Biomedical subjects

S Pontremoli

Publications and source records attributed to S Pontremoli.

At least 55 records · Page 3Linked to original sources

PubMed1994

Differential regulation of mu- and m-calpain in rat hearts perfused with Ca2+ and cAMP.

Reversible interconversion between calpastatin I and calpastatin II, the phosphorylated form of the inhibitor, has been induced in rat heart perfused with the combination Ca2+/A23187 ionophore or with cAMP. In the presence of the ionophore and increasing concentrations of Ca2+, calpastatin II is converted into calpastatin I; whereas the reverse reaction is induced by the addition of cAMP. Both interconversions leave substantially unmodified the amount of calpastatin I which inhibits mu-calpain with higher efficiency and accordingly keeps the proteinase in an permanent inactive state. On the contrary, expression of m-calpain activity is significantly repressed or alternatively largely augmented as a result of a profound increase or decrease in the level of calpastatin II induced by perfusion with cAMP or with Ca2+/ionophore respectively. Taken together our results demonstrate that bidirectional interconversion can take place in rat heart cells and through this mechanism the activity of m-calpain can be efficiently controlled. Ca2+ ions and cAMP are temptatively proposed as the natural stimuli responsible for the modulation of this overall process. Experimental evidences are provided indicating that the transition to the active form of mu-calpain involves association to the plasma membrane, a process competitively antagonized by calpastatin I; thus suggesting that interaction with one or the other ligand can affect the intracellular ratio between inactive and active mu-calpain forms.

Animals↗

PubMed1994

Protein kinase C isoforms in murine erythroleukemia cells and their involvement in the differentiation process.

In addition to alpha, delta and epsilon-protein kinase C, murine erythroleukemia cells contain zeta-PKC and also a c-PKC isoform, named alpha 1, which shows cross-reactivity with an anti-alpha-PKC antipeptide antibody. In a C44 MEL cell clone, characterized by a high rate of differentiation, both c-PKC forms are expressed at a level higher than that of the N23 MEL cell clone which differentiates at a low rate and contains higher levels of epsilon-PKC and particularly of the delta-PKC isozyme. In the course of MEL cell differentiation, delta-PKC in N23 cells and alpha 1-PKC in C44 cells are rapidly down-regulated and the overall process is almost completed before cell commitment. Of the other three PKC isozymes present in both clones, only alpha-PKC is down-regulated to a significant extent. It is proposed that modulation of the signal delivered by each PKC isozyme is one of the biochemical mechanisms involved in MEL cell differentiation.

Acetamides↗

PubMed1994

Modulation of calpastatin specificity in rat tissues by reversible phosphorylation and dephosphorylation.

Two calpastatins, with Mr 110 KD and named calpastatin I and II, have been isolated from rat heart and kidney and displayed distinct inhibitory efficiency with mu- and m-calpain, respectively, as those isolated from rat skeletal muscle. Whereas the level of calpastatin I always exceeds that of mu-calpain, the level of calpastatin II appears to be more closely correlated to the level of m-calpain. As previously shown for skeletal muscle, the two inhibitor proteins can be interconverted by a phosphorylation-dephosphorylation reaction; the enzyme responsible for phosphate incorporation in calpastatin I is now identified in c-AMP dependent protein kinase A. In rat erythrocytes, containing a single calpain form, the single low Mr calpastatin form does not undergo reversible phosphorylation and is equally efficient in respect to typical mu- and m-calpain. The presence of two interconvertible calpastatin forms provides the cells with a highly sensitive mechanism of regulation of the Ca(2+)-dependent proteolytic system.

Animals↗

PubMed1994

The plasma membrane calcium pump is the preferred calpain substrate within the erythrocyte.

The activation of calpain in normal human erythrocytes incubated in the presence of Ca2+ and the Ca2+ ionophore A23187 led to the decline of the Ca(2+)-dependent ATPase activity of the cells. Preloading of the erythrocyte with an anticalpain antibody prevented the decline. The pump was also inactivated by applied to isolated erythrocyte plasma membranes. The decline of the pump activity corresponded to the degradation of the pump protein and was inversely correlated to the amount of the natural inhibitor of calpain, calpastatin, present in the cells. In erythrocytes containing only 50% of the normal level the degradation started at a concentration of Ca2+ significantly lower than in normal cells. A comparison of the concentrations of Ca2+ required for the degradation of a number of erythrocyte membrane proteins showed that the Ca2+ pump and band 3 were the most sensitive. All other membrane proteins tested were attacked at higher levels of intracellular Ca2+. Thus, the degradation of the Ca2+ pump protein may be a simple and sensitive means to monitor calpain activation in vivo. Furthermore, the results have shown that the calpastatin level correlated directly with the amount of activable calpain and with the concentration of Ca2+ required to trigger the activation process.

