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Biomedical subjects

G Miotto

Publications and source records attributed to G Miotto.

At least 19 recordsLinked to original sources
PubMed1999

Ganglioside GM1 protection from apoptosis of rat heart fibroblasts.

Ceramide is involved as a mediator of apoptosis induced by a variety of signaling molecules or stressful events. Ceramide-derived sphingosine 1-phosphate behaves as an antiapoptotic agent. The ganglioside GM1 is known to protect neuronal cell lines from apoptosis induced by serum/growth factor withdrawal and its effect is mediated in part by the direct activation of the trkA NGF receptor [G. Ferrari et al. (1995) J. Biol. Chem. 270, 3074-3080]. We show that GM1, similarly to sphingosine 1-phosphate, protects rat heart fibroblasts from apoptosis induced by the protein kinase C inhibitor staurosporine and by C2-ceramide. Furthermore, we show that GM1 induces the synthesis of sphingosine 1-phosphate and that this effect is partially prevented by the sphingosine kinase inhibitor N,N-dimethylsphingosine. We conclude that the antiapoptotic action of GM1 is largely to be ascribed to an increased sphingosine kinase activity.

Animals↗
PubMed1999

Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence.

The occurrence and the mode of opening of the mitochondrial permeability transition pore (MTP) were investigated directly in intact cells by monitoring the fluorescence of mitochondrial entrapped calcein. When MH1C1 cells and hepatocytes were loaded with calcein AM, calcein was also present within mitochondria, because (i) its mitochondrial signal was quenched by the addition of tetramethylrhodamine methyl ester and (ii) calcein-loaded mitochondria could be visualized after digitonin permeabilization. Under the latter condition, the addition of Ca2+ induced a prompt and massive release of the accumulated calcein, which was prevented by CsA, indicating that calcein release could, in principle, probe MTP opening in intact cells as well. To study this process, we developed a procedure by which the cytosolic calcein signal was quenched by Co2+. In hepatocytes and MH1C1 cells coloaded with Co2+ and calcein AM, treatment with MTP inducers caused a rapid, though limited, decrease in mitochondrial calcein fluorescence, which was significantly reduced by CsA. We also observed a constant and spontaneous decrease in mitochondrial calcein fluorescence, which was completely prevented by CsA. Thus MTP likely fluctuates rapidly between open and closed states in intact cells.

Animals↗
PubMed1998

Imaging the mitochondrial permeability transition pore in intact cells.

The involvement of mitochondrial permeability transition pore (MTP) in cellular processes is generally investigated by indirect means, such as changes in mitochondrial membrane potential or pharmacological inhibition. However, such effects could not be related univocally to MTP. In addition, source of errors could be represented by the increased retention of membrane potential probes induced by cyclosporin A (CsA) and the interactions between fluorescent probes. We developed a direct technique for monitoring MTP. Cells were co-loaded with calcein-AM and CoCl2, resulting in the quenching of the cytosolic signal without affecting the mitochondrial fluorescence. MTP inducers caused a rapid decrease in mitochondrial calcein fluorescence which, however, was not completely prevented by CsA. Besides the large and rapid efflux of calcein induced by MTP agonists, we also observed a constant and spontaneous decrease of mitochondrial calcein which was completely prevented by CsA. Thus, MTP likely fluctuates between open and closed states in intact cells.

Animals↗
PubMed1996

Determination of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine in human plasma by high-performance liquid chromatography after pre-column derivatization with 1-aminoanthracene.

