Biomedical subjects
T Pozzan
Publications and source records attributed to T Pozzan.
Intracellular Ca2+ pools in PC12 cells. A unique, rapidly exchanging pool is sensitive to both inositol 1,4,5-trisphosphate and caffeine-ryanodine.
Release of Ca2+ from intracellular stores was studied in the parent PC12 cell line and in recently isolated clones sensitive or insensitive to caffeine. In the caffeine-sensitive cells the cytosolic free Ca2+ concentration ([Ca2+]i) responses by the xanthine drug and by stimulants of receptors coupled to inositol 1,4,5-trisphosphate (Ins-P3) generation (bradykinin, ATP) depend on separate pathways because 1) caffeine does not stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate and 2) Ca(2+)-induced Ca2+ release, the process activated by caffeine, plays no major role in the Ins-P3-induced Ca2+ mobilization. Although distinct, these two mechanisms converge onto the same Ca2+ store. In fact 1) the [Ca2+]i responses by receptor agonists and caffeine were not additive; 2) either type of agent reduced (up to complete inhibition) the response to a subsequent administration of the same or the other agent; 3) all these responses were prevented by selective Ca2+ ATPase blockers; 4) ryanodine, which affects the intracellular Ca2+ channel sensitive to caffeine, also induced depletion of the receptor-sensitive Ca2+ pool; 5) in the 10 PC12 clones tested, sensitivity to caffeine paralleled ryanodine sensitivity. Therefore, PC12 cells, similar to some smooth muscle fibers but at variance with neurons and other secretory cells, express a single, rapidly exchanging Ca2+ store in which two distinct intracellular Ca2+ channels, i.e. the receptors for caffeine-ryanodine and Ins-P3, appear to be colocalized.
Intracellular Ca2+ pools in PC12 cells. Three intracellular pools are distinguished by their turnover and mechanisms of Ca2+ accumulation, storage, and release.
Three, non-cytosolic Ca2+ pools were characterized in intact PC12 cells. The first pool, sensitive to both inositol 1,4,5-trisphosphate and caffeine (Zacchetti, D., Clementi, E., Fasolato, C., Zottini, M., Grohovaz, F., Fumagalli, G., Pozzan, T., and Meldolesi, J. (1991) J. Biol. Chem. 266, 20152-20158) accounts for approximately equal to 200 microM of Ca2+/liter of cell water (less than 30% of total exchangeable Ca2+) and takes up Ca2+ from the cytosol via a Ca(2+)-ATPase, blocked by thapsigargin. A second pool, approximately equal to 400 microM/liter, is insensitive to both inositol 1,4,5-trisphosphate, caffeine, and thapsigargin and is released by the Ca2+ ionophore ionomycin. This pool is probably heterogeneous and its intracellular localization and physiological roles remain undefined. The third pool, approximately equal to 170 mumoles of Ca2+/liter, was discharged by the combination of ionomycin together with a substance that collapsed intracellular pH gradients, such as monensin or NH4Cl. This indicates that the pool is acidic, at variance with the first two. When exocytosis was stimulated, the size of this pool declined, indicating its primary residence within secretory granules. In the conditions of our experiments no major transfer of Ca2+ among the pools seemed to occur. This is the first comprehensive description of non-cytosolic Ca2+ pools investigated in intact neurosecretory cells by non-invasive procedures.
Identification, kinetic properties and intracellular localization of the (Ca(2+)-Mg2+)-ATPase from the intracellular stores of chicken cerebellum.
The microsomal fraction of chicken cerebellum expresses a large amount of Ca(2+)-ATPase (105 kDa), which is phosphorylated by ATP in the presence of Ca2+. The Ca(2+)-ATPase activity is highly sensitive to temperature and to the presence of detergents. This ATPase has kinetic properties similar to those of chicken skeletal-muscle sarcoplasmic reticulum, as (i) it is activated by low (microM) and inhibited by high (mM) Ca2+ concentrations, (ii) it shows biphasic activation with ATP and (iii) it is inhibited by vanadate. However, the vanadate-sensitivity is at least 10 times greater than that observed in chicken skeletal or cardiac sarcoplasmic-reticulum Ca(2+)-ATPases. Thus, despite cross-reacting with antibodies against the cardiac and skeletal isoforms, the cerebellar microsomal Ca(2+)-ATPase appears to be distinct from both muscle enzymes. The Ca(2+)-ATPase is concentrated in, but not exclusive to, Purkinje neurons. In Purkinje neurons the Ca(2+)-ATPase appears to be expressed throughout the cell body, the dendritic tree (and the spines) and the axons. At the electron-microscope level the Ca(2+)-ATPase is found in smooth and rough endoplasmic-reticulum cisternae as well as in other, yet unidentified, smooth-surfaced structures.
