Biomedical subjects

Tullio Pozzan

Publications and source records attributed to Tullio Pozzan.

At least 37 records · Page 2Linked to original sources

PubMed2004

The presenilin 2 M239I mutation associated with familial Alzheimer's disease reduces Ca2+ release from intracellular stores.

Mutations in presenilin (PS) genes account for the majority of the cases of the familial form of Alzheimer's disease (FAD). PS mutations have been correlated with both over-production of the amyloid-beta-42 (Abeta42) peptide and alterations of cellular Ca(2+) homeostasis. We here show, for the first time, the effect of the recently described PS2 FAD-associated M239I mutation on two major parameters of intracellular Ca(2+) homeostasis: the Ca(2+) storing capacity of the endoplasmic reticulum (ER) and the activation level of capacitative Ca(2+) entry (CCE), the Ca(2+) influx pathway activated by depletion of intracellular stores. Ca(2+) release from intracellular stores was significantly reduced in fibroblasts from FAD patients, compared to that found in cells from healthy individuals or patients affected by sporadic forms of Alzheimer's Disease (AD). No significant difference was however found in CCE between FAD and control fibroblasts. Similar results were obtained in two cell lines (HEK293 and HeLa) stably or transiently expressing the PS2 M239I mutation.

Aged↗

PubMed2003

Mitochondrial pH monitored by a new engineered green fluorescent protein mutant.

We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pK(a) (pK(a)') around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca(2+) ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca(2+) accumulation by this organelle. Finally, we demonstrate that Ca(2+) mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.

Chromatography, Affinity↗

PubMed2003

Calcium and apoptosis: facts and hypotheses.

Although longstanding experimental evidence has associated alterations of calcium homeostasis to cell death, only in the past few years the role of calcium in the signaling of apoptosis has been extensively investigated. In this review, we will summarize the current knowledge, focusing on (i) the effect of the proteins of the Bcl-2 family on ER Ca2+ levels, (ii) the action of the proteolytic enzymes of apoptosis on the Ca2+ signaling machinery, (iii) the ensuing alterations on the signaling patterns of extracellular stimuli, and (iv) the intracellular targets of 'apoptotic' Ca2+ signals, with special emphasis on the mitochondria and cytosolic Ca2+-dependent enzymes.

Animals↗

PubMed2003

Regulation of endoplasmic reticulum Ca2+ dynamics by proapoptotic BCL-2 family members.

Uncontrolled cytosolic Ca(2+) overload is a common cause of cell death in several pathological conditions. Recent evidences reveal a more regulated role for intracellular Ca(2+) stores in controlling cell death. Proteins of the BCL-2 family include anti- and proapoptotic members that control the mitochondrial amplification loop of apoptosis. The antiapoptotic protein BCL-2 prevents this mitochondrial loop, while the "multidomain" proapoptotic proteins BAX and BAK are crucial to initiate it. BCL-2, BAX and BAK localize also to the endoplasmic reticulum (ER), the main intracellular Ca(2+) store. Overexpression of BCL-2 reduces resting ER Ca(2+) and death in response to apoptotic stimuli that mobilize Ca(2+). Our recent data indicate that multidomain proapoptotics also influence Ca(2+) metabolism. Cells deficient for Bax, Bak (DKO) display lowered steady state ER Ca(2+) concentrations ([Ca(2+)](er)) and secondarily decreased mitochondrial Ca(2+) uptake. Genetic and pharmacologic correction of [Ca(2+)](er) indicates that it controls death in response to Ca(2+)-dependent, mitochondria utilizing signals such as oxidative stress and lipid mediators; and that it participates in the regulation of the apoptotic response to most intrinsic stimuli, such as staurosporine. Thus, BAX and BAK control apoptosis not only at the mitochondria, but also at the ER, an obligate checkpoint for Ca(2+)-dependent apoptotic stimuli.

Animals↗

PubMed2003

Glutamate-mediated cytosolic calcium oscillations regulate a pulsatile prostaglandin release from cultured rat astrocytes.