A new sensitive high-performance liquid chromatographic procedure for the determination of L-carnitine (LC), acetyl-L-carnitine (ALC) and propionyl-L-carnitine (PLC) in human plasma has been developed. Precolumn derivatization with 1-aminoanthracene (1AA), performed in phosphate buffer in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as catalyst, is involved. The fluorescent derivatives were isocratically separated on a reversed-phase column (C18). The eluate was monitored with a fluorimetric detector set at 248 nm (excitation wavelength) and 418 nm (emission wavelength). Because of the presence of endogenous carnitines, the validation was performed using dialyzed plasma. The identity of the derivatized compounds was assessed by mass spectrometry and the purity of the chromatographic peaks was confirmed by HPLC-tandem mass spectrometry. The limits of quantitation were 5 nmol/ml for LC, 1 nmol/ml for ALC and 0.25 nmol/ml for PLC. The recovery of the extraction procedure was in the range 82.6%-95.4% for all 3 compounds. Good linearity (R approximately 0.99) was observed within the calibration ranges studied: 5-160 nmol/ml for LC, 1-32 nmol/ml for ALC and 0.25-8 nmol/ml for PLC. Precision was in the range 0.3-16.8% and accuracy was always lower than 10.6%.

Acetylcarnitine↗
PubMed1996

Mitochondrial alterations induced by aspirin in rat hepatocytes expressing mitochondrially targeted green fluorescent protein (mtGFP).

Mitochondria in primary living hepatocytes were visualized in cells transfected with a chimeric plasmid encoding for the green fluorescent protein (GFP) of Aequorea victoria engineered to be specifically targeted to mitochondria, as described recently (Rizutto et al. (1995) Curr. Biol. 5, 635-642). The identification of the fluorescent organelles as authentic mitochondria was confirmed by double labeling with rhodamine 123. Acetylsalicylate treatment of hepatocytes induced in mitochondria typical morphological alterations closely analogous to the swelling promoted by acetylsalicylate in isolated mitochondria. Cyclosporin A, which in isolated mitochondria prevents the changes induced by acetylsalicylate, had no protective action but induced per se specific alterations in the morphology of mitochondria. Moreover, exposure of hepatocytes to cyclosporin A followed by acetylsalicylate caused the same mitochondrial changes induced by each of the two compounds separately. The structural alterations caused by acetylsalicylate were constantly associated with a decrease in mitochondrial urea synthesis and cell viability.

Animals↗
PubMed1996

Autophagy.

Explore the source record for details and available documents.

Animals↗
PubMed1994

Inhibition of macroautophagy and proteolysis in the isolated rat hepatocyte by a nontransportable derivative of the multiple antigen peptide Leu8-Lys4-Lys2-Lys-beta Ala.

The multiple antigen peptide derivative, Leu8-Lys4-Lys2-Lys-beta Ala (Leu8-MAP), was synthesized by attaching the carboxyl of leucine to the NH2 termini of a branched lysine core, termed MAP, creating a molecule of about 1900 Da with 8 leucine residues. On a molar basis (independent of the number of leucine substitutions), Leu8-MAP was as effective as leucine in suppressing macroautophagy and proteolysis; moreover, it exhibited the same apparent Km (about 0.1 mM). The effect was specific for leucine since Ile8-MAP was inactive. It is of interest, though, that Leu8-MAP did not elicit the multiphasic response typical of leucine but instead evoked the single site inhibition normally seen with leucine plus the co-regulator alanine. Some free leucine was produced from Leu8-MAP during hepatocyte incubations, but the amounts were insufficient to account for the inhibition. Although this degradation created species of Leu-MAP that had lost 1-3 residues of leucine, their inhibitory effectiveness was not diminished. Because the extracellular/intracellular distribution ratio of [3H]-Leu8-MAP was 100:1 or greater, the direct transport of Leu8-MAP across the plasma membrane into the cytosolic compartment can be excluded. Hence, cytosolic concentrations of Leu8-MAP will be at least 100-fold smaller than those of leucine under conditions of comparable proteolytic inhibition. For these and related reasons, effects attributable to the recognition of Leu8-MAP cannot be explained by signals generated within the cytosol. They could, however, be mediated from site(s) on the plasma membrane or within associated vesicles.

Animals↗
PubMed1994

Leucine-specific binding of photoreactive Leu7-MAP to a high molecular weight protein on the plasma membrane of the isolated rat hepatocyte.

