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T Pozzan

Publications and source records attributed to T Pozzan.

At least 163 records · Page 9Linked to original sources
PubMed1984

Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease.

Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.

Calcium↗
PubMed1983

Mechanism of activation of pyruvate dehydrogenase by mitogens in pig lymphocytes.

The activity of pyruvate dehydrogenase in extracts of pig mesenteric lymphocytes was measured under different preincubation conditions. The mitogens concanavalin A and ionophore A23187 both increased pyruvate dehydrogenase activity. In both cases activation required extracellular Ca2+. Digitonin-permeabilized cells required 0.5 microM free Ca2+ for half-maximal activation of pyruvate dehydrogenase. The stimulation by concanavalin A in intact cells was probably not due to changes in effectors of pyruvate dehydrogenase kinase. This evidence suggests that activation of pyruvate dehydrogenase is by Ca2+ activation of pyruvate dehydrogenase phosphatase and supports the view that the cytoplasmic free [Ca2+] rises to something less than 1 microM on stimulation with mitogens.

Animals↗
PubMed1983

Is cytosolic ionized calcium regulating neutrophil activation?

The concentration of cytosolic ionized calcium, [Ca2+]i, was measured in intact neutrophils by use of a fluorescent indicator trapped in the icytoplasm. A given rise of [Ca2+]i elicited by the chemotactic peptide formylmethionylleucylphenylalanine (FMLP) was associated with a much greater degree of superoxide generation and myeloperoxidase secretion than was the same or larger [Ca2+]i produced by a specific calcium ionophore, ionomycin, which bypasses cell surface receptors. Thus, FMLP appears to generate some important excitatory signal in addition to a rise in [Ca2+]i and exocytosis and superoxide generation in neutrophils may not be simply dependent on [Ca2+]i as is widely supposed.

Calcium↗
PubMed1983

The nature of the electron spin resonance signal during aerobic uptake of Mn2+ in mitochondria from rat liver.

Rat liver mitochondria take up aerobically large amounts of divalent cations in the absence of exogenous phosphate. The electron spin resonance (ESR) spectrum of matrix Mn2+ reveals the presence of two components: one, a sextet signal, corresponding to hydrated Mn2+; another, a spin exchange signal, attributed either to Mn2+ binding to specific high-energy membrane sites or to complexes of Mn2+ with inorganic phosphate. Identification of the spin exchange signal with a Mn-Pi complex is favoured by the evidence that the spin exchange signal is observed at pH 7.5 but not at pH 6.5 in the absence of exogenous Pi, but at both pH 7.5 and 6.5 in the presence of exogenous Pi. On the other hand both in the absence or presence of exogenous Pi inhibition by N-ethylmaleimide of Pi transport, abolishes the spin exchange signal. This signal is again observed when Pi is generated in the matrix, in the presence of N-ethylmaleimide, by ATP hydrolysis, and again abolished by oligomycin. Finally, addition of uncouplers results in a very slow disappearance of the signal. The amount of Mn2+ participating in the spin exchange signal has been calculated to be in the range of 50-60 nmol X mg protein-1. This amount is compatible with the amount of endogenous Pi present or generated in average mitochondrial preparations. The ESR spectrum obtained by superimposing the spectra of Mn3(PO4)2 precipitate and hydrated Mn2+, in appropriate concentrations and ratios, resembles closely the ESR spectrum during aerobic Mn2+ uptake in mitochondria. The band width of the spin exchange signal of Mn3(PO4)2 is not constant and varies between 40 and 22 mT depending on the state of aggregation of the complex. The kinetics of aggregation can be followed in solution as a function of the concentration of Mn2+, Pi and of pH. Similar kinetics can also be followed during aerobic Mn2+ uptake by controlling the rate of Mn2+ influx. The present data support the previous proposal [Pozzan et al. (1976) Eur. J. Biochem. 71, 93-99] that the spin exchange signal is essentially due to a Mn3(PO4)2 precipitate in the mitochondrial matrix.

Adenosine Triphosphate↗
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↗
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↗
PubMed1982

Effect of ionophores on lymphocyte cellular metabolism.

The effect of valinomycin, nigericin, gramicidin S and D, A23187 and X537A on respiration and cellular ATP content of rat spleen lymphocytes is presented. It has been found that while valinomycin and nigericin interfere with mitochondrial functions gramicidin D does not show an appreciable effect. These results are explained in terms of different ability of ionophores to re-distribute among intracellular membranes. A23187 and X537A, added with Ca2+, strongly enhanced O2 consumption and reduced cellular ATP content.

Animals↗
PubMed1982

Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator.

