Biomedical subjects

S Pontremoli

Publications and source records attributed to S Pontremoli.

At least 73 records · Page 4Linked to original sources

PubMed1990

Isovalerylcarnitine is a specific activator of the high calcium requiring calpain forms.

Isovalerylcarnitine, a product of the catabolism of L-leucine, is a potent activator of rat calpains isolated from erythrocytes, kidney, liver, skeletal and heart muscle. Only calpains II, but not calpains I, are activated by IVC, with the only exception of rat erythrocyte calpain I, the only species present in these cells which has a Ca2+ requirement higher than that of most calpain I isoenzymes. Activation by IVC involves a dual effect: 1) a ten fold increase in the affinity of calpain for Ca2+, and 2) an increase in the Vmax 1.3-1.6 fold above the values observed with the native enzymes at saturating [Ca2+] as well as with the autolyzed fully active calpain form at 5 microM Ca2+. The increased affinity for calcium results in an increased rate of autoproteolysis of calpain II. Activation by IVC is additive to that promoted by interaction (or association) to phospholipids vesicles. Together these results suggest that IVC may operate as a selective activator of calpain both in the cytosol and at the membrane level; in the latter case in synergism with the activation induced by association of the proteinase to the cell membrane.

Animals↗

PubMed1990

Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases.

Two major protein kinase C (PKC) isozymes, accounting for approximately 95% of the total activity in human neutrophils, were separated by hydroxyapatite chromatography and were identified as beta-PKC (60% of the total) and alpha-PKC (35% of the total). No gamma-PKC was detected. A minor Ca2+/phospholipid requiring kinase that eluted from hydroxyapatite after alpha-PKC did not react significantly with any of the specific antisera employed for identification. Modification of beta-PKC or the minor PKC isozyme by calpain yielded Ca2+/phospholipid-independent forms (PKM) that retained only 50% of the original activities. In contrast, PKM formed from alpha-PKC retained full catalytic activity. For each native isozyme the rate of conversion by calpain was accelerated in the presence of Ca2+ and the lipid effectors, and the PKM form generated in each case was resistant to further digestion by calpain. All three PKC isozymes were also modified by a neutral serine proteinase isolated from human neutrophils, with this proteinase the major effect being loss of kinase activity, via a transient production of a Ca2+/phospholipid-independent form. This neutral serine proteinase appears to be localized at sites of interaction of cytoskeletal proteins with the cell membrane. Following stimulation of intact neutrophils with phorbol 12-myristate 13-acetate complete loss of native cytosolic kinase activity was observed, with recovery of approximately 30% of the original activity as a cytosolic Ca+/phospholipid independent form, presumably PKM. Loss of native PKC activity was greatest for the beta-isozyme. In cells stimulated by fMet-Leu-Phe approximately 60% of the original PKC activity was recovered as native cytosolic PKC and 30% as cytosolic PKM. Inhibitors of calpain reduced the extent of down-regulation of PKC, increased the proportion of PKC that remained associated with the plasma membrane and significantly reduced the proteolytically generated fully active PKM. Taken together, the in vitro and in vivo results suggest that calpain is involved primarily in the conversion of the PKC isozymes to the irreversibly activated PKM forms, and that the neutral serine proteinase may be the enzyme responsible for down-regulation, possibly via PKM as an intermediate.

Animals↗

PubMed1990

Identification of the proteolytically activated form of protein kinase C in stimulated human neutrophils.

The proteolytically activated form of protein kinase C has been identified in human neutrophils by using a monoclonal antibody that recognizes both the native kinase and the catalytically active proteolytic fragment (protein kinase M). Stimulation with fMet-Leu-Phe results in the conversion of approximately 30% of native protein kinase C to protein kinase M, with little evidence of further degradation. Stimulation with phorbol 12-myristate 13-acetate, on the other hand, causes only a transient formation of protein kinase M, with complete loss of total kinase activity. These differences are related to the differences in biochemical responses, reported earlier, in neutrophils exposed to these two activators.

Antibodies, Monoclonal↗

PubMed1990

Introduction of the beta isozyme of protein kinase C accelerates induced differentiation of murine erythroleukemia cells.

Induction of differentiation in murine erythroleukemia cells (MELCs) involves a protein kinase C (PKC)-mediated step. Vincristine-resistant cells respond more rapidly to hybrid polar/apolar inducers than the parental cells. These vincristine-resistant MELCs contain elevated levels of the beta isozyme of PKC (PKC-beta). Exogenous homologous murine PKC-beta, incorporated into permeabilized MELCs, accelerates induced differentiation. Neither rat PKC-beta, nor mouse PKC-alpha, nor rat PKC-alpha, incorporated into permeabilized MELCs, is effective in altering the kinetics of induced differentiation. This provides direct evidence for a rate-limiting role for this PKC isozyme during N,N'-hexamethylenebisacetamide-mediated induced differentiation of a transformed cell.

