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Biomedical subjects

T Pozzan

Publications and source records attributed to T Pozzan.

At least 19 recordsLinked to original sources
PubMed2001

Dystrophin muscle enhancer 1 is implicated in the activation of non-muscle isoforms in the skeletal muscle of patients with X-linked dilated cardiomyopathy.

X-linked dilated cardiomyopathy (XLDC) is a dystrophinopathy characterized by severe cardiomyopathy with no skeletal muscle involvement. Several XLDC patients have been described with mutations that abolish dystrophin muscle (M) isoform expression. The absence of skeletal muscle degeneration normally associated with loss of dystrophin function was shown to be due to increased expression of brain (B) and cerebellar Purkinje (CP) isoforms of the gene exclusively in the skeletal muscle of these patients. This suggested that the B and CP promoters have an inherent capacity to function in skeletal muscle or that they are up-regulated by a skeletal muscle-specific enhancer unaffected by the mutations in these patients. In this work we have analyzed the deletion breakpoints of two XLDC patients with deletions removing the M promoter and exon 1, but not affecting the B and CP promoters. Despite the presence of several muscle-specific regulatory motifs, the B and CP promoters were found to be essentially inactive in muscle cell lines and primary cultures. As dystrophin muscle enhancer 1 (DME1), the only known muscle-specific enhancer within the dystrophin gene, is preserved in these patients, we tested its ability to up-regulate the B and CP promoters in muscle cells. B and CP promoter activity was significantly increased in the presence of DME1, and more importantly, activation was observed exclusively in cells presenting a skeletal muscle phenotype. These results point to a role for DME1 in the induction of B and CP isoform expression in the skeletal muscle of XLDC patients defective for M isoform expression.

3T3 Cells↗
PubMed2001

Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

Adenosine Triphosphate↗
PubMed2001

Beat-to-beat oscillations of mitochondrial [Ca2+] in cardiac cells.

The Ca2+-sensitive photoprotein aequorin and the new green fluorescent protein-based fluorescent Ca2+ indicators 'ratiometric-pericam' were selectively expressed in the mitochondria, cytosol and/or nucleus of spontaneously beating ventricular myocytes from neonatal rats. This combined strategy reveals that mitochondrial [Ca2+] oscillates rapidly and in synchrony with cytosolic and nuclear [Ca2+]. The Ca2+ oscillations were reduced in frequency and/or amplitude by verapamil and carbachol and were enhanced by isoproterenol and elevation of extracellular [Ca2+]. An increased frequency and/or amplitude of cytosolic Ca2+ spikes was rapidly mirrored by similar changes in mitochondrial Ca2+ spikes and more slowly by elevations of the interspike Ca2+ levels. The present data unequivocally demonstrate that in cardiac cells mitochondrial [Ca2+] oscillates synchronously with cytosolic [Ca2+] and that mitochondrial Ca2+ handling rapidly adapts to inotropic or chronotropic inputs.

Aequorin↗
PubMed2001

The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action.

The mechanism of action of the anti-apoptotic oncogene Bcl-2 is still largely obscure. We have recently shown that the overexpression of Bcl-2 in HeLa cells reduces the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) by increasing the passive Ca2+ leak from the organelle. To investigate whether this Ca2+ depletion is part of the mechanism of action of Bcl-2, we mimicked the Bcl-2 effect on [Ca2+]er by different pharmacological and molecular approaches. All conditions that lowered [Ca2+]er protected HeLa cells from ceramide, a Bcl-2-sensitive apoptotic stimulus, while treatments that increased [Ca2+]er had the opposite effect. Surprisingly, ceramide itself caused the release of Ca2+ from the endoplasmic reticulum and thus [Ca2+] increased both in the cytosol and in the mitochondrial matrix, paralleled by marked alterations in mitochondria morphology. The reduction of [Ca2+]er levels, as well as the buffering of cytoplasmic [Ca2+] changes, prevented mitochondrial damage and protected cells from apoptosis. It is therefore concluded that the Bcl-2-dependent reduction of [Ca2+]er is an important component of the anti-apoptotic program controlled by this oncogene.

Aequorin↗
PubMed2001

Cytosolic calcium oscillations in astrocytes may regulate exocytotic release of glutamate.

To obtain insights into the spatiotemporal characteristics and mechanism of Ca(2+)-dependent glutamate release from astrocytes, we developed a new experimental approach using human embryonic kidney (HEK) 293 cells transfected with the NMDA receptor (NMDAR), which act as glutamate biosensors, plated on cultured astrocytes. We here show that oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) in astrocytes trigger synchronous and repetitive [Ca(2+)](i) elevations in sensor HEK cells, and that these elevations are sensitive to NMDAR inhibition. By whole-cell patch-clamp recordings, we demonstrate that the activation of NMDARs in HEK cells results in inward currents that often have extremely fast kinetics, comparable with those of glutamate-mediated NMDAR currents in postsynaptic neurons. We also show that the release of glutamate from stimulated astrocytes is drastically reduced by agents that are known to reduce neuronal exocytosis, i.e., tetanus toxin and bafilomycin A(1). We conclude that [Ca(2+)](i) oscillations represent a frequency-encoded signaling system that controls a pulsatile release of glutamate from astrocytes. The fast activation of NMDARs in the sensor cells and the dependence of glutamate release on the functional integrity of both synaptobrevin and vacuolar H(+) ATPase suggest that astrocytes are endowed with an exocytotic mechanism of glutamate release that resembles that of neurons.

