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T Pozzan

Publications and source records attributed to T Pozzan.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cellular calcium handling in brain slices from calbindin D28k-deficient mice.

Cellular calcium handling was examined in brain slices from transgenic antisense mice with a regional deficiency in the neuronal calcium binding protein calbindin D28k and from their non transgenic wild type litter mate controls. Depolarization of brain slices with NMDA or potassium produced a prolonged elevation of neuronal calcium signal in neurons in brain slices from calbindin D28k-deficient transgenic mice. This effect was selective and was seen only in brain areas where the antisense construct produced a significant depletion of calbindin D28k protein. In other regions where calbindin D28k protein was not modified by the construct and in all glial cells whether from wild type or transgenic mice, cellular calcium handling was normal.

Animals↗
PubMed1999

Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells. Studies in aequorin-expressing intact and permeabilized ins-1 cells.

The precise regulation of the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) is important for protein processing and signal transduction. In the pancreatic beta-cell, dysregulation of [Ca2+]er may cause impaired insulin secretion. The Ca2+-sensitive photoprotein aequorin mutated to lower its Ca2+ affinity was stably expressed in the endoplasmic reticulum (ER) of rat insulinoma INS-1 cells. The steady state [Ca2+]er was 267 +/- 9 microM. Both the Ca2+-ATPase inhibitor cyclopiazonic acid and 4-chloro-m-cresol, an activator of ryanodine receptors, caused an almost complete emptying of ER Ca2+. The inositol 1,4,5-trisphosphate generating agonists, carbachol, and ATP, reduced [Ca2+]er by 20-25%. Insulin secretagogues that raise cytosolic [Ca2+] by membrane depolarization increased [Ca2+]er in the potency order K+ >> glucose > leucine, paralleling their actions in the cytosolic compartment. Glucose, which augmented [Ca2+]er by about 25%, potentiated the Ca2+-mobilizing effect of carbachol, explaining the corresponding observation in cytosolic [Ca2+]. The filling of ER Ca2+ by glucose is not directly mediated by ATP production as shown by the continuous monitoring of cytosolic ATP in luciferase expressing cells. Both glucose and K+ increase [Ca2+]er, but only the former generated whereas the latter consumed ATP. Nonetheless, drastic lowering of cellular ATP with a mitochondrial uncoupler resulted in a marked decrease in [Ca2+]er, emphasizing the requirement for mitochondrially derived ATP above a critical threshold concentration. Using alpha-toxin permeabilized cells in the presence of ATP, glucose 6-phosphate did not change [Ca2+]er, invalidating the hypothesis that glucose acts through this metabolite. Therefore, insulin secretagogues that primarily stimulate Ca2+ influx, elevate [Ca2+]er to ensure beta-cell homeostasis.

Adenosine Triphosphate↗
PubMed1999

Mitochondria as biosensors of calcium microdomains.

The notion that the agonist-dependent increases in intracellular Ca2+ concentration, on ubiquitous signalling mechanism, occur with a tightly regulated spatio-temporal pattern has become an established concept in modern cell biology. As a consequence, the concept is emerging that the recruitment of specific intracellular targets and effector system mechanisms depends on exposure to local [Ca2+] that differs substantially from the mean [Ca2+]. A striking example is provided by mitochondria, intracellular organelles that have been overlooked for a long time in the field of calcium signalling due to the low affinity of their Ca(2+)-uptake pathways. We will summarize here some of the evidence indicating that these organelles actively participate in Ca2+ homeostasis in physiological conditions (with consequences not only for the control of their function, but also for the modulation of the complexity of calcium signals) because they have the capability to respond to microdomains of high [Ca2+] transiently generated in their proximity by the opening of Ca2+ channels.

Aequorin↗
PubMed1998

Direct monitoring of the calcium concentration in the sarcoplasmic and endoplasmic reticulum of skeletal muscle myotubes.

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis showed that although both recombinant proteins are distributed in an endomembrane network identifiable with immature SR, the erAEQ protein was retained also in the perinuclear membrane. The difficulty of measuring [Ca2+] in 100-1000 microM range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAEQ is around 300 microM, whereas that measured with erAEQ is significantly lower, i.e. around 200 microM. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with both chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of muscle development, differences exist between SR and ER in their mechanisms of Ca2+ handling. The functional implications of these findings are discussed.

Aequorin↗
PubMed1998

The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum.

