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T Pozzan

Publications and source records attributed to T Pozzan.

At least 109 records · Page 6Linked to original sources
PubMed1989

Interaction of lymphokine-activated killer cells with susceptible targets does not induce second messenger generation and cytolytic granule exocytosis.

CTL activation by specific targets leads to a rapid rise of inositol phosphates (InsPs) and of cytoplasmic-free Ca2+ concentration ([Ca2+]i). While these events are considered necessary to trigger granule secretion, Ca2+-independent cytolytic mechanisms have been recently proposed in addition or as an alternative to the classical Ca2+-dependent exocytosis model. We observed that lymphokine-activated killer (LAK) cells, obtained after stimulation with supraoptimal concentrations of IL-2 in short- or long-term cultures, kill susceptible targets in the absence of a [Ca2+]i rise and InsP3 formation. Moreover, LAK cell-mediated lysis was not associated with an increase in cytotoxic granule exocytosis, as evaluated by BLT-esterase release into the culture supernatant. Furthermore, using an antigen-specific CTL clone, which acquires LAK-like activity when cultured in medium containing high IL-2 doses, second messenger generation and cytolytic granule content secretion were not detected during lysis of unrelated target cells, while killing of specific targets triggered both these processes. These findings suggest that two lytic pathways may coexist in the same effector cells: a second messenger-dependent pathway involving degranulation, which is activated after TCR interaction with specific targets, and another pathway, independent of any known second messenger generation, responsible for unrelated target cell lysis.

Animals↗
PubMed1989

Calciosome, a sarcoplasmic reticulum-like organelle involved in intracellular Ca2+-handling by non-muscle cells: studies in human neutrophils and HL-60 cells.

Calciosomes are intracellular organelles in HL-60 cells, neutrophils and various other cell types, characterized by their content of a Ca2+-binding protein that is biochemically and immunologically similar to calsequestrin (CS) from muscle cells. In subcellular fractionation studies the CS-like protein copurifies with functional markers of the inositol 1,4,5-trisphosphate (IP3) releasable Ca2+-store. These markers (ATP-dependent Ca2+-uptake and IP3-induced Ca2+-release) show a subcellular distribution which is clearly distinct from the endoplasmic reticulum and other organelles. In morphological studies, antibodies against rabbit skeletal muscle CS protein specifically stained hitherto unrecognized vesicles with a diameter between 50 and 250 nm. Thus both, biochemical and morphological studies indicate that the calsequestrin containing intracellular Ca2+-store, now referred to as the calciosome, is distinct from other known organelles such as endoplasmic reticulum. Calciosomes are likely to play an important role in intracellular Ca2+-homeostasis. They are possibly the intracellular target of inositol 1,4,5-trisphosphate and thus the source of Ca2+ that is redistributed into the cytosol following surface receptor activation in non-muscle cells.

Biological Transport, Active↗
PubMed1988

Generation of inositol phosphates, cytosolic Ca2+, and ionic fluxes in PC12 cells treated with bradykinin.

Accumulation of inositol phosphates (Ins-Ps, revealed by high performance liquid chromatography), changes of the cytosolic free Ca2+ [( Ca2+]i, revealed by fura-2), membrane potential and ionic currents (revealed by bis-oxonol and patch clamping) were investigated in PC12 cells treated with bradykinin (BK). The phenomena observed were (a) due to the activation of a B2 receptor (inhibitor studies) and (b) unaffected by pertussis toxin, cAMP analogs, and inhibitors of either cyclooxygenase or voltage-gated Ca2+ channels. During the initial tens of s, three interconnected events predominated: accumulation of Ins-1,4,5-P3, Ca2+ release from intracellular stores and hyperpolarization due to the opening of Ca2+-activated K+ channels. Phorbol myristate acetate partially inhibited Ins-1,4,5-P3 accumulation at all [BK] investigated, and the [Ca2+]i increase at [BK] less than 50 nM. In PC12 cells treated with maximal [BK] in the Ca2+-containing incubation medium, Ins-1,4,5-P3 peaked at 10 s, dropped to 20% of the peak at 30 s, and returned to basal within 5 min; the peak increase of Ins-1,3,4-P3 was slower and was variable from experiment to experiment, while Ins-P4 rose for 2 min, and remained elevated for many min thereafter. Meanwhile, influx of Ca2+ from the extracellular medium, plasma membrane depolarization (visible without delay when hyperpolarization was blocked), and increased plasma membrane conductance were noticed. Evidence is presented that these last three events (which were partially inhibited by phorbol myristate acetate at all [BK]) were due to the activation of a cation influx, which was much more persistent than the elevation of the two Ins-P3 isomers. Our results appear inconsistent with the possibility that in intact PC12 cells the BK-induced activation of cation influx is accounted for entirely by the increases of either Ins-1,3,4-P3 or Ins-1,4,5-P3 (alone or in combination with Ins-1,3,4,5-P4), as previously suggested by microinjection studies in different cell types.

