, some reorganisation of receptor protein at the inner face of the membrane is the sufficient stimulus for the intracellular rearrangements involved in capping.

Animals↗