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T Pozzan

Publications and source records attributed to T Pozzan.

At least 145 records · Page 8Linked to original sources
PubMed1985

Inositol phosphate formation in fMet-Leu-Phe-stimulated human neutrophils does not require an increase in the cytosolic free Ca2+ concentration.

The accumulation of inositol phosphates in myo-[3H]inositol-labelled human neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe was measured. The challenge with the chemotactic peptide caused the generation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3). The formation of the three inositol phosphates followed a differential time course: InsP3 accumulated very rapidly and transiently, whereas InsP increased steadily for more than 2 min. Inositol phosphate formation was only partially decreased by procedures which prevented the fMet-Leu-Phe-dependent increase of cytosolic free Ca2+ concentration.

Calcium↗
PubMed1985

Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. A new, membrane-permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+.

The intracellularly trappable fluorescent Ca2+ indicator quin-2 was used to measure free cytosolic Ca2+, [Ca2+]i, in the two highly dedifferentiated tumor cell lines, Ehrlich and Yoshida ascites carcinomas. It was found that these carcinoma cells can trap quin-2 similarly to normal cells, but [Ca2+]i was apparently significantly lower than in any normal cell tested previously with this method. By using a new lipid-soluble heavy metal chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), which crosses artificial and natural membranes, it was found that endogenous heavy metals are responsible for partially quenching quin-2 fluorescence trapped inside the cells. Although the quenching of intracellular quin-2 fluorescence is quantitatively more relevant in these ascites carcinomas, TPEN was effective also in normal cells like lymphocytes and granulocytes. Both in the normal and especially in the malignant cell lines [Ca2+]i can be grossly underestimated at low intracellular quin-2 concentrations. Endogenous heavy metal quenching is thus a potential source of artifact when [Ca2+]i is measured with quin-2. When corrected for quin-2 fluorescence quenching by intracellular heavy metals, [Ca2+]i and basic regulatory mechanisms of [Ca2+]i homeostasis in Ehrlich and Yoshida carcinomas are similar to those of nontransformed cells.

Aminoquinolines↗
PubMed1985

Tumor promoter phorbol 12-myristate, 13-acetate inhibits phosphoinositide hydrolysis and cytosolic Ca2+ rise induced by the activation of muscarinic receptors in PC12 cells.

Preincubation of PC12 cells (used both before and after differentiation by NGF) with phorbol myristate acetate (PMA) was without effect on the basal concentration of inositol phosphates (metabolites of phosphoinositide hydrolysis) and of free cytosolic Ca2+, but inhibited considerably the increases induced by the cholinergic agonist carbachol via the activation of the muscarinic receptor. Inasmuch as binding was unaffected, this inhibition might occur at the level of receptor coupling to its transduction mechanism(s). Inhibition appeared within 1 min and was maximal after 3 min. The concentrations of PMA needed (10(-9)-10(-8)M) were in the range believed to cause specifically the activation of protein kinase C. The muscarinic receptor, via the hydrolysis of phosphoinositides and the generation of diacylglycerol, participates in the regulation of the latter enzyme. Our data suggest therefore that the receptor operates under stringent feedback control by the metabolites generated as a consequence of its activation.

Animals↗
PubMed1985

Muscarinic receptor-induced phosphoinositide hydrolysis at resting cytosolic Ca2+ concentration in PC12 cells.

In PC12 cells, cultured in the presence of nerve growth factor to increase their complement of muscarinic receptors, treatment with carbachol induces muscarinic receptor-dependent rises in free cytosolic Ca2+ as well as hydrolysis of membrane phosphoinositides. Experiments were carried out to clarify the relationship between these two receptor-triggered events. In particular, since inositol-1,4,5-trisphosphate (the hydrophilic metabolite produced by the hydrolysis of phosphatidylinositol-4,5-bisphosphate) is believed to mediate intracellularly the release of Ca2+ from nonmitochondrial store(s), it was important to establish whether it can be generated at resting cytoplasmic concentration of Ca2+ (approximately 0.1 microM). Cells incubated in Ca2+-free medium were depleted of their cytoplasmic Ca2+ stores by pretreatment with ionomycin. When these cells were then treated with carbachol, their cytosolic concentration of Ca2+ remained at the resting level, whereas inositol-1,4,5-trisphosphate generation was still markedly stimulated. Our results demonstrate that an increase in the concentration of cytosolic Ca2+ is not a necessary intermediate between receptor activation and phosphoinositide hydrolysis, and therefore support the second-messenger role of inositol-1,4,5-trisphosphate.

