Biomedical subjects

Tullio Pozzan

Publications and source records attributed to Tullio Pozzan.

At least 19 recordsLinked to original sources

PubMed2006

Direct in vivo monitoring of sarcoplasmic reticulum Ca2+ and cytosolic cAMP dynamics in mouse skeletal muscle.

Skeletal muscle contraction depends on the release of Ca(2+) from the sarcoplasmic reticulum (SR), but the dynamics of the SR free Ca(2+) concentration ([Ca(2+)](SR)), its modulation by physiological stimuli such as catecholamines, and the concomitant changes in cAMP handling have never been directly determined. We used two-photon microscopy imaging of GFP-based probes expressed in mouse skeletal muscles to monitor, for the first time in a live animal, the dynamics of [Ca(2+)](SR) and cAMP. Our data, which were obtained in highly physiological conditions, suggest that free [Ca(2+)](SR) decreases by approximately 50 microM during single twitches elicited through nerve stimulation. We also demonstrate that cAMP levels rise upon beta-adrenergic stimulation, leading to an increased efficacy of the Ca(2+) release/reuptake cycle during motor nerve stimulation.

Animals↗

PubMed2006

Microdomains of intracellular Ca2+: molecular determinants and functional consequences.

Calcium ions are ubiquitous and versatile signaling molecules, capable of decoding a variety of extracellular stimuli (hormones, neurotransmitters, growth factors, etc.) into markedly different intracellular actions, ranging from contraction to secretion, from proliferation to cell death. The key to this pleiotropic role is the complex spatiotemporal organization of the [Ca(2+)] rise evoked by extracellular agonists, which allows selected effectors to be recruited and specific actions to be initiated. In this review, we discuss the structural and functional bases that generate the subcellular heterogeneity in cellular Ca(2+) levels at rest and under stimulation. This complex choreography requires the concerted action of many different players; the central role is, of course, that of the calcium ion, with the main supporting characters being all the entities responsible for moving Ca(2+) between different compartments, while the cellular architecture provides a determining framework within which all the players have their exits and their entrances. In particular, we concentrate on the molecular mechanisms that lead to the generation of cytoplasmic Ca(2+) microdomains, focusing on their different subcellular location, mechanism of generation, and functional role.

Animals↗

PubMed2005

Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway.

beta-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP]i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving beta3-adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by beta-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the beta-adrenergic pathway. In this, activation of beta3-adrenergic receptors counteracts cAMP generation obtained via stimulation of beta1/beta2-adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.

Adenine↗

PubMed2005

Purinergic receptors mediate two distinct glutamate release pathways in hippocampal astrocytes.

The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain.

Adenosine Triphosphate↗

PubMed2005

Synaptobrevin2-expressing vesicles in rat astrocytes: insights into molecular characterization, dynamics and exocytosis.

The SNARE-dependent exocytosis of glutamate-containing vesicles in astrocytes is increasingly viewed as an important signal at the basis of the astrocyte-to-neurone communication system in the brain. Here we provide further insights into the molecular features and dynamics of vesicles in cultured astrocytes. We found that immunoisolated synaptobrevin2 vesicles are clear vesicles quite heterogenous in size and contain the vesicular glutamate transporter v-Glut-2. Moreover, they are immunopositive for synaptotagmin IV, for AMPA receptor subunits GluR2,3 and, to a lesser extent, for GluR1. We also provide direct evidence for the functional expression of v-Glut-2 in astrocytes and demonstrate that synaptobrevin2-positive vesicles can specifically take up (3H)L-glutamate via a bafilomycin-sensitive mechanism. Finally, by time lapse confocal microscopy, we show that a subpopulation of vesicles (tagged with a synaptobrevin2-EGFP chimera) is highly mobile and can fuse with the plasma membrane, preferentially at the level of the astrocyte processes, in a Ca2+-dependent manner. These latter observations, together with the evidence reported here for the expression of functional v-Glut-2 in synaptobrevin2-positive vesicles, provide a molecular basis for regulated exocytosis in astrocyte.

Animals↗

PubMed2005

Termination of cAMP signals by Ca2+ and G(alpha)i via extracellular Ca2+ sensors: a link to intracellular Ca2+ oscillations.

Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca(2+)-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer-dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin-sensitive Galpha(i) and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca(2+)-mobilizing agonists under physiological conditions.