Calcium↗

PubMed1993

Differentiation of HL60 promyelocytic cells is promoted by a 'differentiation enhancing factor' produced by erythroleukemia cells.

A differentiation enhancing factor isolated from murine erythroleukemia cells is also a potent enhancer of the differentiation of HL60 human promyelocytic leukemia cells, induced by retinoic acid and by phorbol ester. This stimulating effect is the result of a large increase in the sensitivity of HL60 cells for retinoic acid and for phorbol 12-myristate 13-acetate (20-fold and 40-fold, respectively). Accelerated differentiation induced by the protein factor, and monitored by the appearance of marker enzymes, is accompanied by a large increase in the fluctuation of the levels of protein kinase C (PKC) isozymes in HL60 cells. These results provide further support for the role of this new protein factor in cell differentiation and indicate that other cell types are susceptible to its biological effect.

Cell Differentiation↗

PubMed1993

Characterization of the biological role of murine erythroleukemia cells "differentiation enhancing factor" using antisense oligodeoxynucleotides.

On the basis of the amino acid sequence of isolated tryptic peptides, it has been established that the differentiation enhancing factor, produced and active on murine erythroleukemia (MEL) cells, possesses a unique sequence, with no similarity to that of known proteins. Accordingly, this factor can be defined as a novel biologically active peptide. An antisense oligodeoxynucleotide, deduced from the sequence of a non-decapeptide (produced by tryptic digestion of the factor), decreases the rate and the extent of MEL cell differentiation, induced by hexamethylenebisacetamide. In these cells the amount of the factor is reduced to one third of that constitutively present in untreated cells. Exogenous addition of the factor restores cell inducibility to normal values. Taken together, these results demonstrate the presence in MEL cells of a new factor, structurally and functionally unrelated to any of the known biologically active peptides, and suggest its crucial role in the promotion of an initial signal, in chemically induced erythroid differentiation.

Amino Acid Sequence↗

PubMed1993

Role of delta-PKC on the differentiation process of murine erythroleukemia cells.

In murine erythroleukemia (MEL) cells the length of the latent period before the onset of hexamethylenebisacetamide induced terminal erythroid differentiation is inversely correlated to the intracellular level of delta-PKC. This is supported by the following experimental evidence. V3.17[44] MEL cell line, characterized by a very high rate of differentiation, contains an amount of delta-PKC protein one third lower than that present in the N23 MEL cell line, characterized by a very low rate of differentiation. A similar difference in the amount of delta-PKC mRNA is present in the two cell lines. In N23 cells, following addition of HMBA, the amount of delta-PKC protein and delta-PKC mRNA is down-regulated to one third its original value, which now corresponds to that constitutively present in V3.17[44] cells. Furthermore, in these cells the levels of delta-PKC protein and of its specific mRNA are unaffected by treatment with HMBA. Following introduction of homologous purified delta-PKC both MEL cell variants display a longer latent period before the onset of differentiation from 50 to 75 hours in N23 cell line and from 20 to 40 hours in V3.17[44] cells, respectively. Taken together, these results suggest that a delta-PKC related signal plays a negative role in the early stages of MEL cell differentiation and that the level of the kinase is controlled through a down-regulation process upon exposure to the chemical inducer.

Acetamides↗

PubMed1993

Site-directed activation of calpain is promoted by a membrane-associated natural activator protein.

Human erythrocytes contain a calpain activator protein with a molecular mass of approx. 40 kDa. The activator is present in association with the plasma membrane and promotes expression of calpain activity at a concentration of Ca2+ close to physiological values. The initial step of the activating mechanism involves association of the activator with calpain, followed by autoproteolytic activation of the proteinase in the presence of 1 microM Ca2+, at a rate identical to that induced by 1 mM Ca2+. In a reconstituted system, the activator binds to erythrocyte membranes, but not to phospholipid vesicles, suggesting the participation of an intrinsic membrane protein(s). In its membrane-associated form the activator selectively binds calpain, thus favouring interaction of the proteinase with the inner surface of plasma membranes. These results further confirm the importance of a natural activator protein in promoting intracellular activation of calpain under physiological conditions through a site-directed mechanism, which explains the high specificity of the proteinase for membrane of cytoskeletal proteins.

Binding Sites↗

PubMed1992

Modulation of inhibitory efficiency of rat skeletal muscle calpastatin by phosphorylation.