Animals↗
PubMed1985

Leptinotoxin-h action in synaptosomes, neurosecretory cells, and artificial membranes: stimulation of ion fluxes.

Leptinotoxin-h (LPTx), a neurotoxin (otherwise designated beta-leptinotarsin-h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle, Leptinotarsa haldemani, by a simplification of the procedure originally developed by Crosland et al. [Biochemistry 23, 734-741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca2+-containing Ringer medium, at concentrations in the 10(-11) - 10(-10) M range, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage-dependent Na+ and Ca2+ channels (tetrodotoxin or verapamil); (b) large 45Ca influx; and (c) increased free cytosolic Ca2+ concentration. These latter two effects were unaffected by verapamil. In Ca2+-free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (approximately 10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca2+ is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono- and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca2+ channel as the mechanism of action of LPTx. The effects of the toxin resemble those of alpha-latrotoxin (alpha-LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx does not inhibit alpha-LTx binding.

Adrenal Gland Neoplasms↗
PubMed1984

Glyceraldehyde, but not cyclic AMP-stimulated insulin release is preceded by a rise in cytosolic free Ca2+.

The dynamics of changes in membrane potential, cytosolic free Ca2+, [Ca2+]i and immunoreactive insulin release were assessed in RINm5F cells. Membrane depolarization and a rise in [Ca2+]i preceded the stimulation of insulin release by D-glyceraldehyde. Forskolin, which raised the cellular cyclic AMP levels, stimulated insulin release without changing membrane potential or [Ca2+]i. It is concluded that cyclic AMP acts on insulin release not by mobilizing Ca2+ but by another, as yet undefined, mechanism.

Aminoquinolines↗
PubMed1984

Pathway for uncoupler-induced calcium efflux in rat liver mitochondria: inhibition by ruthenium red.

The rate of uncoupler-induced Ca2+ efflux from rat liver mitochondria is increased by acetate and decreased by phosphate. This effect depends on a shift of the apparent Km, which is increased by phosphate and decreased by acetate, while the Vmax is not modified. The modification of the apparent Km by permeant anions presumably reflects changes in the concentration of matrix free Ca2+. A major part of uncoupler-induced Ca2+ efflux is sensitive to Ruthenium Red, the specific inhibitor of the Ca2+ uniporter , but an apparent insensitivity is observed when the H+ permeability is rate limiting in the process of Ca2+ efflux. The titer of uncoupler required for maximal stimulation of Ca2+ efflux increases with the Ca2+ load and may be 1-2 orders of magnitude higher than that required for maximal stimulation of respiration. On the other hand, when the uncoupler concentration is raised above 10(-6) M, the process of Ca2+ efflux becomes again Ruthenium Red insensitive. The Ruthenium Red inhibition of uncoupler-induced Ca2+ efflux is time dependent, in that the degree of inhibition exerted by low amounts of Ruthenium Red increases with the incubation time. Since the inhibition of the rate of Ca2+ influx undergoes a parallel relief, it is possible that Ruthenium Red moves from the cytosolic to the matrix side of the inner membrane. It is concluded that, in native mitochondria, uncoupler-induced Ca2+ efflux occurs via reversal of the uniport Ca2+ carrier, and not through activation of an independent pathway.

Animals↗
PubMed1984

Correlation between cytosolic free Ca2+ and insulin release in an insulin-secreting cell line.