Leu8-MAP (Multiple Antigen Peptide) is an effective inhibitor of macroautophagy and proteolysis in the isolated rat hepatocyte, having an apparent Km (0.1 mM) equaling leucine. Since it is not transported into the cytosolic compartment, it very likely mediates its effect through a plasma membrane site. In an attempt to identify the site we photoreacted intact cells with a biologically active, iodinatable azide derivative of Leu7-MAP. A approximately 340,000 M(r) protein whose labeling was protected 83% with 20 mM Leu was found in plasma membrane fractions when electrophoresed in 7.5-20% gradient gels under nonreducing conditions; addition of 20 mM dithiothreitol generated smaller m.w. products, possibly subunits, of consistent size. No specific labeling was observed with photoreactive derivatives of Ile7-MAP or Val7-MAP.

Amino Acid Sequence↗
PubMed1994

De novo autophagic vacuole formation in hepatocytes permeabilized by Staphylococcus aureus alpha-toxin. Inhibition by nonhydrolyzable GTP analogs.

The role of GTP-binding proteins in autophagic vacuole formation was investigated in isolated rat hepatocytes permeabilized by alpha-toxin from Staphylococcus aureus, an agent which creates stable plasma membrane channels allowing exchange of small (< or = 1000 Da) molecules. Vacuole formation was monitored from the uptake of 125I-tyramine-cellobiitol (125ITC) into osmotically sensitive vacuoles isolated on colloidal silica density gradients. Separation was based on an established observation that autophagic vacuoles are retained in a heavy midgradient band when samples are layered, but are selectively shifted to dense fractions when they are previously dispersed in the gradient material. The vacuolar uptake of 125ITC was concentration-dependent and required exogenous ATP: 94% was directly mediated by sequestration; 6% was acquired by fluid-phase endocytosis as monitored by [carboxyl-14C]dextran-carboxyl. Although the amino acid control of proteolysis was lost, addition of the nonhydrolyzable GTP analog GTP gamma S (as well as GMP-PNP) decreased fractional rates of direct vacuolar 125ITC uptake and long-lived proteolysis by similar amounts (1.02-1.03% h-1), substantiating the notion that the effects were the direct result of autophagic inhibition. These and associated findings, supported by quantitative electron microscopy, indicate the presence of ongoing macro- and microautophagy in alpha-toxin-permeabilized cells and suggest that one or more GTP-binding proteins is required in macroautophagic vacuole formation.

Animals↗
PubMed1994

Multiphasic control of proteolysis by leucine and alanine in the isolated rat hepatocyte.

Autophagically mediated proteolysis in the perfused rat liver is under complex multiphasic control by a small group of amino acids dominated by leucine. Because there have been no prior reports of such regulation in the isolated hepatocyte, our goal was to determine whether it is a manifestation of interactions between diverse cells in the intact liver or, alternatively, the expression of a unique control mechanism within a single population of cells. Hepatocytes were isolated from livers of ad libitum-fed rats and incubated with cycloheximide at low density (approximately 10(6) cells/ml) for the determination of valine release. As in perfusion experiments with synchronously fed rats, proteolytic responses to leucine in cells from fed rats were mediated through two inhibitory mechanisms that alternated randomly on a day-to-day basis. The first (L) represented a typical multiphasic dose-response with low- and high-concentration inhibition separated by a sharp zonal loss of inhibition that could be abolished by alanine. The second (H) mediated inhibition only at high concentrations. It disappeared after 24 h of starvation, leaving L as the prevailing mode. The findings indicate that both macroautophagy and the multiphasic mechanism for regulating it coexist in a single population of hepatocytes, making the cells suitable for studies aimed at defining the putative plasma membrane site of leucine recognition.

Alanine↗
PubMed1992

Control of hepatic proteolysis by leucine and isovaleryl-L-carnitine through a common locus. Evidence for a possible mechanism of recognition at the plasma membrane.