Adrenal Gland Neoplasms↗
PubMed1988

Second-messenger generation in PC12 cells. Interactions between cyclic AMP and Ca2+ signals.

Changes in cyclic AMP concentrations were studied in intact PC12 pheochromocytoma cells exposed to a variety of treatments. A marked increase was triggered by N-(L-2-phenylisopropyl)adenosine, the activator of an adenosine receptor, whereas a decrease (observed even after phosphodiesterase blockade) was induced by carbachol, working through a muscarinic receptor inhibited by the selective muscarinic blocker pirenzepine, only at high concentration (Ki 450 nM). A decrease in cyclic AMP was also induced by clonidine, an alpha 2-adrenergic-receptor agonist. Both the alpha 2-adrenergic and the muscarinic inhibitions were prevented by pretreatment of the cells with pertussis toxin, and were unaffected by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. The latter drug caused a decrease in the resting cyclic AMP concentrations, and a potentiation of the increase induced by adenosine-receptor activation. Except for clonidine, all these treatments were found to be effective in both growing PC12 cells and, although to a smaller degree, in cells that had stopped growing and had acquired a neuron-like phenotype after prolonged treatment with nerve growth factor (NGF). Neither forskolin (a direct activator of adenylate cyclase) nor the activation of adenosine and alpha-adrenergic receptors was able to modify the resting cytosolic Ca2+ concentration [Ca2+]i in PC12 cells. Likewise, the K+-induced [Ca2+]i transients were unchanged after these treatments, whereas the transients induced by carbachol through the activation of a muscarinic receptor highly sensitive to pirenzepine were moderately potentiated by forskolin (and, to a lesser degree, by the adenosine analogue) and attenuated by clonidine. These results characterize in further detail the spectrum and the mutual interrelationships of the intracellular signals induced by receptor activation in PC12 cells, also as a function of the NGF-induced differentiation.

Animals↗
PubMed1988

Second-messenger control of catecholamine release from PC12 cells. Role of muscarinic receptors and nerve-growth-factor-induced cell differentiation.

The role of various intracellular signals and of their possible interactions in the control of neurotransmitter release was investigated in PC12 cells. To this purpose, agents that affect primarily the cytosolic concentration of Ca2+, [Ca2+]i (ionomycin, high K+), agents that affect cyclic AMP concentrations (forskolin; the adenosine analogue phenylisopropyladenosine; clonidine) and activators of protein kinase C (phorbol esters) were applied alone or in combination to either growing chromaffin-like PC12-cells, or to neuron-like PC12+ cells differentiated by treatment with NGF (nerve growth factor). In addition, the release effects of muscarinic-receptor stimulation (which causes increase in [Ca2+]i, activation of protein kinase C and decrease in cyclic AMP) were investigated. Two techniques were employed to measure catecholamine release: static incubation of [3H]dopamine-loaded cells, and perfusion incubation of unlabelled cells coupled to highly sensitive electrochemical detection of released catecholamines. The results obtained demonstrate that: (1) release from PC12 cells can be elicited by both raising [Ca2+]i and activating protein kinases (protein kinase C and, although to a much smaller extent, cyclic AMP-dependent protein kinase); and (2) these various control pathways interact extensively. Activation of muscarinic receptors by carbachol induced appreciable release responses, which appeared to be due to a synergistic interplay between [Ca2+]i and protein kinase C activation. The muscarinic-induced release responses tended to become inactivated rapidly, possibly by feedback desensitization of the receptor mediated by protein kinase C. Muscarinic inactivation was prevented (or reversed) by agents that increase, and accelerated by agents that decrease, cyclic AMP. Agents that stimulate release primarily through the Ca2+ pathway (ionomycin and high K+) were found to be equipotent in both PC12- and PC12+ cells, whereas the protein kinase C activator 12-O-tetradecanoyl-phorbol 13-acetate was approx. 10-fold less potent in PC12+ cells, when administered either alone or in combination with ionomycin. In contrast, the cell binding of phorbol esters was not greatly modified by NGF treatment. Thus control of neurotransmitter release from PC12 cells is changed by differentiation, with a diminished role of the mechanism mediated by protein kinase C.