Changes in the concentration of free cytosolic Ca2+ [( Ca2+]i), membrane potential, and immunoreactive insulin release were measured in suspensions of RINm5F cells. [Ca2+]i was monitored with the intracellularly trapped fluorescent Ca2+ indicator quin 2. Changes in membrane potential were assessed with the fluorescent probe bisoxonol. The effects of depolarizing K+ concentrations and of the Ca2+ ionophore ionomycin were compared with those of the metabolizable stimuli glyceraldehyde and alanine. The mean resting [Ca2+]i was 105 nM +/- 6, n = 35. Of the substances tested, ionomycin caused the most marked increases of [Ca2+]i and insulin release. Membrane depolarization was evoked by K+, glyceraldehyde, or alanine and the agent stimulated insulin release and [Ca2+]i to a similar degree. However, the mechanism by which glyceraldehyde, on the one hand, and K+ and alanine, on the other, elevate [Ca2+]i appears different. Thus, verapamil, a blocker of voltage-sensitive Ca2+ channels, almost abolished the effects of K+ and alanine on [Ca2+]i and insulin release, whereas the responses to glyceraldehyde were only blocked by approximately 50%. Glyceraldehyde may thus, in addition to opening voltage-sensitive Ca2+ channels, alter cellular Ca2+ handling at another step. The present findings provide direct experimental evidence that secretagogue-induced insulin release is accompanied by a rise in [Ca2+]i.

Alanine↗
PubMed1984

Effect of leukotriene B4, prostaglandin E2 and arachidonic acid on cytosolic-free calcium in human neutrophils.

Changes in cytosolic free calcium [Ca2+]i and release of beta-glucuronidase in response to leukotriene B4 (LTB4) were measured in intact neutrophils loaded with the fluorescent Ca2+ indicator, quin 2. LTB4 (10(-10) M or higher) caused a rapid rise in [Ca2+]i due to influx from the extracellular medium and release from intracellular pools as well as enzyme release. PGE2 (3 microM) did not alter [Ca2+]i whereas arachidonic acid (10 microM) raised [Ca2+]i. Pretreatment of cells with the chemotactic peptide FMLP inhibited the subsequent rise of [Ca2+]i induced by LTB4. Since chemotactic peptides activate the lipoxygenase pathway of arachidonic acid metabolism, it may be speculated that endogenous LTB4 generation is involved in neutrophil activation.

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

Alpha-latrotoxin and glycerotoxin differ in target specificity and in the mechanism of their neurotransmitter releasing action.

alpha-Latrotoxin, a high molecular weight protein (130,000) purified from the venom of the black widow spider, and a partially purified neurotoxin, glycerotoxin, prepared from extracts of the jaw glands of the polichaete annelid Glycera convoluta, were previously found to induce similar effects (stimulation of quantal acetylcholine release) at the frog neuromuscular junction. In the present study parallel experiments performed with these two toxins revealed that only glycerotoxin was able to release acetylcholine from Torpedo electric organ synaptosomes, while alpha-latrotoxin did not affect release in this system. In contrast, alpha-latrotoxin stimulated release of dopamine from PC12 cells (a cloned neurosecretory cell line), whereas glycerotoxin was almost inactive. In rat brain synaptosomes both toxins were active. Preincubation of synaptosomal membranes with glycerotoxin was without effect on the subsequent binding of alpha-latrotoxin. Glycerotoxin application induced depolarization of synaptosomal plasma membrane, massive Ca2+ influx, marked increase of the cytosolic Ca2+ concentration, and stimulation of catecholamine release. The latter effect occurred to the same extent when glycerotoxin was applied either in complete medium (containing both Ca2+ and Mg2+), Ca2+-free medium or divalent cation-free medium. Some of these effects of glycerotoxin in rat brain synaptosomes (depolarization, increased Ca2+ influx and increased cytosolic Ca2+ concentration) resemble effects previously reported for alpha-latrotoxin. However, the secretory response induced by the latter was reduced in Ca2+-free, and abolished in divalent cation-free media. The different target specificity and the lack of binding competition of the two toxins could be due to their ability to recognize different receptors whose distribution overlap only in part in the cellular systems we have studied. The differences in action, on the other hand, could depend on postreceptor events, possibly related to the transmembrane insertion of toxin molecules demonstrated by others in artificial lipid membranes.

Animals↗
PubMed1984

Free cytoplasmic Ca2+ and neurotransmitter release: studies on PC12 cells and synaptosomes exposed to alpha-latrotoxin.