Animals↗
PubMed2001

Heterogeneity of second messenger levels in living cells.

Over the last years we have utilised chimeras from aequorin and green fluorescent protein (GFP) to monitor the dynamics of second messenger levels in living cells. In this contribution we address two problems, i.e. the complexity of Ca2+ handling by mitochondria and the localization of cAMP signalling. As to the first, we here demonstrate that physiological increases in mitochondrial Ca2+, monitored with selectively localized recombinant aequorin, concern a sub-population of organelles that is stably and selectively associated with the endoplasmic reticulum. As to cAMP, we describe the use of a novel probe to monitor its changes in living cells, that takes advantage of the phenomenon of fluorescence resonance energy transfer (FRET) between suitable GFPs linked to the regulatory and catalytic subunits of protein kinase A (PKA). When cAMP is low the two fluorophores are in close proximity and generate FRET while increasing levels of cAMP determine progressive reduction of FRET as the two subunits (linked to the GFPs) diffuse apart. We also demonstrate that by using such cAMP sensor, localized increase of this second messenger can be observed upon selective stimulation of plasma membrane receptors.

Animals↗
PubMed2001

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes.

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.

Animals↗
PubMed2000

Mitochondria as all-round players of the calcium game.

Although it has been known for over three decades that mitochondria are endowed with a complex array of Ca2+ transporters and that key enzymes of mitochondrial metabolism are regulated by Ca2+, the possibility that physiological stimuli that raise the [Ca2+] of the cytoplasm could trigger major mitochondrial Ca2+ uptake has long been considered unlikely, based on the low affinity of the mitochondrial transporters and the limited amplitude of the cytoplasmic [Ca2+] rises. The direct measurement of mitochondrial [Ca2+] with highly selective probes has led to a complete reversion of this view, by demonstrating that, after cell stimulation, the cytoplasmic Ca2+ signal is always paralleled by a much larger rise in [Ca2+] in the mitochondrial matrix. This observation has rejuvenated the study of mitochondrial Ca2+ transport and novel, unexpected results have altered long-standing dogmas in the field of calcium signalling. Here we focus on four main topics: (i) the current knowledge of the functional properties of the Ca2+ transporters and of the thermodynamic constraints under which they operate; (ii) the occurrence of mitochondrial Ca2+ uptake in living cells and the key role of local signalling routes between the mitochondria and the Ca2+ sources; (iii) the physiological consequences of Ca2+ transport for both mitochondrial function and the modulation of the cytoplasmic Ca2+ signal; and (iv) evidence that alterations of mitochondrial Ca2+ signalling may occur in pathophysiological conditions.

Animals↗
PubMed2000

Alteration in calcium handling at the subcellular level in mdx myotubes.

In this study, we have tested the hypothesis that augmented [Ca(2+)] in subcellular regions or organelles, which are known to play a key role in cell survival, is the missing link between Ca(2+) homeostasis alterations and muscular degeneration associated with muscular dystrophy. To this end, different targeted chimeras of the Ca(2+)-sensitive photoprotein aequorin have been transiently expressed in subcellular compartments of skeletal myotubes of mdx mice, the animal model of Duchenne muscular dystrophy. Direct measurements of the [Ca(2+)] in the sarcoplasmic reticulum, [Ca(2+)](sr), show a higher steady state level at rest and a larger drop after KCl-induced depolarization in mdx compared with control myotubes. The peaks in [Ca(2+)] occurring in the mitochondrial matrix of mdx myotubes are significantly larger than in controls upon KCl-induced depolarization or caffeine application. The augmented response of mitochondria precedes the alterations in the Ca(2+) responses of the cytosol and of the cytoplasmic region beneath the membrane, which become significant only at a later stage of myotube differentiation. Taking into account the key role played by mitochondria Ca(2+) handling in the control of cell death, our data suggest that mitochondria are potential targets of impaired Ca(2+) homeostasis in muscular dystrophy.

Aequorin↗
PubMed2000

Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) influx in Bcl-2-overexpressing cells.

The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca(2+)-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca(2+) homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca(2+) leak (while leaving Ca(2+) accumulation unaffected), hence reducing the steady-state [Ca(2+)] levels. As a direct consequence, the [Ca(2+)] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca(2+) influx activated by Ca(2+) store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca(2+)-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca(2+) homeostasis may contribute to the protective role of this oncogene against programmed cell death.

Adenosine Triphosphate↗
PubMed2000

High tide of calcium in mitochondria.

Mitochondria in intact cells can transiently accumulate calcium during cell stimulation. The heterogeneity of the response, the extremely high calcium concentrations reached in the mitochondrial matrix, and the ensuing modulation of secretion add further complexity to the spatiotemporal aspects of signalling through calcium ions.