A new, fluorescent, highly selective Ca2+ indicator , "quin2", has been trapped inside intact mouse and pig lymphocytes, to measure and manipulate cytoplasmic free Ca2+ concentrations, [Ca2+]i. Quin2 is a tetracarboxylic acid which binds Ca2+ with 1:1 stoichiometry and an effective dissociation constant of 115 nM in a cationic background mimicking cytoplasm. Its fluorescence signal (excitation 339 nm, emission 492 nm) increases about fivefold going from Ca-free to CA-saturated forms. Cells are loaded with quin2 by incubation with its acetoxymethyl ester, which readily permeates the membrane and is hydrolyzed in the cytoplasm, thus trapping the impermeant quin2 there. The intracellular quin2 appears to be free in cytoplasm, not bound to membranes and not sequestered inside organelles. The fluorescence signal from resting cells indicates a [Ca2+]i of near 120 nM. The millimolar loadings of quin2 needed for accurately calibrated signals do not seem to perturb steady-state [Ca2+]i, but do somewhat slow or blunt [Ca2+]i transients. Loadings of up to 2mM are without serious toxic effects, though above this level some lowering of cellular ATP is observed. [Ca2+]i was well stabilized in the face of large changes in external Ca2+. Alterations of Na+ gradients, membrane potential, or intracellular pH had little effect. Mitochondrial poisons produced a small increase in [Ca2+]i, probably due mostly to the effects of severe ATP depletion on the plasma membrane. Thus intracellulary trapped chelators like quin2 offer a method to measure or buffer [Ca2+]i in hitherto intractable cell types.

Aminoquinolines↗
PubMed1982

Anti-immunoglobulin, cytoplasmic free calcium, and capping in B lymphocytes.

This paper examines, in mouse spleen lymphocytes, the effect of anti-immunoglobulin (anti-Ig) on the cytoplasmic free calcium concentration, [Ca2+]i, measured with the fluorescent indicator quin2, and the relationship of [Ca2+]i to the capping of surface Ig. Anti-Ig causes a rapid rise of [Ca2+], which precedes capping. Assuming that only those 40-50% of the cells which can bind anti-Ig (the B cells) undergo a [Ca2+]i response, [Ca2+]i in those cells approaches 500 nM. It declines to resting levels over many minutes, roughly paralleling the formation of caps. Part of the [Ca2+]i signal is due to stimulated influx across the plasma membrane, since in Ca2+-free medium, anti-Ig gives a smaller and shorter [Ca2+]i rise. The amplitude of this reduced transient now varies inversely with quin2 content, as if some 0.25 mmol Ca per liter of cells was released into the cytoplasm from internal stores. These stores are probably sequestered in organelles since A23187 in Ca2+-free medium also causes a transient [Ca2+]i rise after which anti-Ig has no effect. These organelles seem not to be mitochondria because uncouplers have hardly any effect on [Ca2+]i. Though anti-Ig normally raises [Ca2+]i before causing capping, there seems to be no causal link between the two events. Cells in Ca2+-free medium whose stores have been emptied by A23187, still cap with anti-Ig even though there is no [Ca2+]i rise. Cells loaded with quin2 in the absence of external Ca2+ still cap anti-Ig normally even though their [Ca2+]i remains steady at below 30 nM, four times lower than normal resting [Ca2+]i.

Adenosine Triphosphate↗
PubMed1982

Cytoplasmic pH and free Mg2+ in lymphocytes.

Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31P nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly-trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37 degrees C, pHi was close to 7.0. Addition of physiological levels of HCO3- and CO2 transiently acidified the cells by approximately 0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]i was assessed in three ways: (a) from the external Mg2+ null-point at which the ionophore A23187 produced no net movement of Mg2+ or H+; (b) by Mg-sensitive electrode measurements in freeze-thawed pellets; and (c) from the 31P nuclear magnetic resonance spectrum of the gamma-phosphate of intracellular ATP. Total cell Mg2+ was approximately 12 mmol per liter cell water. The NMR data indicated [Mg2+]i greater than 0.5 mM. The null-point method gave [Mg2+]i approximately 0.9 nM. The electrode measurements gave 1.35 mM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg2+.

Animals↗
PubMed1981

Effect of monovalent cation ionophores on lymphocyte cellular metabolism.

The effect of valinomycin, nigericin and gramicidin on the cellular O2 consumption and on ATP content has been investigation. It has been found that while valinomycin and nigericin interfere with mitochondrial functions, gramicidin D does not show any appreciable effect. These results are explained in terms of the differing abilities of ionophores to redistribute among intracellular membranes.

Adenosine Triphosphate↗
PubMed1980

cis-Unsaturated fatty acids inhibit cap formation on lymphocytes by depleting cellular ATP.

The inhibition of anti-immunoglobulin cap formation on lymphocytes by cis-unsaturated fatty acids, but not saturated or trans-unsaturated fatty acids, which recently been reported (Klausner, R. N., Bhalla, D. K., Dragsten, P., Hoover, R. L., and Karnovsky, M. J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 437-441), can be accounted for quantitatively by the effects of the fatty acids on ATP levels in the intact cells. Only the cis-unsaturated fatty acids lowered the cellular ATP level sufficiently (to < 80%) to inhibit cap formation significantly. The profile for the dependence of cap formation on cellular ATP level obtained with the fatty acids is very similar to the corresponding profile described previously for several capping ligands and a range of metabolic inhibitors used to depress the cellular ATP levels. Oxygen electrode experiments indicate that the unsaturated, but not the saturated, fatty acids can uncouple lymphocyte mitochondria in intact cells, whereas both types of fatty acids can uncouple isolated rat liver mitochondria. The difference in the effect of the two types of fatty acids on ATP levels in the intact cells is attributed to the inability of the saturated fatty acids to penetrate in sufficient concentrations to the mitochondria to cause uncoupling. The protective action of external calcium against inhibition of capping by the free fatty acids is attributed to the effect of calcium in cross-bridging the anionic fatty acids and reducing their effective concentration. These interpretations account for all of the experimental data and are much simpler than the model proposed by Klausner et al.

Adenosine Triphosphate↗
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↗