The relationship between the free cytoplasmic Ca2+ concentration, [Ca2+]i, and neurotransmitter release was investigated in guinea pig brain synaptosomes and the neurosecretory cell line PC12. Release was induced by alpha-latrotoxin, which acts in both Ca2+ -containing and Ca2+ -free incubation media, or by the classical depolarizing agents high K+ and veratridine, which require extracellular Ca2+. Two complementary approaches were used to reveal changes of [Ca2+]i: (i) direct measurement by a fluorescent Ca2+ indicator (quin2) and (ii) study of the Ca2+ -dependent phosphorylation of a protein, synapsin I, located at the cytoplasmic surface of synaptic vesicles. Depolarizing agents, when applied in Ca2+ -containing medium, induced the [Ca2+]i to increase promptly 3- to 6-fold, drastically increased synapsin I phosphorylation, and caused stimulation of transmitter release. With alpha-latrotoxin, the [Ca2+]i increase was delayed and occurred at a slower rate, the increase of synapsin I phosphorylation was less drastic, and the release response was much more pronounced. In Ca2+ -free medium, depolarizing agents released no transmitter and had no effect on [Ca2+]i or synapsin I phosphorylation, whereas with alpha-latrotoxin these processes were dissociated: considerable stimulation of the release without apparent change of [Ca2+]i and synapsin I phosphorylation. We conclude that the relationship between average [Ca2+]i and transmitter release is not straightforward and, in particular, that the release evoked by alpha-latrotoxin in Ca2+ -free medium is mediated by a factor(s) other than bulk redistribution of Ca2+ from intracellular stores.

Aminoquinolines↗
PubMed1984

Monitoring of cytosolic free Ca2+ in C5a-stimulated neutrophils: loss of receptor-modulated Ca2+ stores and Ca2+ uptake in granule-free cytoplasts.

The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intracellular Ca2+ indicator quin2, 2-[[2-bis(acetylamino)-5-methylphenoxy]methyl-6-methoxy-8- bis(acetylamino)]quinoline. Neutrophils at rest have a cytosolic Ca2+ concentration of 85 +/- 5 nM, which in 2-4 min increases to 300-400 nM upon interaction with the complement fragment C5a in a concentration range of 35 pM to 1.2 microM. In the same concentration range, C5a also sequentially activates neutrophil directional migration (ED50 less than 0.5 nM), O-2 production (ED50 = 9 nM), and secretion of the contents of specific granules (ED50 = 39 nM). The selective Ca2+ ionophore ionomycin also increases cytosolic Ca2+ concentration above 1 microM under conditions where it stimulates neutrophil functions. Conversely, phorbol 12-myristate 13-acetate markedly activates secretion and O-2 production without modifying the average cytosolic Ca2+ concentration. In the presence of EGTA (Ca2+out approximately equal to 20 nM), with both C5a and ionomycin, cytosolic Ca2+ increases to less than 200 nM, and functional responses are greatly decreased. Nucleus- and granule-free neutrophil cytoplasts accumulate Ca2+ and produce O-2 when exposed to ionomycin but not to C5a. These results and other considerations suggest that (i) activation of neutrophil functions may occur after cytosolic Ca2+ has exceeded the apparent threshold level of 200 nM; (ii) C5a receptor-mediated activation of Ca2+ influx may require cooperation between the neutrophil surface and some cytoplasmic organelle and/or redistribution of the C5a-receptor complexes on the cell surface; and (iii) the phorbol diester stimulates Ca2+-dependent pathways presumably by directly activating other mechanisms such as protein phosphorylation.

Animals↗
PubMed1984

Ca2+-dependent and -independent release of neurotransmitters from PC12 cells: a role for protein kinase C activation?