Frog brain expresses a 60 KDa Ca2+ binding protein similar to mammalian calreticulin.
The present report was undertaken in an effort to characterize the nature of Ca2+ binding protein(s) in the central nervous system of less evolved vertebrates. In particular we investigated whether the brain microsomal fraction of Rana esculenta expresses calsequestrin, calreticulin and/or other related Ca2+ binding protein(s). We found that a 60 KDa protein having an NH2-terminal amino acid sequence similar to mammalian calreticulin is the major microsomal Ca2(+)-binding protein.
Excitatory amino acid-induced alterations of cytoplasmic free Ca2+ in individual cerebellar granule neurons: role in neurotoxicity.
The effects of glutamate on intracellular free Ca2+, [Ca2+]i, and neurotoxicity were compared in cerebellar granule neurons in vitro. [Ca2+]i was measured with fura-2 and digital fluorescence imaging microscopy; neurotoxicity was monitored using a vital dye and colorimetric analysis. Glutamate produced dose-dependent increases in [Ca2+]i, which tended to be transient for glutamate concentrations in a range of 0.01-0.5 microM and sustained for higher levels of glutamate. The ED50 for the [Ca2+]i response to glutamate was 6 microM. The LD50 for glutamate-induced neurotoxicity was similar, i.e., 10 microM. The effect of glutamate on [Ca2+]i was greatly diminished when external Ca2+ was removed and blocked by Mg2+ or N-methyl-D-aspartate (NMDA)-type receptor antagonists. The latter conditions as well as preloading granule neurons with the intracellular Ca2+ chelator quin2 largely prevented glutamate cytotoxicity. The neurotoxic effect of glutamate required incubations with the stimulus for 10-20 min at 25 degrees C. Withdrawal of glutamate after this period was accompanied by a prolonged alteration in [Ca2+]i. Pretreatment of the cells with the ganglioside GM1 reduced this late increase in [Ca2+]i as well as the neurotoxic effects of glutamate. This indicates that glutamate-induced neurotoxicity results from a composite of diverse temporal alterations in Ca2+ homeostasis and that blunting any of these components reduces excitotoxicity.
Ca2+ influx following receptor activation.
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Intracellular Ca2+ stores in chicken Purkinje neurons: differential distribution of the low affinity-high capacity Ca2+ binding protein, calsequestrin, of Ca2+ ATPase and of the ER lumenal protein, Bip.
To identify intracellular Ca2+ stores, we have mapped (by cryosection immunofluorescence and immunogold labeling) the distribution in the chicken cerebellar cortex of an essential component, the main low affinity-high capacity Ca2+ binding protein which in this tissue has been recently shown undistinguishable from muscle calsequestrin (Volpe, P., B. H. Alderson-Lang, L. Madeddu, E. Damiani, J. H. Collins, and A. Margreth. 1990. Neuron. 5:713-721). Appreciable levels of the protein were found exclusively within Purkinje neurons, distributed to the cell body, the axon, and the elaborate dendritic tree, with little labeling, however, of dendritic spines. At the EM level the protein displayed a dual localization: within the ER (rough- and smooth-surfaced cisternae, including the cisternal stacks recently shown [in the rat] to be highly enriched in receptors for inositol 1,4,5-triphosphate) and, over 10-fold more concentrated, within a population of moderately dense, membrane-bound small vacuoles and tubules, identified as calciosomes. These latter structures were widely distributed both in the cell body (approximately 1% of the cross-sectional area, particularly concentrated near the Golgi complex) and in the dendrites, up to the entrance of the spines. The distribution of calsequestrin was compared to those of another putative component of the Ca2+ stores, the membrane pump Ca2+ ATPase, and of the ER resident lumenal protein, Bip. Ca2+ ATPase was expressed by both calciosomes and regular ER cisternae, but excluded from cisternal stacks; Bip was abundant within the ER lumena (cisternae and stacks) and very low within calciosomes (average calsequestrin/Bip immunolabeling ratios were approximately 0.5 and 36.5 in the two types of structure, respectively). These results suggest that ER cisternal stacks do not represent independent Ca2+ stores, but operate coordinately with the adjacent, lumenally continuous ER cisternae. The ER and calciosomes could serve as rapidly exchanging Ca2+ stores, characterized however by different properties, in particular, by the greater Ca2+ accumulation potential of calciosomes. Hypotheses of calciosome biogenesis (directly from the ER or via the Golgi complex) are discussed.
