NAMI-A inhibits the PMA-induced ODC gene expression in ECV304 cells: involvement of PKC/Raf/Mek/ERK signalling pathway.

Imidazolium trans-imidazole dimethyl sulfoxide tetrachlororuthenate (NAMI-A) is a new compound active against lung metastasis of solid metastasizing tumours. While its in vivo effect has been studied, the molecular insights that underlie its action are largely unknown. Among the possible pathways responsible for malignant transformation, PKC arose as one of the most promising targets for new antineoplastic drugs. We demonstrated the capability of NAMI-A of inhibiting PMA induced-PKC activity in ECV304 in a dose-dependent fashion. Furthermore, NAMI-A through modulation of PKC activity has been proved capable of reducing the phorbol ester induced expression of ornithine decarboxilase (ODC) gene and to abrogate the activation of the Raf/MEK/ERK pathway. Taken together these results suggest that many of the in vivo outcomes of NAMI-A treatment may be the result of a direct action on PKC.

Resveratrol inhibition of lipid peroxidation.

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than alpha-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than alpha-tocopherol; (e) to be a weaker antiradical than alpha-tocopherol in the reduction of the stable radical DPPH*. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like alpha-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.

Spectrophotometric measurement of hydroperoxides at increased sensitivity by oxidation of Fe2+ in the presence of xylenol orange.

The method, developed by modifying the FOX methods described by Wolff (Methods Enzymol. 233, 182-189, 1994), involves the oxidation of Fe2- by peroxides at low pH in the presence of both the ferric-complexing dye xylenol orange and sucrose, the amplifier of the reaction. The method proved to be a convenient, simple and efficient assay for the direct measurement of both water and lipid soluble peroxides. In fact it improves by about 60% the sensitivity of the FOX1 method for water soluble peroxides, and by 7-8 times that of the FOX2 method for lipid soluble peroxides. It allows the detection of 0.1 microM peroxide in the test solution. The method is suitable to measure the lipid hydroperoxides present in phosphatidylcholine liposomes and in human LDL. The data obtained allowed us to define a mathematical expression to calculate the lipid hydroperoxide content of liposomes knowing their oxidation index.

Heparin down-regulates the phorbol ester-induced protein kinase C gene expression in human endothelial cells: enzyme-mediated autoregulation of protein kinase C-alpha and -delta genes.

Overexpression of protein kinase C-alpha and protein kinase C-delta has been shown to modulate a number of biological effects, including the cell growth and differentiation. We hypothesized that heparin, a potent antimitogenic drug, could affect the cell proliferation by inhibiting the expression of specific protein kinase C genes. Heparin, markedly but not completely, inhibited the serum-stimulated protein kinase C-alpha and -delta mRNA expression. Protein kinase C inhibition or down-regulation significantly decreased the serum-induced protein kinase C isoenzyme gene expression. Heparin failed to inhibit the residual effect of serum that was resistant to the above-mentioned treatments. Phorbol 12-myristate 13-acetate elicited an increase of protein kinase C isoenzyme gene expression that was completely prevented by protein kinase C inhibition or down-regulation. Heparin dose-dependently counteracted and ultimately abolished the increase in the protein kinase C isoenzyme gene expression elicited by phorbol 12-myristate 13-acetate. These results suggest that the inhibition of an autoregulatory role wielded by protein kinase C on the protein kinase C-alpha and -delta gene expression might represent a possible mechanism by which glycosaminoglycans modulate the cell growth.

Nuclear opioid receptors activate opioid peptide gene transcription in isolated myocardial nuclei.

Opioid-binding sites were identified in highly purified nuclei isolated from hamster ventricular myocardial cells. A significant increase in the maximal binding capacity for a kappa opioid receptor ligand was observed in myocardial nuclei from BIO 14.6 cardiomyopathic hamsters, as compared with nuclei obtained from normal myocytes of the F1B strain. The exposure of isolated nuclei to dynorphin B, a natural agonist of kappa opioid receptors, markedly increased opioid peptide gene transcription. The transcriptional effect was mediated by nuclear protein kinase C activation and occurred at a higher rate in nuclei from cardiomyopathic myocytes than in nuclei isolated from normal cells. Thus, a nuclear endorphinergic system may play an intracrine role in the regulation of gene transcription under both normal and pathological conditions.

Heparin inhibits phorbol ester-induced ornithine decarboxylase gene expression in endothelial cells.

Glycosaminoglycans regulate angiogenesis by affecting the availability of different growth factors for the endothelial cell (EC). However, little is known about the molecular and functional consequences resulting from direct interaction of these polyelectrolytes with the EC. Here we show that heparin markedly inhibited serum-stimulated DNA synthesis and ornithine decarboxylase (ODC) mRNA expression in human endothelial cells (HEC). About 50% of the serum effect on DNA synthesis and ODC gene expression was prevented by the selective protein kinase C (PKC) inhibitor chelerythrine or by PKC down-regulation. Heparin was ineffective in counteracting that part of the effect of serum that was resistant to PKC inhibition or down-regulation. In serum-free cultured HEC, heparin completely abolished the increase in DNA synthesis and ODC mRNA expression elicited by a number of PKC activators. Cell exposure to difluoromethylornithine, an irreversible inhibitor of ODC enzyme, dramatically antagonised both serum- and phorbol 12-myristate 13-acetate (PMA)-stimulated DNA synthesis. These results suggest that inhibition of PKC-mediated ODC gene expression by glycosaminoglycans may represent an important mechanism in the regulation of HEC proliferation.