Rat skeletal muscle calpastatin form is markedly modified in its inhibitory properties by means of a reverse reaction which involves both phosphorylation and dephosphorylation. Dephospho-calpastatin shows greater inhibitory efficiency versus mu-calpain, whereas phospho-calpastatin shows maximal inhibition versus m-calpain. Both forms are present in fresh rat muscle. Phosphorylation has been reproduced "in vitro" using a homologous Ca2+ independent protein kinase and found to result in the incorporation of approximately one mole of 32P per mole of protein. Dephosphorylation was induced by treatment with alkaline phosphatase and 32P release shown found to correlate with modifications of the inhibitory properties. This reversible covalent modification of calpastatin is considered an important advancement in the understanding of how different calpain isoforms can be more efficiently controlled by a single inhibitor isozyme form.

Alkaline Phosphatase↗

PubMed1992

Different susceptibility of red cell membrane proteins to calpain degradation.

The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca2+], degradation of anion transport protein (band 3) and Ca(2+)-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of 10-50 microM Ca2+. In addition, when exposed to progressive and limited increases in [Ca2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca2+] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca(2+)-ATPase appear to be the substrates with highest susceptibility to digestion by calpain.

Animals↗

PubMed1991

Mechanism of action of the calpain activator protein in rat skeletal muscle.

Rat skeletal muscle contains a calpain activator protein characterized by a high specificity for calpain II, the high Ca(2+)-requiring isoform of this class of proteinases. The activator protein increases the rate of intramolecular conversion of the native 80-kDa catalytic subunit of calpain into the autolysed 75-kDa forms with maximal rate at concentrations of calcium approximately 25 times lower than those required by the native proteinase. The activator protein interacts with native calpain II forming a 1:1 complex; interaction does not occur with the fully activated form, produced by autoproteolysis. Even after immobilization to membranes, the activator binds to calpain, which then undergoes sequential activation and release from its bound form. The activator is itself resistant to digestion by calpain II, whereas it increases the rate at which homologous calpastatin is degraded by the proteinase. Taken together, these results are indicative of the existence in rat skeletal muscle of an activating system specific for calpain II which is potentially involved in the regulation of the inhibitory efficiency of calpastatin, through modulation of its intracellular level.

Animals↗

PubMed1991

Respiratory burst in activated neutrophils is directly correlated to the intracellular level of protein kinase C.

The production of superoxide anion in human and rat neutrophils is directly correlated to the level of protein kinase C. Such correlation has been established on a comparative basis by analysis of neutrophils from normal and hypertensive subjects, characterized by an increased amount of protein kinase C, and of neutrophils from normal and genetically hypertensive rats characterized by low amounts of the kinase. Protein kinase C activity in all these different populations of neutrophils is modulated by specific inhibitors in an identical dose-dependent fashion which results in a linearly correlated decrease in O2- production. Taken together, these results provide a direct demonstration that in neutrophils the intracellular level of protein kinase C represents one of the determinants of the rate and extent of O2- production.

Blotting, Western↗

PubMed1991

Differentiation of murine erythroleukemia cells by hexamethylenebisacetamide involves secretion and binding to membranes of a differentiation enhancing factor.

A protein factor previously shown to enhance terminal differentiation of transformed erythroid cells is synthesized by murine erythroleukemia cells and secreted in the early stages of differentiation induced by hexamethylenebisacetamide (HMBA). Secretion also occurs, constitutively, in the absence of inducer, from a murine erythroleukemia cell variant characterized by an accelerated response to HMBA. The protein factor binds to intact cells following addition of HMBA and enhances translocation of protein kinase C to the nuclear fraction. These results strongly support an important role for this natural protein factor in cell differentiation.

Acetamides↗

PubMed1991

Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains.

Two forms of calpastatin, differing in their specificity for the homologous calpain isozymes I and II, have been separated from rat skeletal muscle extracts and purified to homogeneity. Calpastatin I, the first form to elute in chromatography on DE32, is more effective against calpain I, while calpastatin II is more effective as an inhibitor of calpain II. Based on their molecular mass (approximately 105 kDa) both calpastatin forms belong to the high molecular mass class found in muscles of other animal species (Murachi, T., 1989, Biochem. Int. 18, 263-294). For calpain I, which is active with low (mu-M) concentrations of Ca2+, maximum inhibition with either calpastatin form was observed over a wide range of Ca2+ concentrations. With calpain II, which requires high (mM) concentrations of Ca2+ for activity, maximum inhibition required Ca2+ concentrations above 1 mM. Both calpastatin forms were found to be highly sensitive to degradation by calpain II, but almost completely resistant to degradation by calpain I. Degradation of calpastatin by calpain II is competitively inhibited by the addition of a calpain substrate. Isovaleryl carnitine (IVC), an intermediate product of L-leucine catabolism, previously demonstrated to be a potent and specific activator of rat skeletal muscle calpain II (Pontremoli, S., Melloni, E., Viotti, P. L., Michetti, M., Di Lisa, F., and Siliprandi, N., 1990. Biochem. Biophys. Res. Commun. 167, 373-380) greatly enhances the rate of degradation of calpastatins by calpain II. IVC, which decreases the Ca2+ requirement for maximal calpain II activity, also decreases the concentration of Ca2+ required for digestion of the inhibitor. For calpain II, regulation by either calpastatins may occur only in the presence of high [Ca2+].