Deprivation-induced proteolysis in the perfused rat liver is controlled through the multiphasic action of 7 regulatory amino acids of which L-leucine plays the dominant role. Recently, isovaleryl-L-carnitine (IVC) was shown to mimic the leucine's effects, suggesting that the two molecules share structural features that are recognized at a common site(s). In this study we find that each evokes identical responses consisting of inhibitory effects at 0.08 and 0.8 mM, separated by a sharp zonal loss of inhibition at 0.15 mM. As monitored by density shifts of beta-hexosaminidase in colloidal silica gradients, macroautophagy is suppressed by both. Responses to Leu and IVC at 0.08 and 0.15 mM are stereospecific and require a reactive group at the alpha-carbon (or equivalent) and a high degree of branched chain specificity. In addition, 0.5 mM Ala coregulates with IVC and Leu by decreasing the zonal loss at 0.15 mM. The fact that the multiphasic responses can be duplicated with equimolar mixtures of Leu + IVC indicates that both react at the same site(s). IVC is readily taken up by a saturable process, but owing to its rapid hydrolysis in the cell, the ratio of internal to external IVC remains low over a 4-fold concentration range. These findings, together with a kinetic analysis of concerted responses to regulatory amino acids, suggest that the recognition sites are at a position in the cell, possibly at the plasma membrane, to react reversibly with plasma amino acids.

Alanine↗
PubMed1992

4-Amino-6-methylhept-2-enoic acid: a leucine analogue and potential probe for localizing sites of proteolytic control in the hepatocyte.

A recent analysis of leucine analogues has suggested that the carboxyl group is not required for mediating low concentration proteolytic inhibition in liver cells. In designing a probe to localize the regulatory site(s), we tested this hypothesis by synthesizing an analogue with a 2-carbon insert between the carboxyl and alpha-carbon. The Wittig product, a trans olefin, was fully active. Surprisingly, low concentration activity was lost when the double bond was eliminated by hydrogenation although some inhibitory effectiveness at high concentrations was evident. Since the double bond extends the carboxyl group away from the alpha-carbon, the results support the above hypothesis as well as the feasibility of adding functional groups to the carboxyl end of leucine.

Animals↗
PubMed1991

Amino acid control of proteolysis in perfused livers of synchronously fed rats. Mechanism and specificity of alanine co-regulation.

The primary control of autophagically mediated proteolysis in perfused rat liver is carried out via two alternate mechanisms in response to specific regulatory amino acids. One (L) elicits direct inhibition at low and high plasma levels, but requires a co-regulatory amino acid to express inhibition at normal concentrations. The second (H) is ineffective at normal levels and below, but active at higher concentrations. Because regulation is subject to unpredictable variability with ad libitum feeding, we have utilized rats synchronously fed 4 h day-1 to stabilize responses. Proteolytic control is seen to evolve in stages: H appears 12 h after the start of feeding; by 18 h L emerges, alternating with H in a statistically predictable way; with omission of the 24-h feeding, H disappears and L remains constant through 42 h. In both 18- and 42-h rats, alanine, glutamate, and aspartate exhibit similar inhibitory activity when added singly to the regulatory group at normal plasma concentrations. However, since alanine, but not glutamate or aspartate, evokes proteolytic acceleration when it is deleted from a full plasma mixture, alanine appears to be the sole co-regulator. Alanine yields co-regulatory effects with normal plasma leucine (0.2 mM) in 18- and 42-h animals and interacts synergistically with 0.8 mM leucine in 42-h but not in 18-h rats where leucine alone inhibits strongly. Because the inactivation of alanine amino-transferase by aminooxyacetate (determined from the conversion of [14C]alanine to glucose) does not alter the co-regulatory and synergistic effects of alanine, regulation by alanine must be mediated from a site of recognition before transamination.

Alanine↗
PubMed1989

Ca2+-mediated action of long-chain acyl-CoA on liver mitochondria energy-linked processes.

