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J Meldolesi

Publications and source records attributed to J Meldolesi.

At least 55 records · Page 3Linked to original sources
PubMed1996

Pharmacological and functional properties of voltage-independent Ca2+ channels.

During the last few years, considerable progress has taken place in our knowledge of the molecular and functional properties of the various voltage-independent Ca2+ channels. In addition to the ionotropic receptor-channels (ROCs), that are not discussed in the present review, these channels include the SMOCs, activated via second messengers or other transducing processes directly triggered by receptor activation; and the SOCCs, activated as a consequence of depletion of the rapidly exchanging Ca2+ stores in the cytoplasm. In parallel, a pharmacological approach to the study of these channels has been developed, based primarily on heterogeneous drugs already known for different biological effects, and subsequently recognized as voltage-independent Ca(2+)-channel blockers. From the systematic analysis of the effects of these drugs new information has emerged about SMOCs and SOCCs function. In addition, pharmacological blockade of these channels appears to have beneficial therapeutic effects in pathological conditions such as tumoral cell growth, inflammation and immunity. At the moment the field is rapidly evolving, with major developments expected in the years ahead.

Animals↗
PubMed1995

Survival of Italian biomedical research.

Despite crises in image and funding, Italian biomedical research has reached a turning point. A brighter future is in prospect if the universities successfully reorganize themselves and new competitive institutes are established.

Humans↗
PubMed1995

Nitric oxide action on growth factor-elicited signals. Phosphoinositide hydrolysis and [Ca2+]i responses are negatively modulated via a cGMP-dependent protein kinase I pathway.

The role of nitric oxide (NO) in the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and intracellular Ca2+ release responses induced by epidermal, platelet-derived, and fibroblast growth factors was investigated in three cell lines, a clone of NIH-3T3 fibroblasts overexpressing epidermal growth factor receptors and the tumoral epithelial cells A431 and KB. In all three cell types, pretreatment with NO donors decreased growth factor-induced PIP2 and Ca2+ responses, whereas pretreatment with NO synthase inhibitors increased them. The Ca2(+)-dependent PIP2 hydroysis induced by micromolar concentrations of the Ca2+ ionophore, ionomycin, was also modulated negatively and positively by NO donors and synthase inhibitors, respectively. In contrast, the Ca2+ content of the intracellular stores was unaffected by the various pretreatments employed. NO donors and synthase inhibitors induced an increase and decrease, respectively, of the intracellular cGMP formation in all three cell lines investigated. All of the effects of the NO donors were mimicked by 8-bromo-cGMP administration and abolished by pretreatment with the specific blocker of the cGMP-dependent protein kinase I, KT5823, which by itself mimicked the effects of the synthase inhibitors. Together with previous observations on G protein-coupled receptors, the present results demonstrate that PIP2 hydrolysis and Ca2+ release occur under the feedback control of NO, independently of the phospholipase C (beta, gamma, or delta type) involved and of the mechanism of activation. Such a control, which appears to be effected by the cGMP-dependent protein kinase I acting at the level of the phospholipases C themselves, might ultimately contribute to the inhibitory role of NO on growth previously observed with various cell types.

Alkaloids↗
PubMed1995

Long-term potentiation. The cell biology connection.

The relative importance of pre- and post-synaptic mechanisms in long-term potentiation has been controversial; a cell-biological approach has now provided strong evidence for the involvement of pre-synaptic mechanisms.

Long-Term Potentiation↗
PubMed1995

HIV-1 gp120 glycoprotein induces [Ca2+]i responses not only in type-2 but also type-1 astrocytes and oligodendrocytes of the rat cerebellum.

Cultures of cerebellar cortex cells were exposed to the HIV-1 envelope glycoprotein, gp120, and investigated for cytosolic Ca2+ ion concentration ([Ca2+]i) changes by the fura-2 ratio videoimaging technique while bathed in complete, Na(+)-free or Mg(2+)-free Krebs-Ringer media. At the end of the [Ca2+]i experiments the cells were fixed and immunoidentified through the revelation of markers specific for neurons (microtubule associated protein-2), type-2 (A2B5) or all (glial fibrillary acidic protein) astrocytes, oligodendrocytes (galactocerebroside) or microglia (F4/80 antibody). In complete medium, rapid biphasic (spike-plateau) responses induced by gp120 (0.1-1 nM) were observed in a subpopulation of type-2 astrocytes. In addition, slow but progressive responses were observed in other type-2 cells and oligodendrocytes, whereas type-1 astrocytes showed small responses, if any, and granule neurons did not respond at all. Use of Na(+)-free medium (a condition that blocked another gp120-induced response, cytosolic alkalinization) resulted in an increase in [Ca2+]i response that was appreciable not only in type-2 but also in most type-1 astrocytes, possibly because of the inhibition of the Na+/Ca2+ exchanger and the ensuing decrease in Ca2+ extrusion. Granule neurons, including those in direct contact with responsive astrocytes, remained unresponsive, even when the experiments were carried out in Mg(2+)-free medium supplemented with glycine, a condition that favors activation of the glutamatergic N-methyl-D-aspartate (NMDA) receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