Acetylcysteine↗

PubMed2005

Beta-adrenergic- and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes detected with FRET-based biosensor.

beta-Adrenergic receptor activation regulates cardiac myocyte function through the stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Furthermore, muscarinic receptor activation inhibits as well as facilitates these cAMP-dependent effects. However, it has not always been possible to correlate the muscarinic responses with the direct measurement of changes in cellular cAMP activity. Genetically encoded biosensors have recently been developed, making it possible to monitor real-time changes in cAMP and PKA activity at the single cell level. One such biosensor consists of the regulatory and catalytic subunits of PKA labeled with cyan and yellow fluorescent proteins, respectively. Changes in cAMP activity affecting the association of these labeled PKA subunits can be detected as changes in fluorescence resonance energy transfer. In the present study, an adenovirus-based approach was developed to express this recombinant protein complex in adult cardiac myocytes and use it to monitor changes in cAMP activity produced by beta-adrenergic and muscarinic receptor activation. The biosensor expressed with the use of this system is able to detect changes in cAMP activity produced by physiologically relevant levels of beta-adrenergic receptor activation without disrupting normal functional responses. It was also possible to directly demonstrate the complex temporal pattern of inhibitory and stimulatory changes in cAMP activity produced by muscarinic receptor activation in these cells. The adenovirus-based approach we have developed should facilitate the use of this biosensor in studying cAMP and PKA-dependent signaling mechanisms in a wide variety of cell types.

Adenoviridae↗

PubMed2005

Improved strategies for the delivery of GFP-based Ca2+ sensors into the mitochondrial matrix.

The role of mitochondria in Ca2+ handling has acquired renewed interest in recent years in the field of cell signaling. Detailed studies of Ca2+ dynamics in this organelle at the single cell level have been hampered by technical problems in the available Ca2+ probes. Some of the latest generation GFP-based Ca2+ probes (Camgaroos, Cameleons and Pericams) show great potential to address this issue. Our data show that the choice of targeting sequence influences not only the overall efficiency of subcellular localization of the probes, but also their functional characteristics within the matrix. In particular, we here show that the use of a tandemly duplicated mitochondrial targeting sequence is capable of improving the delivery efficacy of all tested probes into the organelle's matrix, in particular that of Cameleon, a GFP-based Ca2+ probe that is otherwise largely mistargeted to the cytosol. The devised strategy should be generally applicable to other proteins that are characterized by poor targeting. Last, but not least, we also demonstrate that if the targeting sequence is not removed from the imported protein, the fluorescent properties and the Ca2+ affinity of the probe can be grossly affected.

Biosensing Techniques↗

PubMed2005

Reduction of Ca2+ stores and capacitative Ca2+ entry is associated with the familial Alzheimer's disease presenilin-2 T122R mutation and anticipates the onset of dementia.

Mutations in the presenilin genes PS1 and PS2, the major cause of familial Alzheimer's disease (FAD), are associated with alterations in Ca2+ signalling. In contrast to the majority of FAD-linked PS1 mutations, which cause an overload of intracellular Ca2+ pools, the FAD-linked PS2 mutation M239I reduces Ca2+ release from intracellular stores [Zatti, G., Ghidoni, R., Barbiero, L., Binetti, G., Pozzan, T., Fasolato, C., Pizzo, P., 2004. The presenilin 2 M239I mutation associated with Familial Alzheimer's Disease reduces Ca2+ release from intracellular stores. Neurobiol. Dis. 15/2, 269-278]. We here show that in human FAD fibroblasts another PS2 mutation (T122R) reduces both Ca2+ release and capacitative Ca2+ entry. The observation, done in two monozygotic twins, is of note since only one of the subjects showed overt signs of disease at the time of biopsy whereas the other one developed the disease 3 years later. This finding indicates that Ca2+ dysregulation anticipates the onset of dementia. A similar Ca2+ alteration occurred in HeLa and HEK293 cells transiently expressing PS2-T122R. Based on these data, the "Ca2+ overload" hypothesis in AD pathogenesis is here discussed and reformulated.

Aged↗

PubMed2005

Glibenclamide stimulates fluid secretion in rodent cholangiocytes through a cystic fibrosis transmembrane conductance regulator-independent mechanism.