In the past few years, intracellular organelles, such as the endoplasmic reticulum, the nucleus and the mitochondria, have emerged as key determinants in the generation and transduction of Ca2+ signals of high spatio-temporal complexity. Little is known about the Golgi apparatus, despite the fact that Ca2+ within its lumen controls essential processes, such as protein processing and sorting. We report the direct monitoring of the [Ca2+] in the Golgi lumen ([Ca2+]Golgi) of living HeLa cells, using a specifically targeted Ca2+-sensitive photoprotein. With this probe, we show that, in resting cells, [Ca2+]Golgi is approximately 0.3 mM and that Ca2+ accumulation by the Golgi has properties distinct from those of the endoplasmic reticulum (as inferred by the sensitivity to specific inhibitors). Upon stimulation with histamine, an agonist coupled to the generation of inositol 1,4,5-trisphosphate (IP3), a large, rapid decrease in [Ca2+]Golgi is observed. The Golgi apparatus can thus be regarded as a bona fide IP3-sensitive intracellular Ca2+ store, a notion with major implications for the control of organelle function, as well as for the generation of local cytosolic Ca2+ signals.

Aequorin↗
PubMed1998

Ca2+ homeostasis in the agonist-sensitive internal store: functional interactions between mitochondria and the ER measured In situ in intact cells.

Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals.

Adenosine Triphosphate↗
PubMed1998

Ca2+ depletion from granules inhibits exocytosis. A study with insulin-secreting cells.

The secretory compartment is characterized by low luminal pH and high Ca2+ content. Previous studies in several cell types have shown that the size of the acidic Ca2+ pool, of which secretory granules represent a major portion, could be estimated by applying first a Ca2+ ionophore followed by agents that collapse acidic pH gradients. In the present study we have employed this protocol in the insulin-secreting cell line Ins-1 to determine whether the Ca2+ trapped in the secretory granules plays a role in exocytosis. The results demonstrate that a high proportion of ionophore-mobilizable Ca2+ in Ins-1 cells resides in the acidic compartment. The latter pool, however, does not significantly contribute to the [Ca2+]i changes elicited by thapsigargin and the inositol trisphosphate-producing agonist carbachol. By monitoring membrane capacitance at the single cell level or by measuring insulin release in cell populations, we show that Ca2+ mobilization from nonacidic Ca2+ pools causes a profound and long lasting increase in depolarization-induced secretion, whereas breakdown of granule pH had no significant effect. In contrast, releasing Ca2+ from the acidic pool markedly reduces secretion. It is suggested that a high Ca2+ concentration in the secretory compartment is needed to sustain optimal exocytosis.

Animals↗
PubMed1998

On the role of voltage-dependent calcium channels in calcium signaling of astrocytes in situ.

Calcium ions play crucial roles in a large variety of cell functions. The recent proposal that changes in the intracellular calcium concentration ([Ca2+]i) in astrocytes underline a reciprocal communication system between neurons and astrocytes encourages the interest in the definition of the various components participating in this novel Ca2+ signaling system. We investigate here whether functional voltage-operated calcium channels (Ca2+ VOCs), which are clearly expressed in cultured astrocytes, participate in the regulation of [Ca2+]i also in astrocytes in situ. Depolarization with 40-60 mM K+ was used to analyze the activity of Ca2+ VOCs in Indo-1-loaded astrocytes in acute slices from the visual cortex and the CA1 hippocampal region of developing rats. We demonstrate here that the depolarization-induced [Ca2+]i increases in astrocytes are solely attributed to the activation of metabotropic receptors by neurotransmitters, such as glutamate, released by synaptic terminals on depolarization. In fact, (1) the K+-induced [Ca2+]i increases in astrocyte [Ca2+]i were potently reduced by alpha-methyl-4-carboxyphenylglycine, a metabotropic glutamate receptor competitive inhibitor; (2) after emptying intracellular Ca2+ stores with cyclopiazonic acid, none of the astrocytes displayed a [Ca2+]i increase on the depolarizing stimulus; and (3) after inhibiting neurotransmitter secretion in neurons by incubating the slices with tetanus neurotoxin, no [Ca2+]i increase on K+ stimulation was observed in astrocytes. Finally, patch-clamp whole-cell recordings from hippocampal astrocytes in acute brain slices failed to reveal any voltage-dependent calcium currents. On the basis of these results, the various roles proposed for astrocyte Ca2+ VOCs in the CNS should be reconsidered.

Animals↗
PubMed1998

Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses.

The spatial relation between mitochondria and endoplasmic reticulum (ER) in living HeLa cells was analyzed at high resolution in three dimensions with two differently colored, specifically targeted green fluorescent proteins. Numerous close contacts were observed between these organelles, and mitochondria in situ formed a largely interconnected, dynamic network. A Ca2+-sensitive photoprotein targeted to the outer face of the inner mitochondrial membrane showed that, upon opening of the inositol 1,4,5-triphosphate (IP3)-gated channels of the ER, the mitochondrial surface was exposed to a higher concentration of Ca2+ than was the bulk cytosol. These results emphasize the importance of cell architecture and the distribution of organelles in regulation of Ca2+ signaling.