LU52396, an inhibitor of the store-dependent (capacitative) Ca2+ influx.

The effects of 1-[2-(4-fluorophenyl)cyclohexyl]-2-[4-(3-phenylalkyl)-piperazin -1-yl]- ethanol, LU52396, on a) Ca2+ influx across the plasma membrane and b) Ca2+ mobilization from intracellular rapidly-exchanging Ca2+ stores were investigated in HeLa cells and in isolated microsomal fractions derived from the cerebellum and the skeletal muscle. LU52396 was found to be a potent inhibitor (Ki of about 2 microM) of the Ca2+ influx activated by depletion of intracellular Ca2+ stores, a phenomenon referred to as store-dependent or capacitative Ca2+ influx. Such an effect, which was reversed by cell washing, was mediated neither by a depolarization of the cell, with decrease in the driving force for cation influx, nor by a change of the intracellular pH, and might therefore be due to a direct action of the drug on either the responsible channel in the plasma membrane or, less likely, on its regulatory mechanisms. Additional effects, i.e. inhibition of receptor-mediated Ca2+ influx, of Ca2+ release from intracellular stores via either inositol 1,4,5-trisphosphate or ryanodine receptors, and of Ca2+ reuptake into the stores via sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPases, were also induced by the drug, however at concentrations 20-fold or more than those effective on the store-dependent influx. To our knowledge LU52396 is the first pharmacological tool that is found to be addressed with some preference to the store-dependent Ca2+ influx. It promises, therefore, to be useful for the characterization of the process, the identification of the responsible channel and, possibly, also of the molecular mechanisms through which these channels operate.

Animals↗
PubMed1995

Shear stress-induced [Ca2+]i transients and oscillations in mouse fibroblasts are mediated by endogenously released ATP.

The effects of ATP, U-73122, apyrase, and saline shear stress on [Ca2+]i homeostasis were studied in fura-2 loaded, mouse fibroblast cells (L929), both in suspension and plated on glass. Release of internal Ca2+ was induced by ATP, via a receptor identified pharmacologically as a P2U type. In single cells, low concentrations of ATP evoked [Ca2+]i oscillations. These events were blocked by the putative phospholipase C inhibitor, U-73122 (but not by the inactive analog U-73343) and by the ATP/ADPase, apyrase. In addition, both these agents reduced the [Ca2+]i of unstimulated cells, especially after stirring, and blocked spontaneously occurring [Ca2+]i oscillations, which suggested an already activated state of the ATP receptor, independent from exogenous stimulations. Moreover, it was found that stirring of the cells was correlated with a steady accumulation of inositol phosphates, also blockable by apyrase, and that [Ca2+]i mobilization could be induced by puffs of saline in single cells. The transition to a Ca(2+)-free environment also provoked [Ca2+]i oscillations, most likely via the increase in ATP4- concentration. This evidence suggests that endogenous ATP is released from L fibroblasts in response to fluid shear stress, and this results in an autocrine, tonic up-regulation of the phosphoinositide signaling system and an ensuing alteration in Ca2+ homeostasis. Up until now, such a response to shear stress was believed to be unique to endothelial cells.

Adenosine Triphosphate↗
PubMed1995

Ageing affects cytosolic Ca2+ binding proteins and synaptic markers in the retina but not in cerebral cortex neurons of the rat.