BACKGROUND & AIMS: Progressive liver disease is a severe complication of cystic fibrosis, a genetic disease characterized by impaired epithelial adenosine 3',5'-cyclic monophosphate-dependent secretion caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). In the liver, CFTR is expressed in cholangiocytes and regulates the fluid and electrolyte content of the bile. Glibenclamide, a sulfonylurea and a known CFTR inhibitor, paradoxically stimulates cholangiocyte secretion. We studied the molecular mechanisms underlying this effect and whether glibenclamide could restore cholangiocyte secretion in cystic fibrosis. METHODS: NRC-1 cells, freshly isolated rat cholangiocytes, isolated rat biliary ducts, and isolated biliary ducts from CFTR-defective mice (Cftr tm1Unc ) were used to study fluid secretion (by video-optical planimetry), glibenclamide-induced secretion (by high-performance liquid chromatography in cell culture medium), intracellular pH and intracellular Ca 2+ concentration transients [2'7'-bis(2-carboxyethyl)-5,6,carboxyfluorescein-acetoxymethylester and Fura-2 f-AM (5-Oxazolecarboxylic acid, 2-(6-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(2-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)-2-benzofuranyl)-, (acetyloxy)methyl ester) microfluorometry], gene expression (by reverse-transcription polymerase chain reaction), and changes in membrane capacitance (by patch-clamp experiments). RESULTS: Stimulation of cholangiocyte secretion by glibenclamide and tolbutamide required Cl - and was mediated by the sulfonylurea receptor 2B. Glibenclamide-induced secretion was blocked by inhibitors of exocytosis (colchicine, wortmannin, LY294002, and N -ethylmaleimide) and by inhibitors of secretory granule acidification (vanadate, bafilomycin A1, and niflumic acid) but was Ca 2+ and depolarization independent; membrane capacitance measurements were consistent with stimulation of vesicular transport and fusion. Glibenclamide, unlike secretin and forskolin, was able to stimulate secretion in Cftr tm1Unc mice, thus indicating that this secretory mechanism was preserved. CONCLUSIONS: The ability of glibenclamide to stimulate secretion in CFTR-defective mice makes sulfonylureas a model class of compounds to design drugs useful in the treatment of cystic fibrosis with liver impairment and possibly of other cholestatic diseases.

ATP-Binding Cassette Transporters↗

PubMed2004

Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum.

Proapoptotic BCL-2 family members BAX and BAK are required for the initiation of mitochondrial dysfunction during apoptosis and for maintaining the endoplasmic reticulum (ER) Ca(2+) stores necessary for Ca(2+)-dependent cell death. Conversely, antiapoptotic BCL-2 has been shown to decrease Ca(2+) concentration in the ER. We found that Bax(-/-)Bak(-/-) double-knockout (DKO) cells have reduced resting ER Ca(2+) levels because of increased Ca(2+) leak and an increase in the Ca(2+)-permeable, hyperphosphorylated state of the inositol trisphosphate receptor type 1 (IP3R-1). The ER Ca(2+) defect of DKO cells is rescued by RNA interference reduction of IP3R-1, supporting the argument that this channel regulates the increased Ca(2+) leak in these cells. BCL-2 and IP3R-1 physically interact at the ER, and their binding is increased in the absence of BAX and BAK. Moreover, knocking down BCL-2 decreases IP3R-1 phosphorylation and ER Ca(2+) leak rate in the DKO cells. These findings support a model in which BCL-2 family members regulate IP3R-1 phosphorylation to control the rate of ER Ca(2+) leak from intracellular stores.

Animals↗

PubMed2004

Impaired permeability to Ins(1,4,5)P3 in a mutant connexin underlies recessive hereditary deafness.

Connexins are membrane proteins that assemble into gap-junction channels and are responsible for direct, electrical and metabolic coupling between connected cells. Here we describe an investigation of the properties of a recombinantly expressed recessive mutant of connexin 26 (Cx26), the V84L mutant, associated with deafness. Unlike other Cx26 mutations, V84L affects neither intracellular sorting nor electrical coupling, but specifically reduces permeability to the Ca(2+)-mobilizing messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)). Both the permeability to Lucifer Yellow and the unitary channel conductance of V84L-mutant channels are indistinguishable from those of the wild-type Cx26. Injection of Ins(1,4,5)P(3) into supporting cells of the rat organ of Corti, which abundantly express Cx26, ensues in a regenerative wave of Ca(2+) throughout the tissue. Blocking the gap junction communication abolishes wave propagation. We propose that the V84L mutation reduces metabolic coupling mediated by Ins(1,4,5)P(3) to an extent sufficient to impair the propagation of Ca(2+) waves and the formation of a functional syncytium. Our data provide the first demonstration of a specific defect of metabolic coupling and offer a mechanistic explanation for the pathogenesis of an inherited human disease.

Animals↗

PubMed2004

Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors.

Fast excitatory neurotransmission is mediated by activation of synaptic ionotropic glutamate receptors. In hippocampal slices, we report that stimulation of Schaffer collaterals evokes in CA1 neurons delayed inward currents with slow kinetics, in addition to fast excitatory postsynaptic currents. Similar slow events also occur spontaneously, can still be observed when neuronal activity and synaptic glutamate release are blocked, and are found to be mediated by glutamate released from astrocytes acting preferentially on extrasynaptic NMDA receptors. The slow currents can be triggered by stimuli that evoke Ca2+ oscillations in astrocytes, including photolysis of caged Ca2+ in single astrocytes. As revealed by paired recording and Ca2+ imaging, a striking feature of this NMDA receptor response is that it occurs synchronously in multiple CA1 neurons. Our results reveal a distinct mechanism for neuronal excitation and synchrony and highlight a functional link between astrocytic glutamate and extrasynaptic NMDA receptors.