Adenosine Triphosphate↗
PubMed1998

Cooperation between the RING + B1-B2 and coiled-coil domains of PML is necessary for its effects on cell survival.

PML/RARalpha is the abnormal protein product of the Acute Promyelocytic Leukemia-specific 15;17 translocation. Both the PML and RARalpha components are required for the PML/RARalpha biological activities, namely its capacity to block differentiation and to increase survival of haematopoietic precursors. The physiological role of PML and its contribution to the function of the fusion protein are unknown. PML localizes to the cytoplasm and within specific nuclear bodies (NBs). In vitro, overexpression of PML correlates with suppression of cell transformation. The PML aminoterminal portion retained within the PML/RARalpha protein contains the RING finger, two newly defined cystein/histidine-rich motifs called B-boxes (B1 and B2) and a coiled-coil region. We report here that PML has a growth suppressive activity in all the cell lines tested, regardless of their transformed phenotype, and that the cellular basis for the PML growth suppression is induction of apoptotic cell death. Analysis of various nuclear and cytoplasmic PML isoforms showed that the PML growth suppressive activity correlates with its nuclear localization. Analysis of the localization and growth suppressive activity demonstrated that: (i) the Ring + B1-B2 and coiled-coil regions are both indispensable and sufficient to target PML to the NBs; (ii) individual deletions of the various PML domains have no effect on its growth suppressor activity; (iii) the Ring + B1-B2 region exerts a partial growth suppressor activity but its fusion with the coiled-coil region is sufficient to recapitulate the suppressive function of wild type PML. These results indicate that PML is involved in cell survival regulation and that the PML component of the fusion protein (Ring + B1-B2 and coiled-coil regions) retains intact biological activity, thereby suggesting that the effects of PML/RARalpha on survival derive from the activation of the incorporated PML sequence.

3T3 Cells↗
PubMed1998

Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process.

Free [Ca2+] in agonist-sensitive internal stores of single intact cells was measured in situ in order to examine the role of [Ca2+] in modulating the store refilling process. BHK-21 fibroblasts were loaded with the low-affinity fluorescent calcium indicator mag-fura-2-AM such that >80% of the dye was trapped in organelles, where it reported [Ca2+] changes solely in an agonist- and thapsigargin-sensitive internal store. The rates of store reloading following stimulation by 100 nM bradykinin were essentially unchanged when cytosolic [Ca2+] was clamped to resting values with BAPTA-AM. In control cells, recharging of stores totally depended on the presence of external Ca2+, but pre-loading the cells with BAPTA-AM permitted efficient refilling in Ca2+-free, EGTA-containing external medium. Our results show: (i) Ca2+ stores normally are recharged by Ca2+ which must first transit the cytoplasm; (ii) an elevation in cytoplasmic [Ca2+] is not required to replenish Ca2+ stores; (iii) the activation of the plasma membrane Ca2+ pump during the Ca2+ spike ordinarily results in complete extrusion of released Ca2+; and (iv) the buffering capacity of the cytoplasm is an essential component of the store refilling process. An interesting finding was that acute treatment of cells with BAPTA-AM activated capacitative Ca2+ entry at the plasma membrane, due to its efficient hydrolysis in the stores, and the ensuing decrease in the endoplasmic reticulum [Ca2+].

Animals↗
PubMed1998

Capacitative Ca2+ entry is closely linked to the filling state of internal Ca2+ stores: a study using simultaneous measurements of ICRAC and intraluminal [Ca2+].

ICRAC (the best characterized Ca2+ current activated by store depletion) was monitored concurrently for the first time with [Ca2+] changes in internal stores. To establish the quantitative and kinetic relationship between these two parameters, we have developed a novel means to clamp [Ca2+] within stores of intact cells at any level. The advantage of this approach, which is based on the membrane-permeant low-affinity Ca2+ chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), is that [Ca2+] within the ER can be lowered and restored to its original level within 10-15 s without modifications of Ca2+ pumps or release channels. Using these new tools, we demonstrate here that Ca2+ release-activated Ca2+ current (ICRAC) is activated (a) solely by reduction of free [Ca2+] within the ER and (b) by any measurable decrease in [Ca2+]ER. We also demonstrate that the intrinsic kinetics of inactivation are relatively slow and possibly dependent on soluble factors that are lost during the whole-cell recording.

Animals↗
PubMed1998

Prostaglandins stimulate calcium-dependent glutamate release in astrocytes.