Two cerebral cortex areas (frontal and occipital) and the retina of rats varying in age from 0.4 to 30 months were investigated for the expression levels and distribution of two cytosolic high affinity Ca2+ binding proteins, calbindin-28 and calretinin, and of two presynaptic protein markers. Of these latter proteins, one is integral (synaptophysin) the other peripheral (synapsin I) to the synaptic vesicle membranes. In the cortex areas, no significant changes of the markers were observed, except for a drop of calretinin from 0.4 to 2 months, probably related to a stage of neuronal development. In the retina, calbindin-28 decreased progressively during ageing (-40% at 30 months) while calretinin remained unchanged. Concomitantly, the two synaptic vesicle proteins dropped, synaptophysin > 50% and synapsin I > 85%. The role of these changes in sustaining the functional alterations previously described in the retina of aged animals remains to be investigated.

Aging↗
PubMed1995

Differential localization and functional role of calsequestrin in growing and differentiated myoblasts.

Calsequestrin (CSQ) is the low affinity, high capacity Ca(2+)-binding protein concentrated within specialized areas of the muscle fiber sarcoplasmic reticulum (a part of the ER) where it is believed to buffer large amounts of Ca2+. Upon activation of intracellular channels this Ca2+ pool is released, giving rise to the [Ca2+]i increases that sustain contraction. In order to investigate the ER retention and the functional role of the protein, L6 rat myoblasts were infected with a viral vector with or without the cDNA of chicken CSQ, and stable clones were investigated before and after differentiation to myotubes. In the undifferentiated L6 cells, expression of considerable amounts of heterologous CSQ occurred with no major changes of other ER components. Ca2+ release from the ER, induced by the peptide hormone vasopressin, remained however unchanged, and the same occurred when other treatments were given in sequence to deplete the ER and other intracellular stores: with the Ca2+ pump blocker, thapsigargin; and with the Ca2+ ionophore, ionomycin, followed by the Na+/H+ ionophore, monensin. The lack of effect of CSQ expression on the vasopressin-induced [Ca2+]i responses was explained by immunocytochemistry showing the heterologous protein to be localized not in the ER but in large vacuoles of acidic content, positive also for the lysosomal enzyme, cathepsin D, corresponding to a lysosomal subpopulation. After differentiation, all L6 cells expressed small amounts of homologous CSQ. In the infected cells the heterologous protein progressively decreased, yet the [Ca2+]i responses to vasopressin were now larger with respect to both control and undifferentiated cells. This change correlated with the drop of the vacuoles and with the accumulation of CSQ within the ER lumen, where a clustered distribution was observed as recently shown in developing muscle fibers. These results provide direct evidence for the contribution of CSQ, when appropriately retained, to the Ca2+ capacity of the rapidly exchanging, ER-located Ca2+ stores; and for the existence of specific mechanism(s) (that in L6 cells develop in the course of differentiation) for the ER retention of the protein. In the growing L6 myoblasts the Ca(2+)-binding protein appears in contrast to travel along the exocytic pathway, down to post-Golgi, lysosome-related vacuoles which, based on the lack of [Ca2+]i response to ionomycin-monensin, appear to be incompetent for Ca2+ accumulation.

Animals↗
PubMed1995

Ca2+ waves in PC12 neurites: a bidirectional, receptor-oriented form of Ca2+ signaling.

Spatial and temporal aspects of Ca2+ signaling were investigated in PC12 cells differentiated with nerve growth factor, the well known nerve cell model. Activation of receptors coupled to polyphosphoinositide hydrolysis gave rise in a high proportion of the cells to Ca2+ waves propagating non decrementally and at constant speed (2-4 microns/s at 18 degrees C and approximately 10-fold faster at 37 degrees C) along the neurites. These waves relied entirely on the release of Ca2+ from intracellular stores since they could be generated even when the cells were incubated in Ca(2+)-free medium. In contrast, when the cells were depolarized with high K+ in Ca(2+)-containing medium, increases of cytosolic Ca2+ occurred in the neurites but failed to evolve into waves. Depending on the receptor agonist employed (bradykinin and carbachol versus ATP) the orientation of the waves could be opposite, from the neurite tip to the cell body or vice versa, suggesting different and specific distribution of the responsible surface receptors. Cytosolic Ca2+ imaging results, together with studies of inositol 1,4,5-trisphosphate generation in intact cells and inositol 1,4,5-trisphosphate-induced Ca2+ release from microsomes, revealed the sustaining process of the waves to be discharge of Ca2+ from the inositol 1,4,5-trisphosphate- (and not the ryanodine-) sensitive stores distributed along the neurites. The activation of the cognate receptor appears to result from the coordinate action of the second messenger and Ca2+. Because of their properties and orientation, the waves could participate in the control of not only conventional cell activities, but also excitability and differential processing of inputs, and thus of electrochemical computation in nerve cells.