Carvedilol inhibition of lipid peroxidation. A new antioxidative mechanism.

To define the molecular mechanism(s) of carvedilol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Carvedilol inhibits the peroxidation of sonicated phosphatidylcholine liposomes triggered by FeCl2 addition whereas atenolol, pindolol and labetalol are ineffective. The inhibition proved not to be ascribable (a) to an effect on Fe2+ autoxidation and thus on the generation of oxygen derived radical initiators; (b) to the scavenging of the inorganic initiators O2*- and *OH; (c) to an effect on the reductive cleavage of organic hydroperoxides by FeCl2; (d) to the scavenging of organic initiators. The observations that (a) carvedilol effectiveness is inversely proportional to the concentration of FeCl2 and lipid hydroperoxides in the assay; (b) the drug prevents the onset of lipid peroxidation stimulated by FeCl3 addition and; (c) it can form a complex with Fe3+, suggest a molecular mechanism for carvedilol action. It may inhibit lipid peroxidation by binding the Fe3+ generated during the oxidation of Fe2+ by lipid hydroperoxides in the substrate. The lag time that carvedilol introduces in the peroxidative process would correspond to the time taken for carvedilol to be titrated by Fe3+; when the drug is consumed the Fe3+ accumulates to reach the critical parameter that stimulates peroxidation. According to this molecular mechanism the antioxidant potency of carvedilol can be ascribed to its ability to bind a species, Fe3+, that is a catalyst of the process and to its lipophilic nature that concentrates it in the membranes where Fe3+ is generated by a site specific mechanism.

Opioid Peptide gene expression in the myocardial cell.

Both κ and δ opioid receptors have been identified in the myocardial cell. These receptors are coupled to phosphoinositide turnover and protein kinase C (PKC) activation, and their stimulation affects the cytosolic Ca(2+) and pH homeostasis as well as the contractility and the myofilament responsiveness to Ca(2+). Both the proenkephalin and the prodynorphin gene are expressed in cardiac myocytes. These cells are also able to synthetize and secrete dynorphin B, a biologically active end product of the prodynorphin gene binding selectively the κ opioid receptor. Prodynorphin mRNA and dynorphin B expression are markedly increased in ventricular myocytes isolated from Syrian cardiomyopathic hamsters (the hypertrophic BIO 14.6 strain), as compared with normal cells. Nuclear PKC activation and intracellular Ca(2+) overload have been shown to act as the two major signaling mechanisms involved in the increase in prodynorphin gene transcription observed in cardiomyopathic myocytes. In these cells, secreted dynorphin B activates κ opioid receptors at the cell surface and elicits an autocrine loop, leading to an increase in nuclear PKC activity and to a tonic feed-forward stimulation of prodynorphin gene transcription. The possibility that opioid genes may act in an autocrine fashion to affect myocardial Ca(2+) homeostasis, growth, and differentiation is also discussed.

Opioid peptide gene expression in the primary hereditary cardiomyopathy of the Syrian hamster. II. Role of intracellular calcium loading.

We have previously shown that prodynorphin gene expression was markedly increased in adult myocytes of BIO 14.6 cardiomyopathic hamsters and that nuclear protein kinase C (PKC) may be involved in the induction of this opioid gene. Here we report that the cytosolic Ca2+ concentration was significantly increased in resting and in KCl-depolarized cardiomyopathic myocytes compared with normal cells. In normal and in cardiomyopathic cells, KCl significantly increased prodynorphin mRNA levels and prodynorphin gene transcription. These effects were abolished by the Ca2+ channel blocker verapamil. In control myocytes, the KCl-induced increase in prodynorphin mRNA expression was in part attenuated by chelerythrine or calphostin C, two selective PKC inhibitors. In these cells, KCl induced the translocation of PKC-alpha into the nucleus, increasing nuclear PKC activity. In resting cardiomyopathic myocytes, the increase in prodynorphin mRNA levels and gene transcription were significantly attenuated by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxy-methylester being completely abolished when the chelating agent was administered in the presence of PKC inhibitors. KCl and the PKC activator 1,2-dioctanoyl-sn-glycerol additively stimulated prodynorphin gene expression both in normal and in cardiomyopathic cells. Therefore, we conclude that PKC activation and intracellular Ca2+ overload may represent the two major signaling mechanisms involved in the induction of the prodynorphin gene in cardiomyopathic cells.

Iron (III) stimulation of lipid hydroperoxide-dependent lipid peroxidation.

In an experimental system where both Fe2+ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl2 and FeCl3 on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe2+ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl2/FeCl3 ratio when PC liposomes deprived of LOOH by triphenylphosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl2. The FeCl2 concentration at which Fe2+ oxidation was maximal, defined as critical Fe2+ concentration [Fe2+]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl3 addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe2+]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl3 addition. The experimental data obtained indicate the kinetics of LOOH-dependent lipid peroxidation depends on the Fe2+/Fe3+ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl3 does not affect the LOOH-independent but the LOOH-dependent lipid peroxidation; and suggest that the Fe3+ endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.