Calcium↗
PubMed2000

A genetically encoded, fluorescent indicator for cyclic AMP in living cells.

Cyclic AMP controls several signalling cascades within cells, and changes in the amounts of this second messenger have an essential role in many cellular events. Here we describe a new methodology for monitoring the fluctuations of cAMP in living cells. By tagging the cAMP effector protein kinase A with two suitable green fluorescent protein mutants, we have generated a probe in which the fluorescence resonance energy transfer between the two fluorescent moieties is dependent on the levels of cAMP. This new methodology opens the way to the elucidation of the biochemistry of cAMP in vivo.

Adrenergic alpha-Agonists↗
PubMed2000

The renaissance of mitochondrial calcium transport.

Although the capacity of mitochondria for accumulating Ca2+ down the electrical gradient generated by the respiratory chain has been known for over three decades, the physiological significance of this phenomenon has been re-evaluated only recently. Indeed, it was long believed that the low affinity of the mitochondrial Ca2+ transporters would allow significant uptake only in conditions of cellular Ca2+ overload. Conversely, the direct measurement of [Ca2+] in the mitochondrial matrix revealed major [Ca2+] increases upon agonist stimulation. In this review, we will summarize: (a) the mechanisms that allow this large response, reconciling the biochemical properties of the transporters and the large amplitude of the mitochondrial [Ca2+] rises, and (b) the biological role of mitochondrial Ca2+ signalling, that encompasses the regulation of mitochondrial function and the modulation of the spatio-temporal pattern of cytosolic [Ca2+] increases.

Animals↗
PubMed2000

Imaging signal transduction in living cells with GFP-based probes.

Since the cloning and the eterologous expression of the Green Fluorescence Protein (GFP), a number of applications have been reported where protein location within the cell or gene expression is revealed by fluorescent imaging of living cells. Modified GFPs, however, can now be exploited not only as a fluorescent reporter but also as a dynamic marker of intracellular signalling events, such as fluctuations in the levels of the second messengers Ca2+ and cAMP, or as a probe for detecting changes in pH in various cell compartments. These genetically manipulated GFPs allow monitoring of the biochemistry of the cell in real time and thus offer the possibility to gain a more precise view of the functioning of live cells.

Animals↗
PubMed1999

Targeted recombinant aequorins: tools for monitoring [Ca2+] in the various compartments of a living cell.

In the last decade, the study of Ca2+ homeostasis within organelles in living cells has been greatly enhanced by the utilisation of a recombinant Ca(2+)-sensitive photoprotein, aequorin. Aequorin is a Ca2+ sensitive photoprotein of a coelenterate that, in the past, was widely employed to measure Ca2+ concentration in living cells. In fact, the purified protein was widely used to monitor cytoplasmic [Ca2+] changes in invertebrate muscle cells after microinjection. However, due to the time-consuming and traumatic procedure of microinjection, the role of aequorin in the study of Ca2+ homeostasis remained confined to a limited number of cells (giant cells) susceptible to microinjection. Thus, in most instances, it was replaced by the fluorescent indicators developed by Roger Tsien and coworkers. The cloning of aequorin cDNA [Inouye et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3154-3158] and the explosive development of molecular biology offered new possibilities in the use of aequorin, as microinjection has been replaced by the simpler technique of cDNA transfection. As a polypeptide, aequorin allows the endogenous production of the photoprotein in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localise it within the cell by including defined targeting signals in the amino acid sequence. Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles. In this review, we will not discuss the procedure of aequorin microinjection and its use as purified protein but we will present the new advances provided by recombinant aequorin in the study of intracellular Ca2+ homeostasis, discussing in greater detail the advantages and disadvantages in the use of this probe.

Aequorin↗
PubMed1999

Steady-state free Ca(2+) in the yeast endoplasmic reticulum reaches only 10 microM and is mainly controlled by the secretory pathway pump pmr1.

Over recent decades, diverse intracellular organelles have been recognized as key determinants of Ca(2+) signaling in eukaryotes. In yeast however, information on intra-organellar Ca(2+) concentrations is scarce, despite the demonstrated importance of Ca(2+) signals for this microorganism. Here, we directly monitored free Ca(2+) in the lumen of the endoplasmic reticulum (ER) of yeast cells, using a specifically targeted version of the Ca(2+)-sensitive photoprotein aequorin. Ca(2+) uptake into the yeast ER displayed characteristics distinctly different from the mammalian ER. At steady-state, the free Ca(2+) concentration in the ER lumen was limited to approximately 10 microM, and ER Ca(2+) sequestration was insensitive to thapsigargin, an inhibitor specific for mammalian ER Ca(2+) pumps. In pmr1 null mutants, free Ca(2+) in the ER was reduced by 50%. Our findings identify the secretory pathway pump Pmr1, predominantly localized in the Golgi, as a major component of ER Ca(2+) uptake activity in yeast.

ATP-Binding Cassette Transporters↗