Animals↗
PubMed1995

Overexpression of calreticulin increases the Ca2+ capacity of rapidly exchanging Ca2+ stores and reveals aspects of their lumenal microenvironment and function.

A molecularly tagged form of calreticulin (CR), a low affinity-high capacity Ca2+ binding protein that resides in the ER lumen, was transiently transfected into HeLa cells to specifically modify the Ca2+ buffering capacity of the intracellular Ca2+ stores. Fluorescence and confocal microscope immunocytochemistry revealed the tagged protein to be expressed by over 40% of the cells and to overlap in its distribution the endogenous CR yielding a delicate cytoplasmic network, i.e., the typical pattern of ER. In contrast, no signal was observed associated with the plasmalemma (marked by ConA) and within the nucleus. One- and two-dimensional Western blots revealed the transfected to exceed the endogenous CR of approximately 3.5-fold and to maintain its Ca2+ binding ability, whereas the expression of other ER proteins was unchanged. Ca2+ homeostasis in the transfected cells was investigated by three parallel approaches: (a) 45Ca equilibrium loading of cell populations; (b) [Ca2+]c measurement with fura-2 followed by quantitative immunocytochemistry of single cells and iii) [Ca2+]c measurement of cell population upon cotransfection with the Ca(2+)-sensitive photoprotein, aequorin. The three approaches revealed different aspects of Ca2+ homeostasis, yielding results which were largely complementary. In particular, the following conclusions were established: (a) both endogenous and transfected CR participate in Ca2+ buffering within the IP3-sensitive, rapidly exchanging, Ca2+ stores; the other pools of the cells were in contrast unaffected by CR transfection; (b) the Ca2+ capacity of the stores is not the main limiting factor of individual IP3-mediated Ca2+ release responses triggered by receptor agonists; (c) in control cells, the contribution of CR to Ca2+ buffering within the IP3-sensitive stores accounts for approximately 45% of the total, the rest being probably contributed by the other lumenal (and also membrane) Ca2+ binding proteins; (d) the free [Ca2+] within the lumen of the IP3-sensitive stores, revealed by the degree of Ca2+ binding to the transfected CR protein, amounts to values in (or approaching) the millimolar range; and (e) Ca2+ influx across the plasmalemma activated by depletion of the stores is directly dependent on the lumenal [Ca2+].

Amino Acid Sequence↗
PubMed1995

Calcium homeostasis in mouse fibroblast cells: affected by U-73122, a putative phospholipase C beta blocker, via multiple mechanisms.

1. The inhibitory effects of the putative phospholipase C beta inhibitor, U-73122, on ligand-induced and thapsigargin-induced [Ca2+]i transients were investigated in mouse fibroblast cells (the L line). 2. Ca2+ release from intracellular stores was stimulated either by ATP (and also by UTP or ADP) working through the activation of a P2U receptor, or by lysophosphatidic acid, which elicited a more pronounced response. 3. U-73122 inhibited the Ca2+ mobilization produced by all the agonists in a dose-dependent manner, consistent with a mode of action involving phospholipase C inhibition. 4. In addition, however, U-73122 slowed the kinetics of intracellular Ca2+ release induced by the Ca(2+)-ATPase inhibitor, thapsigargin, and reduced the influx of Ca2+ across the plasma membrane, following stimulation of store-dependent influx by the latter. 5. We conclude that U-73122 has multiple sites of action, all of which can lead to a change in Ca2+ homeostasis. Thus, particular caution is recommended when employing this agent and when interpreting the results obtained.

Animals↗
PubMed1994

Oscillations of cytosolic calcium in rat chromaffin cells: dual modulation in frequency and amplitude.

Rat chromaffin cells in primary culture have a high tendency to exhibit [Ca2+]i oscillations, spontaneously or after moderate stimulation with treatments that induce polyphosphoinositide hydrolysis or mild depolarization. Previous studies in a variety of cell systems had shown that strengthening of the above treatments increases the frequency and not the amplitude of the oscillations. We now demonstrate that in cultured chromaffin cells either one of these oscillation reinforcements can be elicited, depending on whether the Ca2+ influx induced by the applied stimulus is asynchronous with or timed by the [Ca2+]i spikes of the oscillations. In excitable cells the encoding of the oscillation activity appears therefore to operate according to not only digital (modulation in frequency) but also analog (modulation in amplitude) models.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Expression of muscle calsequestrin in epithelial HeLa cells: distribution and functional role.