Astrocytes in the brain form an intimately associated network with neurons. They respond to neuronal activity and synaptically released glutamate by raising intracellular calcium concentration ([Ca2+]i), which could represent the start of back-signalling to neurons. Here we show that coactivation of the AMPA/kainate and metabotropic glutamate receptors (mGluRs) on astrocytes stimulates these cells to release glutamate through a Ca2+-dependent process mediated by prostaglandins. Pharmacological inhibition of prostaglandin synthesis prevents glutamate release, whereas application of prostaglandins (in particular PGE2) mimics and occludes the releasing action of GluR agonists. PGE2 promotes Ca2+-dependent glutamate release from cultured astrocytes and also from acute brain slices under conditions that suppress neuronal exocytotic release. When applied to the CA1 hippocampal region, PGE2 induces increases in [Ca2+]i both in astrocytes and in neurons. The [Ca2+]i increase in neurons is mediated by glutamate released from astrocytes, because it is abolished by GluR antagonists. Our results reveal a new pathway of regulated transmitter release from astrocytes and outline the existence of an integrated glutamatergic cross-talk between neurons and astrocytes in situ that may play critical roles in synaptic plasticity and in neurotoxicity.

Animals↗
PubMed1998

New light on mitochondrial calcium.

The possibility of specifically addressing recombinant probes to mitochondria is a novel, powerful way to study these organelles within living cells. We first showed that the Ca(2+)-sensitive photoprotein aequorin, modified by the addition of a mitochondrial targeting sequence, allows to monitor specifically the Ca2+ concentration in the mitochondrial matrix ([Ca2+]m) of living cells. With this tool, we could show that, upon physiological stimulation, mitochondria undergo a major rise in [Ca2+]m, well in the range of the Ca2+ sensitivity of the matrix dehydrogenases, in a wide variety of cell types, ranging from non excitable, e.g., HeLa and CHO, and excitable, e.g., cell lines to primary cultures of various embryological origin, such as myocytes and neurons. This phenomenon, while providing an obvious mechanism for tuning mitochondrial activity to cell needs, appeared at first in striking contrast with the low affinity of mitochondrial Ca2+ uptake mechanisms. Based on indirect evidence, we proposed that the mitochondria might be close to the source of the Ca2+ signal and thus exposed to microdomains of high [Ca2+], hence allowing the rapid accumulation of Ca2+ into the organelle. In order to verify this intriguing possibility, we followed two approaches. In the first, we constructed a novel aequorin chimera, targeted to the mitochondrial intermembrane space (MIMS), i.e., the region sensed by the low-affinity Ca2+ uptake systems of the inner mitochondrial membrane. With this probe, we observed that, upon agonist stimulation, a portion of the MIMS is exposed to saturating Ca2+ concentrations, thus confirming the occurrence of microdomains of high [Ca2+] next to mitochondria. In the second approach, we directly investigated the spatial relationship of the mitochondria and the ER, the source of agonist-releasable Ca2+ in non-excitable cells. For this purpose, we constructed GFP-based probes of organelle structure; namely, by targeting to these organelles GFP mutants with different spectral properties, we could label them simultaneously in living cells. By using an imaging system endowed with high speed and sensitivity, which allows to obtain high-resolution 3D images, we could demonstrate that close contacts (< 80 nm) occur in vivo between mitochondria and the ER.

Aequorin↗
PubMed1998

The endoplasmic reticulum Ca2+ store: a view from the lumen.

The endoplasmic reticulum (ER) and its specialized subcompartments such as the sarcoplasmic reticulum, is the main dynamic Ca2+ storage compartment of the cell. Key cellular functions are regulated, either directly or indirectly, by the free Ca2+ concentration in the ER. This article discusses the properties of Ca2+ storage in the ER and considers the functions that appear to be regulated by the Ca2+ stores within the ER, both in and around the ER and at a distance from it.

Animals↗
PubMed1998

Delayed activation of the store-operated calcium current induced by calreticulin overexpression in RBL-1 cells.

Calreticulin (CRT) is a high-capacity, low-affinity Ca2+-binding protein located in the lumen of the endoplasmic reticulum (ER) of all eukaryotic cells investigated so far. Its high level of conservation among different species suggests that it serves functions fundamental to cell survival. The role originally proposed for CRT, i.e., the main Ca2+ buffer of the ER, has been obscured or even casted by its implication in processes as diverse as gene expression, protein folding, and cell adhesion. In this work we seek the role of CRT in Ca2+ storing and signaling by evaluating its effects on the kinetics and amplitude of the store-operated Ca2+ current (ICRAC). We show that, in the rat basophilic leukemia cell line RBL-1, overexpression of CRT, but not of its mutant lacking the high-capacity Ca2+-binding domain, markedly retards the ICRAC development, however, only when store depletion is slower than the rate of current activation. On the contrary, when store depletion is rapid and complete, overexpression of CRT has no effect. The present results are compatible with a major Ca2+-buffering role of CRT within the ER but exclude a direct, or indirect, role of this protein on the mechanism of ICRAC activation.

Animals↗