The synaptic release of glutamate evokes in astrocytes periodic increases in [Ca2+]i, due to the activation of metabotropic glutamate receptors (mGluRs). The frequency of these [Ca2+]i oscillations is controlled by the level of neuronal activity, indicating that they represent a specific, frequency-coded signalling system of neuron-to-astrocyte communication. We recently found that neuronal activity-dependent [Ca2+]i oscillations in astrocytes are the main signal that regulates the coupling between neuronal activity and blood flow, the so-called functional hyperaemia. Prostaglandins play a major role in this fundamental phenomenon in brain function, but little is known about a possible link between [Ca2+]i oscillations and prostaglandin release from astrocytes. To investigate whether [Ca2+]i oscillations regulate the release of vasoactive prostaglandins, such as the potent vasodilator prostaglandin E2 (PGE2), from astrocytes, we plated wild-type human embryonic kidney (HEK)293 cells, which respond constitutively to PGE2 with [Ca2+]i elevations, onto cultured astrocytes, and used them as biosensors of prostaglandin release. After loading the astrocyte-HEK cell co-cultures with the calcium indicator Indo-1, confocal microscopy revealed that mGluR-mediated [Ca2+]i oscillations triggered spatially and temporally coordinated [Ca2+]i increases in the sensor cells. This response was absent in a clone of HEK cells that are unresponsive to PGE2, and recovered after transfection with the InsP3-linked prostanoid receptor EP1. We conclude that [Ca2+]i oscillations in astrocytes regulate prostaglandin releases that retain the oscillatory behaviour of the [Ca2+]i changes. This finely tuned release of PGE2 from astrocytes provides a coherent mechanistic background for the role of these glial cells in functional hyperaemia.

Animals↗

PubMed2003

Stable interactions between mitochondria and endoplasmic reticulum allow rapid accumulation of calcium in a subpopulation of mitochondria.

To better understand the functional role of the mitochondrial network in shaping the Ca2+ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca2+ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca(2+)-sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca2+ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca2+ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca2+ release channels (and thus responsible for the fast uptake of Ca2+ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum.

Aequorin↗

PubMed2003

Paradoxical Ca2+ rises induced by low external Ca2+ in rat hippocampal neurones.

Confocal Ca2+ imaging of rat hippocampal slices shows a paradoxical effect of acute reductions of the [Ca2+]o. Upon slice perfusion with low-Ca2+ media, a prompt intracellular Ca2+ rise selectively occurs in neurones. This response is observed only in slices challenged with agonists of group I metabotropic glutamate or M1 muscarinic receptors. In contrast, the intracellular Ca2+ level of non-stimulated neurones is insensitive to reductions of [Ca2+]o. The phenomenon is observed in 20-25 % of cultured cortical neurones. Evidence is provided demonstrating that: (1) this paradoxical response is not due to a non-specific decrease in divalent cation concentration but it is selectively activated by a reduction in [Ca2+]o, being maximal with [Ca2+]o between 0.25 and 0.5 mM; (2) upon maximal stimulation, 70-90 % of CA1-CA3 pyramidal neurones sense a reduction in [Ca2+]o; a weaker response is observed in neurones from the neocortex, whereas neurones from the dentate gyrus and granule cells from the cerebellum fail to respond; (3) conditions that elicit paradoxical Ca2+ responses cause depolarisation and increase the firing rate of hippocampal neurones; (4) paradoxical Ca2+ rises depend, primarily, on Ca2+ influx through L-type voltage-operated Ca2+ channels and to a lesser extent on release from intracellular Ca2+ stores. Inhibition of phospholipase C or protein kinase C failed to suppress the neuronal response, whereas a selective inhibitor of the Src-family of tyrosine kinases abolishes the paradoxical neuronal Ca2+ rise. A model is presented to explain how this response is elicited by contemporaneous reduction of the [Ca2+]o and metabotropic receptor stimulation; implications for the pathophysiology of the CNS are also discussed.

Animals↗

PubMed2003

Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem.

Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

Animals↗

PubMed2003

BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis.