Acetamides↗

PubMed1989

Differential expression of protein kinase C isozymes and erythroleukemia cell differentiation.

Hexamethylene bisacetamide (HMBA) and other polar/apolar chemical agents are potent inducers of erythroid differentiation in murine erythroleukemia cells (MELC), as well as other transformed cell lines. Although the mechanism of action of HMBA is not yet known, evidence has been obtained that protein kinase C (PKC) plays a role in this process. In this study we provide further evidence that establishes this relationship. MELC contain two principal PKC activities, PKC beta and PKC alpha. MELC variants, selected for resistance to vincristine (VC), which display acceleration of their rates of induced differentiation, are enriched in PKC beta activity. When MELC are exposed to HMBA there is a fall in PKC activity, largely accounted for by a decline in PKC beta. This decline in PKC activity is faster in the VC-resistant, rapidly differentiating MELC. We previously demonstrated that VC-resistant MELC are resistant to the inhibition of differentiation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In both VC-sensitive and -resistant MELC, PMA causes rapid membrane translocation and then a decline in PKC activity, accompanied by a generation of a Ca2+- and phospholipid-independent protein kinase activity. In VC/PMA-resistant variants, this Ca2+/phospholipid-independent protein kinase activity persists considerably longer than in the VC-sensitive variants. This correlates with the resistance to PMA and provides additional evidence for a role for the Ca2+/phospholipid-independent protein kinase activity during induced differentiation.

Acetamides↗

PubMed1989

Activation of neutrophil calpain following its translocation to the plasma membrane induced by phorbol ester or fMet-Leu-Phe.

Stimulation of human neutrophils with phorbol myristate acetate or fMet-Leu-Phe results in translocation to the plasma membrane of approximately 25-40% of the cellular calpain activity. In the membrane-bound form the Ca2+-requirement for proteolytic activity is substantially reduced. An anti-calpain monoclonal antibody that is internalized by stimulated neutrophils is recovered in the same subcellular fraction that contains the membrane-bound calpain, apparently in the form of pinocytotic vesicles. When both monoclonal antibody and calpain were present in these vesicles, a pronounced inhibition of the membrane bound proteinase activity was observed. These results provide an explanation for the previously observed inhibitory effect of the monoclonal antibody on intracellular calpain activity and on the concomitant inhibition of granule exocytosis. The activated calpain associated with the plasma membrane compartment is therefore identified as the form specifically involved in mediating the physiological responses.

Antibodies, Monoclonal↗

PubMed1989

Enhanced activation of the respiratory burst oxidase in neutrophils from hypertensive patients.

In neutrophils of patients with essential hypertension the NADPH-dependent O2- production elicited by stimulation with f-Met-Leu-Phe is three to four fold higher in comparison with neutrophils of normotensive control subjects. Neutrophils from hypertensive patients are less responsive to priming, by non-stimulating doses of the agonist, as compared to control cells, which following this pretreatment augment superoxide anion production up to levels close to those expressed by neutrophils from hypertensive patients. No difference in NADPH oxidase activity, between neutrophils from the two groups of subjects, was observed when the rate of O2- production was evaluated in a reconstructed cell-free system containing the membrane fraction and the cytosolic cofactors. These results are consistent with the hypothesis that differences in the functional organization of the oxidase at the membrane level in neutrophils of hypertensive are responsible for the enhanced O2- production following agonist stimulation.

Cell-Free System↗

PubMed1989

The calpains.

In recent years interest has increased concerning the characterization of the structural-functional properties and the identification of the physiological role of non-lysosomal intracellular proteinases. Among these, calpain, a calcium-dependent cysteine proteinase ubiquitously present in a variety of tissues and cells, has been most extensively investigated in terms of activation, regulatory mechanisms, specificity and biological function. This review discusses each of these points on the basis of the most recent results concerning the general characteristics of calpain activity, and its preferential site of action within the cell as related to the specific functions of the proteinase in different cell types. As with other proteinases, calpain has to be under a continuous spatial and temporal control, and the structural and functional properties of the natural calpain inhibitor, calpastatin, must also be considered. The calpain-calpastatin system is the functional proteolytic unit that governs the activity of this intracellular proteolytic system, which is tightly correlated to the control of calcium homeostasis and thereby to the biological process of transmembrane signalling.

Calpain↗

PubMed1989

The role of intracellular proteinases in human neutrophil activation.