The decrease of steady-state transmembrane potential (delta psi) and loss of accumulated Ca2+ are magnified if palmitoyl-CoA is added to rat liver mitochondria exposed to Ca2+ and phosphate. The extent of this damage increases with increasing concentration of long-chain acyl-CoA. Addition of L-carnitine with or without the addition of palmitoyl-CoA considerably delays the deenergization. In the latter case, there is a substantial decrease in the assayed endogenous long-chain acyl-CoA content. This protective action of L-carnitine is abolished by L-aminocarnitine, a powerful inhibitor of carnitine palmitoyl transferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21.). The removal of Ca2+ by EGTA, or the inhibition of its uptake by Ruthenium red or Mg2+ further enhances the degree of protection.

Acyl Coenzyme A↗
PubMed1989

Inhibitory action of isovaleryl-L-carnitine on proteolysis in perfused rat liver.

Isovaleryl-l-carnitine inhibits the proteolysis induced by amino acid deprivation in the perfused rat liver to an extent equivalent, or, below 0.4 mM, even greater than that previously found for 1-leucine (Ref. 1). Also the typical concentration-response curve previously found for leucine (Ref. 1) is mimicked by isovaleryl-l-carnitine. The maximum inhibition (approximately 50% of the control) occurred for both l-leucine and isovaleryl-l-carnitine above 0.8 mM. Only at these high concentrations also 1-carnitine and isobutyryl-l-carnitine exhibit a significant, albeit lower, degree of inhibition. The possible mechanism of this proteolysis inhibition is discussed.

Animals↗
PubMed1988

On the mechanism of spermine transport in liver mitochondria.

Spermine penetrates the mitochondrial matrix at significant rates which increase sharply and non-ohmically with membrane potential. In this respect, spermine uptake is qualitatively similar to that of other cations whose electrophoretic transport has been studied in mitochondria. At 200 mV and 1 mM spermine, the observed rate of spermine uptake was about 7 nmol x mg-1 x min-1, and the rate constant was about 8 times greater than that of tetraethylammonium cation. These rates are remarkably rapid considering that spermine is largely tetravalent at the pH of the experiment. The fluxes of spermine and tetraethylammonium are log-linear with membrane potential. The slope of the tetraethylammonium plot is consistent with leakage of this ion across a sharp Eyring barrier located in the middle of the membrane. The slope of the spermine plot is half that predicted by such a leak pathway, raising the possibility that spermine may cross the inner membrane by means of a channel. Whatever its mechanism of penetration, if comparable rates of uptake obtain in vivo and if spermine is not metabolized within the mitochondrial matrix, then a separate efflux mechanism would appear to be required to prevent unlimited spermine loading.

Animals↗
PubMed1987

Transport and function of L-carnitine and L-propionylcarnitine: relevance to some cardiomyopathies and cardiac ischemia.

Carnitine, an essential cofactor in fatty acid oxidation, plays a central role in myocardial metabolism. Interpretation of the biochemical features of disturbed myocardial function, particularly in ischemia, may be facilitated by understanding carnitine biosynthesis, transport and function. Biosynthesis: In man, deoxycarnitine, the immediate precursor of carnitine, is synthesized in all tissues, whereas the last step, the conversion of deoxycarnitine into carnitine may only take place in liver, kidney and brain (Figs. 1 and 2). Deoxycarnitine formed by organs like muscle or heart is released into the plasma, taken up by liver and kidney, converted into carnitine which is secreted into the bloodstream to be taken up by heart or muscle (Fig. 2). Carnitine transport and cellular function: The myocardial uptake of carnitine against a large concentration gradient (Table 1) occurs in an 1:1 exchange-diffusion process. Under physiological conditions, intracellular deoxycarnitine is exported and extracellular carnitine is imported. According to this model, myocardial carnitine deficiency may be due either to a functional alteration of the sarcolemmal carnitine carrier or to a deficient synthesis of deoxycarnitine. D-carnitine, acetylcarnitine and long-chain acylcarnitine esters are also transported by the carrier at different rates. This might account for the release of endogenous acylcarnitines accumulated in anoxic or ischemic conditions, contributing to the cardioprotective effect of carnitine by reduction in intracellular long-chain acyl-coenzyme A.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