The intracellular mechanisms regulating the process of evoked neurotransmitter release were studied in the cloned neurosecretory cell line PC12. Various agents were employed that were known, from previous studies in other systems, to stimulate release in a manner either strictly dependent or independent of the concentration of extracellular Ca2+, [Ca2+]o. Three parameters were investigated in cells suspended in either Ca2+-containing or Ca2+-free Krebs-Ringer media: release of previously accumulated [3H]dopamine; average free cytoplasmic Ca2+ concentration, [Ca2+]i (measured by the quin2 technique); and cell ultrastructure, with special reference to the number and structure of secretion granules. The release induced by the ionophores transporting monovalent cations, X537A and monensin, occurred concomitantly with profound alterations of secretory granule structure (swelling and dissolution of the dense core). These results suggest that the effect of these drugs is due primarily to leakage of dopamine from granules to the cytoplasm and extracellular space. In contrast, the changes induced by other stimulatory drugs used concerned not the structure but the number of secretory granules, indicating that with these drugs stimulation of exocytosis is the phenomenon underlying the increased transmitter release. The release response induced by the Ca2+-ionophore ionomycin was dependent on [Ca2+]o, occurred rapidly, was concomitant with a marked rise of [Ca2+]i, and ceased after 1-2 min even though [Ca2+]i remained elevated for many minutes. 12-O-tetradecanoylphorbol, 13-acetate and diacylglycerol (both of which are known as activators of protein kinase C) induced slow responses almost completely independent of [Ca2+]o and not accompanied by changes of [Ca2+]i. Combination of an activator of protein kinase C with a low concentration of ionomycin failed to modify the [Ca2+]i rise induced by the ionophore, but elicited a marked potentiation of the release response, which was two- to fourfold larger than the sum of the responses elicited separately by either drugs. Thus, activation of protein kinase C seems to play an important role in the regulation of exocytosis in neurosecretory cells, possibly by increasing and maintaining the sensitivity to Ca2+ of the intracellular apparatus regulating granule discharge by exocytosis.

Adrenal Gland Neoplasms↗
PubMed1984

Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils.

The intracellularly trapped fluorescent calcium indicator, quin 2, was used not only to monitor changes in cytosolic-free calcium, [Ca2+]i, but also to assess the role of [Ca2+]i in neutrophil function. To increase cytosolic calcium buffering, human neutrophils were loaded with various quin 2 concentrations, and [Ca2+]i transients, granule content release as well as superoxide [O2-] production were measured in response to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) and the calcium ionophore ionomycin. Receptor-mediated cell activation induced by fMLP caused a rapid rise in [Ca2+]i. The extent of [Ca2+]i rise and granule release were inversely correlated with the intracellular concentration of quin 2, [quin 2]i. These effects of [quin 2]i were more pronounced in the absence of extracellular Ca2+. The initial rate and extent of fMLP-induced O2- production were also inhibited by [quin 2]i. The rates of increase of [Ca2+]i and granule release elicited by ionomycin were also inversely correlated with [quin 2]i in Ca2+-containing medium. As the effects of ionomycin, in contrast to those of fMLP, are sustained, the final increase in [Ca2+]i and granule release were not affected by [quin 2]i. A further reduction of fMLP effects was seen when intracellular calcium stores were depleted by incubating the cells in Ca2+-free medium with ionomycin. The specificity of quin 2 effects on cellular calcium were confirmed by loading the cells with Anis/AM, a structural analog of quin 2 with low affinity for calcium which did not inhibit granule release. In addition, functional responses to phorbol myristate acetate (PMA), which stimulates neutrophils without raising [Ca2+]i, were not affected by [quin 2]i. The findings indicate that rises in [Ca2+]i control the rate and extent of granule exocytosis and O2-generation in human neutrophils exposed to the chemotactic peptide fMLP.

Aminoquinolines↗
PubMed1984

Phorbol myristate acetate stimulates ATP-dependent calcium transport by the plasma membrane of neutrophils.

We studied the effect of phorbol myristate acetate (PMA) on the plasma membrane ATP-dependent calcium pump in neutrophils. Plasma membrane-enriched fractions ("podosomes") from PMA-stimulated guinea pig neutrophils exhibited a twofold stimulation of ATP-dependent calcium transport when compared with control podosomes. The stimulatory effect was rapid (beginning less than 2 min after exposure to PMA) and reached maximal values within 5 min. PMA increased the maximum velocity but not the affinity of the calcium pump for Ca++. Pump activation was not preceded by a rise in cytosolic free calcium concentration [Ca++]i, as assessed by the intracellularly trapped fluorescent calcium indicator Quin 2, but instead slightly lowered [Ca++]i and prevented the rise in [Ca++]i normally induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine. These results suggest that the calcium pump in the plasma membrane of neutrophils may be stimulated by calcium-independent pathways, and that this activation could be one of the earliest events mediating some of the effects of phorbol esters.

Adenosine Triphosphate↗