Adrenal Gland Neoplasms↗
PubMed1988

D-myo-inositol 1,4,5-trisphosphate phosphatase in skeletal muscle.

The presence and subcellular distribution of D-myo-inositol 1,4,5-trisphosphate phosphatase (InsP3ase) in rabbit fast-twitch skeletal muscle were investigated. A specific InsP3ase was found in both sarcotubular-membrane and soluble fractions. Membrane-bound InsP3ase accounted for 60-65% of total activity. The InsP3ase was detected both on the surface membranes and on the InsP3-sensitive intracellular Ca2+ store, i.e. the sarcoplasmic reticulum. The Km for inositol 1,4,5-trisphosphate (InsP3) ranged between 15 and 18 microM, and the highest Vmax. (19.6 nmol of InsP3 hydrolysed/min per mg of protein) was measured in a membrane fraction enriched in transverse tubules. Several known inhibitors of InsP3ase, e.g. 2,3-bisphosphoglycerate, CdCl2 and EDTA, were active on skeletal-muscle InsP3ase. Total InsP3ase activity of both rabbit and frog skeletal muscle was comparable with that of rabbit brain, liver and main pulmonary artery (smooth muscle). The present results are consistent with the hypothesis that InsP3 plays a role in excitation-contraction coupling in skeletal muscle [Volpe, Salviati, Di Virgilio & Pozzan (1985) Nature (London) 316, 347-349].

Animals↗
PubMed1988

Activators of protein kinase C depolarize insulin-secreting cells by closing K+ channels.

Carbohydrate stimuli of insulin secretion depolarize the pancreatic B cell and the B-cell line RINm5F by inhibiting ATP-sensitive K+ channels. We examined the possibility that this effect is mediated by activation of protein kinase C. In RINm5F cells, the triose D-glyceraldehyde evoked a rapid increase of the mass of 1,2-diacylglycerol, the endogenous activator of protein kinase C. This effect is mainly due to de novo synthesis of the lipid from glycolytic intermediates, as glyceraldehyde carbon was incorporated into 1,2-diacylglycerol within 1 min of exposure to 14C-labelled glyceraldehyde. The effects of two exogenous activators of kinase C, 4-beta-12-phorbol-myristate 13-acetate (PMA) and 1,2-didecanoylglycerol (DC10) on single K+ channel currents were examined in RINm5F cell-attached membrane patches. Both PMA and DC10 depolarized the cells and decreased the open-state probability of the ATP-sensitive K+ channels. These actions were not due to changes in cellular ATP content, since PMA, like glyceraldehyde, failed to alter cellular ATP. As is the case for glyceraldehyde, PMA and DC10 raised cytosolic free Ca2+ [( Ca2+]i) and stimulated insulin secretion. Both of these effects are inhibited in the absence of external Ca2+. This, and the attenuation of the [Ca2+]i rise by verapamil, suggest that all three stimuli raise [Ca2+]i by promoting Ca2+ influx through voltage-gated channels in turn leading to insulin secretion. As the exogenous activators of protein kinase C mimic the effects of glyceraldehyde, it is proposed that the carbohydrate-mediated production of 1,2-diacylglycerol constitutes the link between metabolism and membrane depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1988

"Calciosome," a cytoplasmic organelle: the inositol 1,4,5-trisphosphate-sensitive Ca2+ store of nonmuscle cells?

Calsequestrin (CS) is the protein responsible for the high-capacity, moderate affinity binding of Ca2+ within the terminal cisternae of the sarcoplasmic reticulum, believed up to now to be specific for striated muscle. The cells of two nonmuscle lines (HL-60 and PC12) and of two rat tissues (liver and pancreas) are shown here to express a protein that resembles CS in many respects (apparent mass and pH-dependent migration in NaDodSO4/PAGE; blue staining with StainsAll dye; Ca2+ binding ability) and is specifically recognized by affinity-purified antibodies against skeletal muscle CS. In these cells, the CS-like protein is shown by immunofluorescence and immunogold procedures to be localized within peculiar, heretofore unrecognized structures distributed throughout the cytoplasm. These structures appear to be discrete organelles, which we propose to be named "calciosomes." By cell fractionation (Percoll gradient and free-flow electrophoresis), the CS-like protein of HL-60 cells is shown to copurify with the markers of the inositol 1,4,5-trisphosphate (Ins-P3)-sensitive Ca2+ store, whereas the markers of other organelles (endoplasmic reticulum, Golgi complex, mitochondria, endosomes) and of the plasma membrane do not. Calciosome might thus be the intracellular target of Ins-P3--i.e., the source of the Ca2+ redistributed to the cytosol following receptor-triggered generation of the messenger.