BAX and BAK are "multidomain" proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca2+]er) that results in decreased uptake of Ca2+ by mitochondria after Ca2+ release from the ER. Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca2+ adenosine triphosphatase) corrected [Ca2+]er and mitochondrial Ca2+ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca2+ from intracellular stores (such as arachidonic acid, C2-ceramide, and oxidative stress). In contrast, targeting of BAX to mitochondria selectively restored apoptosis to "BH3-only" signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca2+ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signals.

Animals↗

PubMed2003

CAMP and Ca2+ interplay: a matter of oscillation patterns.

Ca(2+) and cAMP signalling pathways are tightly interconnected and the cellular effects mediated by the two second messengers depend strictly on reciprocal modulation. The signalling network that derives from such interplay holds the potential for expanding the information content of the signal enormously and, thus, can contribute substantially to the specificity and diversity of the response. Recent work points to interdependent Ca(2+) and cAMP oscillation patterns as a new paradigm for signal transduction.

Animals↗

PubMed2003

When calcium goes wrong: genetic alterations of a ubiquitous signaling route.

In all eukaryotic cells, the cytosolic concentration of calcium ions ([Ca2+]c) is tightly controlled by complex interactions among transporters, pumps, channels and binding proteins. Finely tuned changes in [Ca2+]c modulate a variety of intracellular functions, and disruption of Ca2+ handling leads to cell death. Here we review the human genetic diseases associated with perturbations in the Ca2+ signaling machinery. Despite the importance of Ca2+ in physiology and pathology, the number of known genetic diseases that can be attributed to defects in proteins directly involved in Ca2+ homeostasis is limited to few examples, which will be discussed. This paucity in contrast with the wide molecular repertoire may depend on the extreme severity of the phenotype (leading to death in utero) or, conversely, on functional compensation due to redundancy. In the latter case, it stands to reason that other genetic defects in calcium signaling have yet to be identified owing to their subtle phenotype.

Calcium Channels↗

PubMed2003

Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation.

The cellular mechanisms underlying functional hyperemia--the coupling of neuronal activation to cerebral blood vessel responses--are not yet known. Here we show in rat cortical slices that the dilation of arterioles triggered by neuronal activity is dependent on glutamate-mediated [Ca(2+)](i) oscillations in astrocytes. Inhibition of these Ca(2+) responses resulted in the impairment of activity-dependent vasodilation, whereas selective activation--by patch pipette--of single astrocytes that were in contact with arterioles triggered vessel relaxation. We also found that a cyclooxygenase product is centrally involved in this astrocyte-mediated control of arterioles. In vivo blockade of glutamate-mediated [Ca(2+)](i) elevations in astrocytes reduced the blood flow increase in the somatosensory cortex during contralateral forepaw stimulation. Taken together, our findings show that neuron-to-astrocyte signaling is a key mechanism in functional hyperemia.

Afferent Pathways↗

PubMed2003

Looking forward to seeing calcium.

From molecules, single cells and tissues to whole organisms, our insights into Ca2+ signalling and the corresponding physiological phenomena are growing exponentially. Here, we describe the improvements that have been made in the development of the probes and instrumentation that are used for Ca2+ imaging and the expanding applications of Ca2+ imaging in basic and applied research.

Animals↗

PubMed2003

The Theodore Bücher lecture. Investigating signal transduction with genetically encoded fluorescent probes.

Ca2+ and cAMP are ubiquitous second messengers in eukaryotes and control numerous physiological responses ranging from fertilization to cell death induction. To distinguish between these different responses, their subtle regulation in time, space and amplitude is needed. Therefore, the characterization of the signalling process requires measurement of second messengers with tools of precise localization, high dynamic range and as little disturbance of cell physiology as possible. Recently, fluorescent proteins of marine jellyfish have given rise to a set of genetically encoded biosensors which fulfil these criteria and which have already led to important new insights into the subcellular handling of Ca2+ and cAMP. The use of these probes in combination with new microscopical methods such as two-photon microscopy now enables researchers to study second messenger signalling in intact tissues. In this review, the genetically encoded measurement probes and their origin are briefly introduced and some recent insights into the spatio-temporal complexity of both Ca2+ and cAMP signalling obtained with these tools are discussed.