Calsequestrin is a Ca2+ binding protein expressed by a few cell types (mostly muscle fibers). In these cells the distribution of the protein is within the endoplasmic/sarcoplasmic reticulum, however, not uniformly throughout but at discrete sites of the lumen. In order to investigate the mechanisms of this unusual intracellular distribution together with the possible functions of the protein, we have studied stable transfected clones of epithelial HeLa cells. Treatment of these cells with butyric acid induced a rapid (24 h) and massive (approx. 10-fold) increase of the transfected protein, whereas the other lumenal and membrane proteins of the endoplasmic reticulum were either modified slightly or unchanged. When butyric acid treatment was interrupted the calsequestrin levels returned rapidly (within 24 h) to the pre-treatment level. Such a rapid turnover was due in part to secretion, sustained by both spontaneous and Ca(2+)-dependent release of calsequestrin to the extracellular medium. From the physiological point of view, the transfected cells exhibited only moderate increases of the Ca2+ release responses triggered by either ATP (a ligand addressed to the P2u receptor and working through IP3 generation) or thapsigargin (a blocker of the endoplasmic reticulum Ca2+ ATPase), with no further increase after butyric acid induction of calsequestrin. This result appears to correlate with the occurrence of only small amounts of calsequestrin within the endoplasmic reticulum lumen of all transfected cells. The bulk of calsequestrin, in contrast, was found sequestered within large vacuoles distributed both near the cell surface and, after butyric acid treatment, also in the deep cytoplasm. These vacuoles (possibly a lysosomal subcompartment) appear to contain no Ca2+ as no difference in 45Ca release from transfected cells was observed without or with butyric acid pretreatment when exposed to ionomycin, alone or combined with monensin. We conclude that HeLa cells possess no adequate mechanisms to keep calsequestrin in its physiologically relevant location, the endoplasmic reticulum. In the transfected cell the protein seems therefore to be diverted (possibly by default) to vacuoles destined to be rapidly eliminated by the cell.

Butyrates↗
PubMed1994

Calstorin, a new Ca2+ binding protein of the microsome lumen which is abundant in the rat brain.

A new Ca2+ binding protein (apparent Mr: 54 KDa; pI: 4.37; Stains All positive) which, based on N terminal sequence and immunological criteria, appears different from calsequestrin, calreticulin and the chaperonins, has been identified in the rat brain and recovered primarily in the microsome fraction. Carbonate extraction and trypsin digestion experiments suggest the protein to be located within the microsome lumen. Its expression levels are considerable especially in the cerebellum (65% with respect to calreticulin). 45Ca binding experiments on 2D blots suggest the protein to be of high capacity (higher than calreticulin) and low affinity (apparent Kd: 3 mM). These properties are typical of the proteins participating in the storage of Ca2+ within rapidly exchanging organelles. The tentative name of calstorin (calcium-storage-protein, CST) is therefore proposed.

Amino Acid Sequence↗
PubMed1994

Cytosolic Ca2+ binding proteins during rat brain ageing: loss of calbindin and calretinin in the hippocampus, with no change in the cerebellum.

The expression of two cytosolic, high affinity Ca(2+)-binding proteins, calbindin-28 and calretinin, has been investigated in the cerebellum and hippocampus of young and old rats (from 12 days to 30 months) by combining immunofluorescence and Western blotting. Three markers, calreticulin (the major Ca2+ binding protein within the lumen of the endoplasmic reticulum), MAP-2 (a microtubule binding protein concentrated in neuronal dendrites) and synaptophysin (an integral protein of synaptic vesicles), were studied in parallel. In the cerebellar cortex a rise from 12 to 60 days was observed with calbindin-28 and, especially, calretinin, concentrated in the Purkinje and granule neurons, respectively. The level of expression of the two proteins subsequently remained high and the distribution was unchanged, even in the cerebellum of old animals. A completely different pattern was observed in the hippocampus. Here calretinin, present especially in fibres and interneurons, was abundant in the young, decreased in the adult and reached low values in the old rats. Calbindin-28 accumulated during growth, especially in a subpopulation of CA1 pyramidal cells and in the mossy fibres of CA3, then declined, although irregularly, during ageing. These changes of the two proteins were more marked in the dorsal and central parts than in the ventral part of the hippocampus. In the same brain areas the levels of expression of the three additional markers and their distribution within neurons and synapses were unchanged by ageing.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