Animals↗
PubMed1988

Immunocytochemistry of calciosomes in liver and pancreas.

Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K. H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi, and D. P. Lew. Proc. Natl. Acad. Sci. USA. 85:1091-1095). Immunoperoxidase and immunogold experiments carried out in both thick and ultrathin cryosections of rat hepatocytes and pancreatic acinar cells by using antimuscle CS antibodies revealed a specific labeling widely distributed in the entire cytoplasm, while nuclei were negative. Individual calciosomes appeared as small (105 nm) membrane-bound vacuoles intermingled with, and often apposed to ER cisternae and mitochondria. Other calciosomes were scattered in the Golgi area, in between zymogen granules and beneath the plasma membrane. The cumulative volume of the CS-positive organelles was measured to account for the 0.8 and 0.45% of the cytoplasm in liver and pancreas cells, respectively. The real total volume of the calciosome compartment is expected to be approximately twice as large. In hepatocytes, structures similar to CS-positive calciosomes were decorated by antibodies against the Ca2+ ATPase of muscle SR, while ER cisternae were not. By dual labeling, colocalization was revealed in 53.6% of the organelles, with 37.6% positive for the ATPase only. CS appeared preferentially confined to the content, and the Ca2+ ATPase to the contour of the organelle. The results suggested a partial segregation of the two antigens, reminiscent of their well-known segregation in muscle SR. Additional dual-label experiments demonstrated that hepatic calciosomes express neither two ER markers (cytochrome-P450 and NADH-cytochrome b5 reductase) nor the endolysosome marker, luminal acidity (revealed by 3-[2,4-dinitroanilino]-3'-amino-N-methyl dipropylamine). Calciosomes appear as unique cytological entities, ideally equipped to play a role in the rapid-scale control of the cytosolic-free Ca2+ in nonmuscle cells.

Animals↗
PubMed1988

The Ins(1,4,5)P3-sensitive Ca2+ store of non-muscle cells: endoplasmic reticulum or calciosomes?

The binding of a number of extracellular ligands (hormones, growth factors, neurotransmitters etc.) to their plasma membrane receptors causes hydrolysis of phosphatidylinositol bisphosphate to initiate the formation of two second messengers, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol, DAG. DAG has been shown to activate protein kinase C, whereas Ins(1,4,5)P3 induces the release of Ca2+ from an intracellular pool. This rapidly mobilizable, Ins(1,4,5)P3-sensitive Ca2+ store has until now been identified as the endoplasmic reticulum, ER. We demonstrate that this is untenable and provide evidence for the existence of an unrecognized organelle, the 'calciosome'. This conclusion is based on the following experimental evidence. (1) There is no correlation between the abundance of ER and the amount Ins(1,4,5)P3-sensitive Ca2+ release. (2) There is no correlation between ER markers and those for the Ca2+ store [Ins(1,4,5)P3 binding and sensitivity, Ca2+ uptake]. (3) A protein similar to striated muscle calsequestrin, CS, has been identified in microsomal fractions from a number of tissues; it copurifies with markers of the Ca2+ store, but not with those of ER. (4) Subcellular localization of the CS-like protein by electron microscopy reveals that in all cells so far analysed this protein is localized in small, membrane-enclosed structures, calciosomes, which are also stained by an anti-Ca2+-ATPase antibody. Calciosomes appear to be morphologically distinct from any other known cell organelle. (5) Although they stain different portions of the calciosomes (membrane and lumen, respectively), anti-Ca2+-ATPase and anti-CS antibodies do not recognize any antigen in ER cysternae; antibodies directed against known components of ER do not bind to calciosomes.

Animals↗
PubMed1988

Immunocytological identification of the microsomal calcium store of nonmuscle cells.

The biochemical and functional similarities between skeletal muscle sarcoplasmic reticulum and the microsomal Ca2+ store of nonmuscle cells are discussed. It is shown that antibodies raised against two characteristic proteins of sarcoplasmic reticulum, Ca2+ ATPase and calsequestrin, recognize similar proteins in nonmuscle cells. The subcellular distribution of these two antigens was studied at the subcellular levels in ultrathin cryosections. In a variety of cell types these two proteins were found to be localized in small membrane enclosed vesicles, apparently distinct from other known organelles. We propose that these newly recognized structures (calciosomes) represent the functional equivalent of sarcoplasmic reticulum in nonmuscle cells.