Aequorin↗

PubMed2003

Actin reorganization is abnormal and cellular ATP is decreased in Hailey-Hailey keratinocytes.

Actin reorganization and the formation of adherens junctions are necessary for normal cell-to-cell adhesion in keratinocytes. Hailey-Hailey disease (HHD) is blistering skin disease, resulting from mutations in the Ca2+ ATPase ATP2C1, which controls Ca2+ concentrations in the cytoplasm and Golgi of human keratinocytes. Because actin reorganization is among the first responses to raised cytoplasmic Ca2+, we examined Ca2+-induced actin reorganization in normal and HHD keratinocytes. Even though HHD keratinocytes display raised baseline cytoplasmic Ca2+, we found that actin reorganization in response to Ca2+ was impaired in HHD keratinocytes. Defects in actin reorganization were linked to a marked decrease in cellular ATP in HHD keratinocytes, which persists, in vivo, in HHD epidermis. Defective actin reorganization was reproduced in normal keratinocytes in which the intracellular ATP concentration had been lowered pharmacologically. ATP concentrations in undifferentiated keratinocytes markedly declined after extracellular Ca2+ was increased, but then recovered to a new baseline that was approximately 150% of the previous baseline. In contrast, ATP concentrations in HHD keratinocytes did not change in response to increased extracellular Ca2+. This report provides new insights into how the ATP2C1-controlled ATP metabolism mediates Ca2+-induced cell-to-cell adhesion in normal keratinocytes. In addition, these findings implicate inadequate ATP stores as an additional cause in the pathogenesis of HHD and suggest novel therapeutic options.

Acantholysis↗

PubMed2003

Human keratinocyte ATP2C1 localizes to the Golgi and controls Golgi Ca2+ stores.

Hailey-Hailey disease (MIM16960) is a blistering skin disease caused by mutations in the Ca2+ ATPase ATP2C1. We found that the abnormal Ca2+ signaling seen in Hailey-Hailey disease keratinocytes correlates with decreased protein levels of ATP2C1. Human ATP2C1 protein approximated 115 kDa in size. The ATP2C1 is localized to the Golgi apparatus in human keratinocytes, similar to its localization in yeast and Caenorhabditis elegans. To test whether the ATP2C1 controls Golgi Ca2+ stores, we measured intraorganelle Ca2+ concentrations using specifically targeted aequorins. Whereas normal keratinocytes display Golgi Ca2+ levels comparable to other epithelial cells, Hailey-Hailey disease keratinocyte Golgi Ca2+ refill is slower, and the maximum Ca2+ concentration reached is significantly lower. These findings were replicated in vivo, because clinically normal Hailey-Hailey disease epidermis contained lower Ca2+ stores and displayed an abnormal Ca2+ gradient. In this report we localize the ATP2C1, demonstrate its physiologic relevance in mammalian cells, and measure intraorganelle Golgi Ca2+ in keratinocytes.

Adult↗

PubMed2002

Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells.

The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.

Animals↗

PubMed2002

Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes.

The second messenger cyclic adenosine monophosphate (cAMP) is the most important modulator of sympathetic control over cardiac contractility. In cardiac myocytes and many other cell types, however, cAMP transduces the signal generated upon stimulation of various receptors and activates different cellular functions, raising the issue of how specificity can be achieved. In the general field of signal transduction, the view is emerging that specificity is guaranteed by tight localization of signaling events. Here, we show that in neonatal rat cardiac myocytes, beta-adrenergic stimulation generates multiple microdomains with increased concentration of cAMP in correspondence with the region of the transverse tubule/junctional sarcoplasmic reticulum membrane. The restricted pools of cAMP show a range of action as small as approximately 1 micrometer, and free diffusion of the second messenger is limited by the activity of phosphodiesterases. Furthermore, we demonstrate that such gradients of cAMP specifically activate a subset of protein kinase A molecules anchored in proximity to the T tubule.

1-Methyl-3-isobutylxanthine↗