In addition to other proteinases human neutrophils contain two non granular neutral endopeptidases: a serine proteinase and a cysteine Ca2+ dependent proteinase named calpain. Serine proteinase localized in association with the cytoskeleton-membrane proteins, apparently exerts a dual role: it is partially released into the medium during neutrophil stimulation by phorbol myristate acetate (PMA), presumably acting as one of the cytotoxic factors; and in its intracellular localization is presumably involved in the process of down regulation of native protein kinase C (PKC). Calpain, predominantly present in resting conditions in an inactive form, becomes activated in the course of neutrophil stimulation and appears to be involved both in the down regulation of native PKC as well as in the formation of a proteinase-activated kinase form, presumably derived from PKC and defined as PKC-M. Calpain once activated appears to be also involved in cytoskeleton rearrangement, through proteolytic degradation specifically oriented by substrate phosphorylation. Activation, down regulation of PKC, formation of the proteinase-activated kinase, as well as proteolytic processing of cytoskeleton have been demonstrated to be correlated to those biochemical responses which characterize neutrophil activation.

Calpain↗

PubMed1988

Characterization of the calpastatin defect in erythrocytes from patients with essential hypertension.

In erythrocytes of patients with essential hypertension the level of calpastatin activity was found to be significantly lower than in red cells of normotensive subjects (1). We now demonstrate, by Western blot analysis, that the decreased inhibitory activity is due to a corresponding decrease in the amount of the inhibitor protein. This is also supported by the observation that calpastatins isolated and purified from erythrocytes of normotensive and hypertensive patients, have identical specific activity. Data are presented indicating that the decreased level of calpastatin cannot be ascribed to an accelerated decay of the inhibitor during the erythrocyte life span. Taken together the previous and present results further emphasize that an umbalanced proteolytic system may represent one of the molecular mechanisms responsible for those membrane abnormalities underlying the development of essential hypertension and its clinical complications.

Antibodies, Monoclonal↗

PubMed1988

Carbon tetrachloride-induced inhibition of protein kinase C in isolated rat hepatocytes.

Isolated rat hepatocytes exposed to CCl4 showed a dramatic decrease in [32P] incorporation into proteins which was evident as early as 5 min after the haloalkane addition. DEAE cellulose separation of protein kinases present in both particulated and cytosolic fractions of hepatocytes revealed that only the calcium and phospholipids dependent protein kinase C was affected by the treatment with CCl4, while kinases not requiring these factors for their activity were unmodified. Several 4-hydroxyunsaturated aldehydes known to be produced during CCl4-stimulated lipid peroxidation were found to inhibit protein kinase C at micromolar concentrations, suggesting the possibility that peroxidative events might be responsible for the impairment of protein kinase C during CCl4 intoxication.

Aldehydes↗

PubMed1988

The role of calpain in the selective increased phosphorylation of the anion-transport protein in red cell of hypertensive subjects.

The phosphorylation of the anion-transport protein (band 3) is selectively increased in human red cell membrane, following exposure of intact cells to ionophore and micromolar calcium. The phosphorylation is catalyzed by a membrane associated protein kinase distinct from either protein kinase C or Ca2+/calmodulin dependent protein kinase. We show that the increase in phosphorylation of band 3 is abolished if red cells had been pre-loaded with an inhibitor of calpain or with an anticalpain monoclonal antibody. Our findings suggest that calpain activity may control, both at a functional and at a structural level, the activity of this important transmembrane protein through the modulation of its susceptibility as a substrate of membrane bound protein kinase(s). Based on previous observations indicating the presence in erythrocytes from hypertensive patients of an uncontrolled intracellular calpain-mediated proteolytic system accompanied by an increased phosphorylation of band 3 protein(s), we suggest that our results may shed light on the type of molecular alteration which is associated with the hypertensive state.

Anion Transport Proteins↗

PubMed1988

Effects of a monoclonal anti-calpain antibody on responses of stimulated human neutrophils. Evidence for a role for proteolytically modified protein kinase C.

A monoclonal antibody directed against the Ca2+-requiring proteinase (calpain) of human neutrophils was employed to assess the role of this proteinase in mediating the responses to stimuli such as phorbol 12-myristate 13-acetate or fMet-Leu-Phe. In the presence of either phorbol 12-myristate 13-acetate or fMet-Leu-Phe the antibody is taken up by the neutrophils, and a marked inhibition of intracellular calpain is observed. The decreased calpain activity is accompanied by (a) a significant decrease in the proteolytic conversion of native protein kinase C (Ca2+/phospholipid-dependent enzyme) to the soluble form that does not require Ca2+ or phospholipids for activity; (b) a marked increase in the production of superoxide anion; and (c) a decrease in the exocytosis of granule contents. The increase in superoxide production can be attributed to a more prolonged association of native protein kinase C with the plasma membrane, thus enhancing the phosphorylation of membrane proteins that precedes O(2-) production (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Sacco, O., and Horecker, B. L. (1986), Biochem. Biophys. Res. Commun. 140, 1121-1126). The decreased exocytosis can be attributed to a decreased phosphorylation of certain cytoskeletal proteins, catalyzed by the soluble form of protein kinase C (Pontremoli, S., Melloni, E., Michetti, M., Sparatore, B., Salamino, F., Sacco, O., and Horecker, B. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3604-3608); the subsequent reorganization of the cytoskeleton appears to be related to degranulation. These effects of the monoclonal anti-calpain provide direct evidence for an essential role for calpain in the activation of human neutrophils.