Animals↗
PubMed1987

Dopamine inhibits cytosolic Ca2+ increases in rat lactotroph cells. Evidence of a dual mechanism of action.

Single rat lactotroph cells were studied after loading with the cytosolic free Ca2+ concentration ([Ca2+]i) indicator fura-2 either 1 or 3 days after cell dispersion. Under unstimulated conditions, two groups of lactotrophs were observed, the first (predominant at day 1) with large [Ca2+]i fluctuations (peaks up to 300 nM) probably due to spontaneous action potentials and the second (predominant at 3 days) with stable [Ca2+]i (values variable between 65 and 200 nM). The effect of dopamine on the resting [Ca2+]i was different in the two groups. Even at high dopamine concentrations, no change occurred in the second group; whereas in the first, disappearance of fluctuations and marked decrease of [Ca2+]i were observed. These effects of dopamine appear to be due to hyperpolarization that was demonstrated by the use of a specific fluorescent indicator, bis(oxonol). Two types of triggered [Ca2+]i transients were studied in detail: those due to redistribution of Ca2+ from the intracellular stores (induced by thyrotropin-releasing hormone) and those due to Ca2+ influx through voltage-gated Ca2+ channels (induced by high [K+]). Dopamine (1 microM) markedly inhibited both these transients by the action of D2 receptors (blocked by 1-sulpiride and domperidone). All effects of dopamine were prevented by treatment of the cells with pertussis toxin, indicating the involvement of one (or more) GTP-binding protein(s). Another consequence of D2 receptor activation is the inhibition of adenylate cyclase. Treatments (cholera toxin, forskolin), known to raise cAMP levels, were found to dissociate the effects of dopamine on [Ca2+]i inasmuch as they markedly relieved the inhibition of the redistributive transients by thyrotropin-releasing hormone but left hyperpolarization and inhibition of K+ transients unaffected. The spectrum of intracellular signals elicited by the activation of D2 receptors is therefore complex and includes at least two mechanisms that involve [Ca2+]i, one related and the other independent of the decrease of cAMP levels.

Adenylate Cyclase Toxin↗
PubMed1987

Antibody-induced modulation of the CD3/T cell receptor complex causes T cell refractoriness by inhibiting the early metabolic steps involved in T cell activation.

We investigated the mechanism involved in T cell unresponsiveness that follows the monoclonal antibody-induced surface modulation of the CD3-TCR complex. We determined whether modulation of CD3-TCR affected the early metabolic steps such as [Ca2+]i rise and InsP3 formation. A strong inhibition of the increase on [Ca2+]i mediated by either anti-TCR or anti-CD2 mAbs was detected. In contrast, surface modulation of CD2 molecules did not prevent the [Ca2+]i increase induced by anti-TCR mAb. Similarly, InsP3 increase was strongly reduced only after modulation of CD3-TCR complex (but not of CD2 molecules). Therefore, it appears that surface modulation of CD3-TCR complex causes T cell refractoriness by inhibiting the very early metabolic events that follow receptor-ligand interactions.

Antibodies, Monoclonal↗
PubMed1987

Voltage-dependent activation and inactivation of calcium channels in PC12 cells. Correlation with neurotransmitter release.

The existence and mechanisms of inactivation of voltage-gated Ca2+ channels are important, but still debatable, physiological problems. By using the Ca2+ indicators quin2 and fura-2, we demonstrate that in PC12 cells voltage-gated Ca2+ channels undergo inactivation dependent on both voltage and [Ca2+]i. Inactivation, however, is never complete and a small number of channels remains open during prolonged depolarization, explaining the steady state elevation of [Ca2+]i observed in cells depolarized with high KCl. A close parallel exists between Ca2+ channel inactivation and the transient nature of neurotransmitter release: secretion is rapidly stimulated during the first 30 s of depolarization, when a transient overshoot in [Ca2+]i can be demonstrated, while it is negligible during the following period, despite the persistence of an elevated [Ca2+]i; predepolarization in Ca2+-free medium and subsequent addition of Ca2+ (a condition which allows the development of the voltage inactivation) abolishes the fast phase of secretion, while not modifying the steady state [Ca2+]i eventually attained; and increases in the intracellular Ca2+ buffering decreases the amplitude of the fast secretion phase induced by KCl without altering the steady state [Ca2+]i. We suggest that localized [Ca2+]i gradients form close to the plasma membrane shortly after depolarization and that the [Ca2+]i reached in these regions is the relevant parameter in the regulation of secretion.

Adrenal Gland Neoplasms↗