Internalization pathway of C3b receptors in human neutrophils and its transmodulation by chemoattractant receptors stimulation.
On the surface of phagocytes, C3b receptors (CR1) bind C3b-coated particles and promote their ingestion after activation by appropriate stimuli such as lymphokines or the chemoattractant formyl methionyl leucyl phenylalanine (fMLP) and fibronectin. The aims of the present study were 1) to define at the electron microscopic level the nature of the process responsible for CR1 internalization and 2) to dissect the mechanism by which a physiological activator (fMLP) stimulates this process. CR1 was visualized either by the immunogold technique or by quantitative electron microscopic autoradiography using a monoclonal anti-CR1 antibody. Both techniques revealed that after anti-CR1 binding, CR1 cluster on the neutrophil surface in a time-, temperature-, and antibody-dependent fashion, but do not concentrate in coated pits. CR1 internalization requires receptor cross-linking (does not occur in the presence of Fab fragments of anti-CR1) and intact microfilaments. It results in the association of the internalized material with large flattened vacuoles, organized in stacks. Together with the surface localization of CR1 close to cytoplasmic projections (ruffles), these observations suggest that uptake of CR1 occurs through a macropinocytotic process. Eventually, CR1 concentrate in lysosomal structures. fMLP markedly stimulates this pattern of CR1 internalization without affecting their clustering or their lack of association with coated pits. Stimulation by fMLP is inhibited by pertussis toxin, unaffected by preventing receptor-triggered cytosolic free calcium [Ca2+]i elevations, and mimicked by phorbol myristate acetate. Taken together our data demonstrate 1) that, in neutrophils, CR1 is internalized via a coated pit independent macropinocytotic process, dependent on intact microfilaments and receptor cross-linking; 2) that, in the same cells, fMLP is internalized via the classical coated pits pathway; and 3) that fMLP amplifies CR1 uptake possibly via protein kinase C stimulation.
Receptor-mediated calcium influx in PC12 cells. ATP and bradykinin activate two independent pathways.
In the neurosecretory cell line PC12 the cytosolic free Ca2+ concentration, [Ca2+]i, and membrane potential were affected by both external ATP and the nonapeptide bradykinin, BK. The latter caused a rapid and large release of Ca2+ from intracellular stores (Ca2+ redistribution) and, in the presence of external Ca2+, a long lasting, but moderate Ca2+ influx, which was insensitive to dihydropyridine blockers. On the contrary, ATP evoked a [Ca2+]i rise which rapidly inactivated. At least three different mechanisms accounted for the ATP-induced increase in [Ca2+]i: less than 20% of the total response was due to intracellular Ca2+ redistribution, consistent with a small increase in inositol 1,4,5-trisphosphate level; the rest (over 80%) was equally accounted for by ATP-activated cation channels and voltage-gated Ca2+ channels. ATP and BK (the latter after K+ channel blockade) caused plasma membrane depolarization. With both agonists the inward current was carried by both Na+ and Ca2+, although the BK-activated current appeared to be more selective for Ca2+. Channels triggered by ATP and BK differed not only in their cation selectivity, but also in modulation by both [Ca2+]i and drugs such as the phorbol ester phorbol 12-myristate 13-acetate and the new antagonist of ligand-activated Ca2+ influx, SK&F 96365.