Animals↗

PubMed1988

Vincristine-resistant erythroleukemia cell line has marked increased sensitivity to hexamethylenebisacetamide-induced differentiation.

Hexamethylenebisacetamide (HMBA)-induced murine erythroleukemia (MEL) differentiation is a multistep process. Commitment is the capacity to express terminal cell division and characteristics of the differentiated phenotype even after the cells are removed from culture with inducer. Culture of MEL cell line 745A.DS19 (DS19) with HMBA causes commitment to terminal differentiation after a latent period of about 10-12 hr. Previous studies have shown that during this latent period, HMBA causes a number of metabolic changes, including modulation in expression of certain protooncogenes. We now report the development of a MEL cell line (designated V3.17) derived from DS19 that is resistant to vincristine and is (i) markedly more sensitive to HMBA, (ii) induced to commitment without a detectable latent period, and (iii) resistant to the effects of phorbol ester and dexamethasone, which are potent inhibitors of HMBA-mediated DS19 differentiation. We suggest that this V3.17 MEL cell line may express a factor that circumvents HMBA-mediated early events, which prepare the cells for commitment to terminal differentiation.

Acetamides↗

PubMed1988

An endogenous activator of the Ca2+-dependent proteinase of human neutrophils that increases its affinity for Ca2+.

An endogenous activator of the Ca2+-dependent proteinase (calpain) has been identified in human neutrophils. In the presence of the activator, the affinity of calpain for Ca2+ is increased by greater than 100-fold and maximum catalytic activity is observed with Ca2+ concentration below 1 microM. The activator is a heat-stable protein having an apparent molecular mass of approximately equal to 40 kDa. It appears to be associated with the cytoskeletal fraction of human neutrophils. Neutrophils also contain an endogenous cytosolic calpain inhibitor (calpastatin), which is readily separated from the activator by size-exclusion chromatography. The effects of the activator and inhibitor appear to be antagonistic and may constitute a physiological mechanism for modulating intracellular calpain activity.

Calcium↗

PubMed1988

Erythrocyte deficiency in calpain inhibitor activity in essential hypertension.

The calpain-calpain inhibitor system was evaluated in erythrocytes of patients with essential hypertension and normotensive controls, either with or without a family history of hypertension. Calpain levels were similar in the controls and hypertensive patients, whereas the inhibitor activity level was significantly reduced in the latter (301.8 +/- 26.4 vs 220 +/- 14 U/mg hemoglobin, p less than 0.001). Borderline hypertensive patients and a few controls with a history of hypertension showed low inhibitor activity. Similar results have recently been reported in genetically hypertensive rats of the Milan strain. A significant inverse correlation (r = -0.43, p less than 0.001) was found between mean arterial pressure and calpain inhibitor. Although the pathophysiological significance of these observations is not yet clear, they suggest a new area of investigation into the molecular mechanisms underlying essential hypertension and its complications.

Adult↗

PubMed1988

The role of calpain and protein kinase C in activation of human neutrophils.

These and earlier findings suggest that certain neutrophil biochemical responses induced by external stimuli are mediated by the reorganization of the cytoskeletal-membrane interactions. This rearrangement of the intracellular architecture appears to result from a coordinated and integrated operation of two enzymes, namely PKC and calpain. This conclusion is schematically represented in a comprehensive model, discussed in the introduction and derived from a number of experimental observations. In focusing the importance of cytoskeletal reorganization, signal-directed proteolysis, induced by specific PKC-mediated phosphorylation, appears to play a fundamental role in cell functions such as positioning and fusion of organelles with plasma membranes and possibly locomotion and transport.

Calpain↗

PubMed1987

Isovalerylcarnitine is a specific activator of calpain of human neutrophils.

Isovalerylcarnitine (IVC) a product of the catabolism of L-leucine, is a potent activator of the Ca2+-dependent proteinase (calpain) of human neutrophils. At concentrations of Ca2+ in the low micromolar range, activation was 12 to 15-fold, and the activity exceeded that observed with millimolar concentrations of Ca2+ in the absence of the activator. Of the acylcarnitine derivatives tested, IVC was most active; D-isovalerylcarnitine was much less effective and palmitylcarnitine was ineffective. IVC did not increase the activity of calpain that was fully activated by an endogenous cytoskeleton-associated activator protein, but at low concentrations of the latter synergistic effects of the two activators were observed. Activation of neutrophil calpain by IVC is fully reversible. Inhibition by calpastatin was also reversed by IVC.

Calcium↗