Animals↗

PubMed1991

The calpastatin defect in hypertension is possibly due to a specific degradation by calpain.

Calpastatin activity, significantly reduced in erythrocytes of patients affected by essential hypertension, is restored to normal values by appropriate therapeutical treatments in a time-dependent fashion and in parallel with the decline in blood pressure. Evidence is also presented indicating that red cell calpastatin is degraded in human and rat red cells by homologous calpain, and that the rate of degradation is approx. 5-times higher in rat erythrocytes. Thus, increased proteolytic degradation catalyzed by calpain could explain both the decrease in the amount of calpastatin activity and the profound difference between the intracellular level of the calpain inhibitor observed in erythrocytes from patients with essential hypertension and the genetically hypertensive rats.

Animals↗

PubMed1990

A vincristine-resistant murine erythroleukemia cell line secretes a differentiation enhancing factor.

A clone of vincristine resistant murine erythroleukemia cells V3.17[44], characterized by high sensitivity to terminal erythroid differentiation induced by hexamethylene bisacetamide, secretes into the extracellular medium a protein factor which partially reduces the latent period before commitment and accelerates the expression of the terminal differentiated phenotype in a slow responding murine erythroleukemia N23 cell variant. This differentiation enhancing factor increases the rate of protein kinase C down-regulation which occurs at slower rate during cell differentiation. The activity of the factor is detected either by coculture of the two cell line variants or by addition of conditioned medium from V3.17[44] cells to a culture of N23 cells in the presence of the inducer. In addition to being secreted by V3.17[44] cells, this factor can also be detected in the cytoplasm of both V3.17[44] and N23 cells, associated with a particulate fraction from which it can be released by sonication.

Animals↗

PubMed1990

Identification of a protein kinase C activating factor from murine erythroleukemia cells: characterization of the activation kinetics.

A protein kinase C (PKC) activating factor (AF) has been identified in the extracellular medium of V3.17 vincristine resistant murine erythroleukemia (MEL) cells clone. The factor is a protein that stimulates the activity of PKC alpha and beta isozymes isolated from MEL cells, rat and mouse brain approximately 2 to 2.5 fold over the Vmax, respectively. AF promotes an identical activation in the presence of all the effectors but also when the amount of Ca2+ is reduced to microM concentration and in the absence of diacylglycerol (DAG). The factor shows a greater activating efficiency with PKC beta isozymes. AF binds to PKC presumably at the DAG binding site as suggested by the competition between phorbol dibutyrate and AF for binding to the kinase. Moreover, AF promotes the selective binding of PKC beta to natural or artificial membranes in the presence of microM concentrations of Ca2+. Altogether these results suggest the presence in MEL cells of a protein factor that can promote association of PKC to the membranes together with activation of the kinase, without the requirement for DAG formation. This could be visualized as a new mechanism for prolonged and selective activation of PKC.

Animals↗

PubMed1990

Identification of an endogenous activator of calpain in rat skeletal muscle.

An additional component of the regulatory system of rat skeletal muscle calpain has been identified. It exerts a potent activating effect on calpain activity and is a heat stable small molecular weight protein. Of the two calpain isozymes present in muscle, the activator is specific for calpain II, being uneffective with calpain I. It promotes activation of the proteinase by reducing 50 fold, from 1 mM to of 20 microM, the requirement of Ca2+ for maximum catalytic activity of the proteinase. However in the presence of the activator calpain II expresses a consistent fraction of the maximum activity even at significantly lower concentrations of Ca2+ (below 5 microM Ca2+). The activator effect follows kinetics that are consistent with the presence of specific binding sites on the calpain molecules. The activator not only removes in a dose dependent fashion the inhibition of calpain by calpastatin, but also prevents inhibition of the proteinase upon the addition of calpastatin. Competition experiments revealed that the proteinase contains distinct sites for the activator and the inhibitor, and that both ligands can bind to calpain with the formation of an almost fully active ternary complex.

Animals↗