Animals↗

PubMed2004

In vivo monitoring of Ca(2+) uptake into mitochondria of mouse skeletal muscle during contraction.

Although the importance of mitochondria in patho-physiology has become increasingly evident, it remains unclear whether these organelles play a role in Ca(2+) handling by skeletal muscle. This undefined situation is mainly due to technical limitations in measuring Ca(2+) transients reliably during the contraction-relaxation cycle. Using two-photon microscopy and genetically expressed "cameleon" Ca(2+) sensors, we developed a robust system that enables the measurement of both cytoplasmic and mitochondrial Ca(2+) transients in vivo. We show here for the first time that, in vivo and under highly physiological conditions, mitochondria in mammalian skeletal muscle take up Ca(2+) during contraction induced by motor nerve stimulation and rapidly release it during relaxation. The mitochondrial Ca(2+) increase is delayed by a few milliseconds compared with the cytosolic Ca(2+) rise and occurs both during a single twitch and upon tetanic contraction.

Animals↗

PubMed2004

The inner side of T cell lipid rafts.

A key question in understanding the functional role of lipid rafts is whether lipid microdomains at the plasma membrane outer leaflet are coupled to lipid microdomains at the inner leaflet. By using a cyan-fluorescent protein (CFP) targeted to inner plasma membrane rafts of Jurkat T cells, we found that raft domains at the outer and inner leaflets are physically coupled and that this coupling requires cholesterol. Interestingly, TCR/CD3 cross-linking induces co-capping of the raft bilayer independently of cholesterol or signaling events, indicating that cholesterol-extracting drugs are unable to destroy TCR-lipid rafts interaction.

CD3 Complex↗

PubMed2004

Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases.

Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes. By performing real-time imaging of cAMP in situ, we establish the hierarchy of these PDEs in controlling cAMP levels in basal conditions and on stimulation with a beta-adrenergic receptor agonist. PDE4, rather than PDE3, appears to be responsible for modulating the amplitude and duration of the cAMP response to beta-agonists. PDE3 and PDE4 localize to distinct compartments and this may underpin their different functional roles. Our findings indicate the importance of distinctly localized PDE isoenzymes in determining compartmentalized cAMP signaling.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2004

Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis.

Phosphorylation of BCL-2 within an unstructured loop inhibits its antiapoptotic effect. We found that phosphorylated BCL-2 predominantly localized to the endoplasmic reticulum (ER) and tested whether phosphorylation would control its activity at this organelle, where Ca(2+) dynamics serve as a critical control point for apoptosis. Phosphorylation greatly inhibits the ability of BCL-2 to lower [Ca(2+)](er) and protect against Ca(2+)-dependent death stimuli. Cells expressing nonphosphorylatable BCL-2(AAA) exhibited increased leak of Ca(2+) from the ER and further diminished steady-state [Ca(2+)](er) stores when compared to cells expressing BCL-2(wt). Consequently, when BCL-2 is phosphorylated, Ca(2+) discharge from the ER is increased, with a secondary increase in mitochondrial Ca(2+) uptake. We also demonstrate that phosphorylation of BCL-2 inhibits its binding to proapoptotic family members. This inhibitory mechanism manifested at the ER, where phosphorylated BCL-2 was unable to bind proapoptotic members. [Ca(2+)](er) proved coordinate with the capacity of BCL-2 to bind proapoptotic BH3-only members, further integrating the apoptotic pathway and Ca(2+) modulation. Unexpectedly, the regulation of ER Ca(2+) dynamics is a principal avenue whereby BCL-2 phosphorylation alters susceptibility to apoptosis.

Animals↗

PubMed2004

Flirting in little space: the ER/mitochondria Ca2+ liaison.

Mitochondria have long been known to accumulate Ca2+; the apparent inconsistency between the low affinity of mitochondrial Ca2+ uptake mechanisms, the low concentration of global Ca2+ signals observed in cytoplasm, and the efficiency in intact cells of mitochondrial Ca2+ uptake led to the formulation of the "hotspot hypothesis." This hypothesis proposes that mitochondria preferentially accumulate Ca2+ at microdomains of elevated Ca2+ concentration ([Ca2+]) that exist near endoplasmic reticulum (ER) Ca2+ release sites and other Ca2+ channels. Physiological Ca2+ signals may affect mitochondrial function--both by stimulating key metabolic enzymes and, under some conditions, by promoting apoptosis. Mitochondria in turn may affect both Ca2+ release from the ER and capacitative Ca2+ entry across the plasma membrane, thereby shaping the size and duration of the intracellular Ca2+ signal. Interactions between mitochondria and the ER are critically dependent on the spatial localization of mitochondria within the cell. The molecular mechanisms that define the organization of mitochondria with regard to the ER and other Ca2+ sources, and the extent to which mitochondrial function varies among different cell types, are open questions whose answers remain to be determined.

Animals↗