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Inhibitory action of isovaleryl-L-carnitine on proteolysis in perfused rat liver.
Full text linkIsovaleryl-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
Calpain activation and death of isolated cardiomyocytes exposed to intracellular calcium overload
No full textThe causal relationships linking cell death with calpain activation are still elusive. To this aim calpain activation was investigated in Hl-5 cardiomyocyte added with 1 μM A23187, a calcium ionophore. To obtain a persistent intracellular Ca2+ overload, as detected by fluo-3 fluorescence, NaCl of the incubation buffer was replaced with KCl and 1 mM vanadate was added to inhibit Ca2+ ATPases. Fluo3 fluorescence dropped immediately to background levels when EGTA was added. In this way it was possible to expose HL-5 cells to various durations of calcium overload. Calpain activation was investigated by means of immunoblot analyses of desmin degradation and fluorescence increases reflecting the hydrolysis of the synthetic peptide Suc-LLVY-AMC, a calpain substrate. Cell death was assessed as lactic dehydrogenase (LDH) release. Calpain activation became detectable after 20 min of calcium overload and was followed by the increase in LDH release, which approached to plateau after 40 min. The addition of EGTA after 30 min was no longer able to block the progression in cell death and calpain activation. More importantly, calpain inhibition by 10 μM PD150606 or 100 μM calpeptin reduced significantly LDH release although at a lesser extent than calpain mediated proteolysis.
In conclusion, the present findings suggest that (i) calpain activation precedes the onset of cell death; (ii) intracellular calcium overload hampers cell viability in a process that eventually becomes independent of Ca2+; (iii) calpain activation is causally related to cell death, although the severity of the present protocol limits the protective efficacy of calpain inhibition
Inhibition of macroautophagy and proteolysis in the isolated rat hepatocyte by a nontransportable derivative of the multiple antigen peptide Leu8-Lys4-Lys2-Lys-βAla
Full text linkThe multiple antigen peptide derivative, Leu8-Lys4-Lys2-Lys-βAla (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 K(m) (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
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
Full text linkDeprivation-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 β-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 α- 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
Mechanism of autophagy in permeabilized hepatocytes: evidence for regulation by GTP binding proteins.
No full textDE-NOVO AUTOPHAGIC VACUOLE FORMATION IN HEPATOCYTES PERMEABILIZED BY STAPHYLOCOCCUS-AUREUS ALPHA-TOXIN - INHIBITION BY NONHYDROLYZABLE GTP ANALOGS
Full text linkThe role of GTP-binding proteins in autophagic vacuole formation was investigated in isolated rat hepatocytes permeabilized by α-toxin from Staphylococcus aureus, an agent which creates stable plasma membrane channels allowing exchange of small (≤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 GTPγS (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 α-toxin-permeabilized cells and suggest that one or more GTP-binding proteins is required in macroautophagic vacuole formation
Photoreactive- Leu7-MAP-biotin: a strategic tool for the isolation of leucine receptor involved in regulation of autophagy in isolated hepatocytes
No full textAmong amino acids leucine is the most effective inhibitor of liver autophagy induced by nutrient deprivation. To elucidate the site of action of leucine we used a branched leucine-mimetic peptide named Leu8-MAP, it was synthesized by attaching 8 residues of leucine to the 4 alfa + 4 epsilon amino termini of the branched Lys core (termed MAP for Multiple Antigen Peptide), thus creating a compact molecule of about 1900 Da with leucine groups arranged peripherally. When compared on a molar basis Leu8-MAP was as effective in suppressing autophagy as leucine and had the same apparent Km (0.1 mM). Inhibition was specific for leucine since Ile8-MAP evoked no response. Because it is not transported into the cytosolic compartment, it very likely mediates its effect through a plasma membrane site.
In an attempt to isolate the site of interaction we photoreacted intact hepatocytes with a biologically active, biotin conjugated, azide derivative of Leu8-MAP [Leu7-(ASA)MAP- Biotin]. An approximately 103 kD protein whose labeling was protected 90% with 5 mM Leucine was found in plasma membrane enriched fractions when electrophoresed in 4-8% gradient gels, blotted on PVDF membrane and detected by chemiluminescence after labeling with Avidin-HRP. Valine and isoleucine did not compete, thus the photobinding was leucine specific.
We exploited the biotin moiety to set up a protocol for isolation/purification of the photolabeled protein(s). The photolabeled plasma membrane enriched fractions were solubilized with zwitterionic detergents and incubated with a support immobilized monomeric avidin, then the biotinilated proteins were recovered under mild conditions with a yield of about 50% and with a high degree of purification. Furthermore the recovered proteins were compatible with bidimensional electrophoresis protocols.
These findings show that Leu7-(ASA)MAP-Biotin is a valuable tool for isolation of the putative plasmalemma leucine receptor
Ganglioside GM1 protection from apoptosis of rat heart fibroblasts
No full textCeramide 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
Multiphasic control of proteolysis by leucine and alanine in the isolated rat hepatocyte.
Full text linkAutophagically 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 (approximate to 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
Autophagy.
No full textAutophagy or autophagocytosis are terms given to a membrane-mediated process in eukaryotic cells in which portions of cytoplasm are sequestered within vacuoles and degraded by acid hydrolases that are acquired by fusion with lysosomes. Although vacuoles of this type may be formed under pathologic conditions, autophagy is fundamentally a physiologic process which plays indispensible roles in cell restructuring and in the ongoing turnover of cytoplasmic macromolecules. Sequestration is the one step in this pathway that separates autophagy from other degradative processes in the cell. As a volume uptake mechanism it is relatively nonselective (one exception is discussed insection 3.1.2.) and, accordingly, will sequester most organelles and macromolecules in proportion to their cytoplasmic abundance. Moreover, it permits the simultaneous handling of more than one class of macromolecule. This is illustrated in the perfused rat liver by the striking similarity in the accelerated responses of protein and RNA degradation to amino acid deprivation (see Table I).
Although the appearance of the vacuoles varies widely among cells, it is, nevertheless, highly conserved and found in nearly all lower plants and animals as well as in higher species. In yeast, for example, a vacuole that expresses autophagic function (Takeshige et al., 1992; reviewed by Jones and Murdock, 1994) plays a major role in the supply of endogenous amino acids. A similar role for the vacuole is found in germinating seeds (Nishimura and Beevers, 1979) and in the turnover of intracellular proteins in protoplasts of cultured plant cells (Canut et al., 1985). An interesting variant of autophagy is utilized in cell remodeling where irreversible alterations are involved (Marty, 1978; Paavola, 1978 a,b), and an enzymatically unique type degrades intracellular membranes in the amoeba Tetrahymena pyriformis to provide lipid substrate for gluconeogenesis by the glyoxalate pathway (May et al., 1982). Finally, in the mammalian heptocyte, where both protein turnover and the need for endogenous amino acids are large, autophagy is highly expressed and closely regulated by complex amino acid feedback and hormonal mechanisms (reviewed by Mortimore and Pösö, 1987). Taken together, these findings attest to a fundamental role of autophagy in cellular homeostasis.
In this chapter the authors will discuss the main features of general intracellular protein and RNA degradation and the major classes of autophagy and present a current overview of autophagic regulation and its mechanism, focusing primarily on the mammalian hepatocyte which has been extensively studied as a model for the pathway
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