Structural and functional aspects of calcium homeostasis in eukaryotic cells.
The maintenance of a low cytosolic free-Ca2+ concentration, ([Ca2+]i) is a common feature of all eukaryotic cells. For this purpose a variety of mechanisms have developed during evolution to ensure the buffering of Ca2+ in the cytoplasm, its extrusion from the cell and/or its accumulation within organelles. Opening of plasma membrane channels or release of Ca2+ from intracellular pools leads to elevation of [Ca2+]i; as a result, Ca2+ binds to cytosolic proteins which translate the changes in [Ca2+]i into activation of a number of key cellular functions. The purpose of this review is to provide a comprehensive description of the structural and functional characteristics of the various components of [Ca2+]i homeostasis in eukaryotes.
Intracellular Ca2+ storage organelles in non-muscle cells: heterogeneity and functional assignment.
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Calreticulin is a candidate for a calsequestrin-like function in Ca2(+)-storage compartments (calciosomes) of liver and brain.
In a search for the non-muscle equivalent of calsequestrin (the low-affinity high-capacity Ca2(+)-binding protein responsible for Ca2+ storage within the terminal cisternae of the sarcoplasmic reticulum), acidic proteins were extracted from rat liver and brain microsomal preparations and purified by column chromatography. No calsequestrin was observed in these extracts, but the N-terminal amino acid sequence of the major Ca2(+)-binding protein of the liver microsomal fraction was determined and found to correspond to that of calreticulin. This protein was found to bind approx. 50 mol of Ca2+/mol of protein, with low affinity (average Kd approx. 1.0 mM). A monoclonal antibody, C6, raised against skeletal-muscle calsequestrin cross-reacted with calreticulin in SDS/PAGE immunoblots, but polyclonal antibodies reacted with native calreticulin only weakly, or not at all, after SDS denaturation. Immuno-gold decoration of liver ultrathin cryosections with affinity-purified antibodies against liver calreticulin revealed luminal labelling of vacuolar profiles indistinguishable from calciosomes, the subcellular structures previously identified by the use of anti-calsequestrin antibodies. We conclude that calreticulin is the Ca2(+)-binding protein segregated within the calciosome lumen, previously described as being calsequestrin-like. Because of its properties and intraluminal location, calreticulin might play a critical role in Ca2+ storage and release in non-muscle cells, similar to that played by calsequestrin in the muscle sarcoplasmic reticulum.
Correlation between plasma membrane potential and second messenger generation in the promyelocytic cell line HL-60.
The effects of plasma membrane depolarization on cytosolic free calcium ([Ca2+]i) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation were investigated in the human promyelocytic cell line HL-60 differentiated with either dimethyl sulfoxide or retinoic acid into neutrophil-like cells. Increases in [Ca2+]i and accumulation of Ins(1,4,5)P3 were triggered by two chemoattractants fMet-Leu-Phe and leukotriene B4. Plasma membrane potential was depolarized by isoosmotic substitution of NaCl with KCl, by the pore-forming ionophore gramicidin D, or by long term treatment with ouabain. Both Ca2+ mobilization from intracellular stores and Ca2+ influx across the plasma membrane were reduced by prior depolarization of plasma membrane potential regardless of the procedure employed to collapse it. Agonist-induced generation of Ins(1,4,5)P3 was also reduced in parallel in pre-depolarized HL-60 cells. The present findings provide further evidence suggesting that plasma membrane potential can be an important modulator of agonist-activated second messenger generation in myelocytic cells.
An anti-HLA class I monoclonal antibody alters the progression in the cell cycle of phytohemagglutinin-activated human T lymphocytes.
The monomorphic anti-HLA Class I monoclonal antibody 01.65 inhibits the incorporation of tritiated thymidine ([3H]TdR) in Phytohemagglutinin (PHA)-activated human T lymphocytes. Our data indicate that 01.65 affects the average duration of the cell cycle by increasing the length of the early S subphase. As a consequence of the increase in the doubling time of the cell population, the absolute number of cells at harvesting time was reduced in 01.65-treated cultures compared to that of untreated cultures. The lengthening of the S-phase and the decrease in the cell number can together quantitatively account for the reduction of [3H]TdR incorporation observed in 01.65-treated cultures.
The inositol 1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: quantitative immunogold labeling reveals concentration in an ER subcompartment.
The Ca2+ mobilization effect of inositol 1,4,5-trisphosphate, the second messenger generated via receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate, is mediated by binding to intracellular receptors, which are expressed in high concentration in cerebellar Purkinje cells. Partially conflicting previous reports localized the receptor to various subcellular structures: elements of ER, both rough and smooth-surfaced, the nuclear envelope, and even the plasma membrane. We have now reinvestigated the problem quantitatively by using cryosections of rat cerebellar tissue immunolabeled with polyclonal monospecific antibodies against the inositol 1,4,5-trisphosphate receptor. By immunofluorescence the receptor was detected only in Purkinje cells, whereas the other cells of the cerebellar cortex remained negative. In immunogold-decorated ultrathin cryosections of the Purkinje cell body, the receptor was concentrated in cisternal stacks (piles of up to 12 parallel cisternae separated by regularly spaced bridges, located both in the deep cytoplasm and beneath the plasma membrane; average density, greater than 5 particles/micron of membrane profile); in cisternal singlets and doublets adjacent to the plasma membrane (average density, approximately 2.5 particles/micron); and in other apparently smooth-surfaced vesicular and tubular profiles. Additional smooth-surfaced elements were unlabeled. Perinuclear and rough-surfaced ER cisternae were labeled much less by themselves (approximately 0.5 particles/micron, two- to threefold the background), but were often in direct membrane continuity with heavily labeled, smooth-surfaced tubules and cisternal stacks. Finally, mitochondria, Golgi cisternae, multivesicular bodies, and the plasma membrane were unlabeled. In dendrites, approximately half of the nonmitochondrial, membrane-bound structures (cisternae, tubules, and vesicles), as well as small cisternal stacks, were labeled. Dendritic spines always contained immunolabeled cisternae and vesicles. The dendritic plasma membrane, of both shaft and spines, was consistently unlabeled. These results identify a large, smooth-surfaced ER subcompartment that appears equipped to play a key role in the control of Ca2+ homeostasis: in particular, in the generation of [Ca2+]i transients triggered by activation of specific receptors, such as the quisqualate-preferring trans(+/-)-1-amino-1,3-cyclopentamedicarboxylic acid glutamatergic receptors, which are largely expressed by Purkinje cells.
Rapidly exchanging Ca2+ stores of non-muscle cells.
The rapid and transient redistribution of calcium from intracellular stores is a key event of cell activation. The nature and molecular composition of intracellular Ca2+ stores of non-muscle cells are the object of intense investigation. In this paper, we review: (a) the experimental evidence in favor of the existence of intracellular, membrane-bound compartments specialized for uptake, storage and release of calcium, (b) the main protein components of rapidly exchanging Ca2+ stores, i.e. Ca2+ pump, intralumenal Ca2+ binding proteins (calsequestrin, calreticulin, etc.) and Ca2+ channels sensitive to either inositol 1,4,5-trisphosphate or Ca2+, caffeine and ryanodine, and (c) the relationship between Ca2+ stores and the endoplasmic reticulum.
Effect of membrane potential on divalent cation transport catalyzed by the "electroneutral" ionophores A23187 and ionomycin.
Depolarization of plasma membrane potential has a potent inhibitory effect on divalent cation influx catalyzed by the carboxylic ionophores ionomycin and A23187. This effect is observed in different cell models and does not depend on either inhibition of Ca2+-activated cation channels or activation of Ca2+ extrusion mechanisms as suggested previously. A dependence of divalent cation influx on the magnitude of membrane potential is observed also in artificial liposomes. The inhibition of ionophore-dependent divalent cation transport by membrane potential depolarization can be modified varying the ionophore concentration and the external pH. These findings suggest that both neutral and positively charged ionophore-cation complexes can cross the plasma membrane and that their contribution to the overall transport process can be varied according to the experimental conditions.