The mechanism of iron (III) stimulation of lipid peroxidation.

A study conducted on Fe2+ autoxidation showed that its rate was extremely slow at acidic pH values and increased by increasing the pH; it was stimulated by Fe3+ addition but the stimulation did not present a maximum at a Fe2+/Fe3+ ratio approaching 1:1. The species generated during Fe(3+)-catalyzed Fe2+ autoxidation was able to oxidize deoxyribose; the increased Fe2+ oxidation observed at higher pHs was paralleled by increased deoxyribose degradation. The species generated during Fe(3+)-catalyzed Fe2+ autoxidation could not initiate lipid peroxidation in phosphatidylcholine liposomes from which lipid hydroperoxides (LOOH) had been removed by treatment with triphenylphosphine. Neither Fe2+ oxidation nor changes in the oxidation index of the liposomes due to lipid peroxidation were observed at pHs where the Fe3+ effect on Fe2+ autoxidation and on deoxyribose degradation was evident. In our experimental system, a Fe2+/Fe3+ ratio ranging from 1:3 to 2:1 was unable to initiate lipid peroxidation in LOOH-free phosphatidylcholine liposomes. By contrast Fe3+ stimulated the peroxidation of liposomes where increasing amounts of cumene hydroperoxide were incorporated. These results argue against the participation of Fe3+ in the initiation of LOOH-independent lipid peroxidation and suggest its possible involvement in LOOH-dependent lipid peroxidation.

Phorbol ester regulation of opioid peptide gene expression in myocardial cells. Role of nuclear protein kinase.

Opioid peptide gene expression was characterized in adult rat ventricular cardiac myocytes that had been cultured in the absence or the presence of phorbol 12-myristate 13-acetate. The phorbol ester induced a concentration- and time-dependent increase of prodynorphin mRNA, the maximal effect being reached after 4 h of treatment. The increase in mRNA expression was suppressed by incubation of cardiomyocytes with staurosporine, a putative protein kinase C inhibitor, and was not observed when the cells were cultured in the presence of the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate. Incubation of cardiac myocytes with phorbol 12-myristate 13-acetate also elicited a specific and staurosporine-sensitive increase in immunoreactive dynorphin B, a biologically active end product of the precursor, both in the myocardial cells and in the culture medium. In vitro run-off transcription assays indicated that transcription of the prodynorphin gene was increased both in nuclei isolated from phorbol ester-treated myocytes and in nuclei isolated from control cells and then exposed to phorbol 12-myristate 13-acetate. No transcriptional effect was observed when cardiac myocytes or isolated nuclei where exposed to 4 alpha-phorbol 12,13-didecanoate. The phorbol ester-induced increase in prodynorphin gene transcription was prevented by pretreatment of myocytes or isolated nuclei with staurosporine, suggesting that myocardial opioid gene expression may be regulated by nuclear protein kinase C. In this regard, cardiac myocytes expressed protein kinase C-alpha, -delta, -epsilon, and -zeta, as shown by immunoblotting. Only protein kinase C-delta and protein kinase C-epsilon were expressed in nuclei that have been isolated from control myocytes, suggesting that these two isotypes of the enzyme may be part of the signal transduction pathway involved in the effect elicited by the phorbol ester on opioid gene transcription in isolated nuclei. The incubation of myocardial nuclei isolated from control cells in the presence of a protein kinase C activator induced the phosphorylation of the myristoylated alanine-rich protein kinase C substrate peptide, a specific fluorescent substrate of the enzyme. The possibility that prodynorphin gene expression may control the heart function through autocrine or paracrine mechanisms is discussed.

Effects of taurine and hypotaurine on lipid peroxidation.

It has been suggested that hypotaurine might inhibit lipid peroxidation in vivo by scavenging the initiator OH. The results presented demonstrate that hypotaurine affects other reactions relevant to the initiation, propagation and termination phases of lipid peroxidation. Hypotaurine a) decreases Fe2+ autoxidation, either spontaneous or catalyzed by Fe3+, that may generate perferryl iron; b) decreases Fe2+ oxidation, by cumene hydroperoxide, that forms the alkoxy radical; c) inhibits the lipid hydroperoxide dependent lipid peroxidation, favoring the onset of the termination phase. Hypotaurine does not affect the autoxidation of Fe2+ bound to phosphatidic acid containing liposomes. Taurine is ineffective in all the experimental systems tested.

Effect of age and extent of dietary restriction on hepatic microsomal lipid peroxidation potential in mice.

Lipid peroxidation potential in hepatic microsomes from young and old mice following two different caloric restriction regimens was measured by a colorimetric thiobarbituric acid method under conditions where Fe2+ autoxidation and free oxygen radical production were undetectable. Peroxidation was highest in the young (3.5-month-old) slightly restricted group (caloric intake 75% of ad libitum mice) but very low in young severely restricted (caloric intake 50% of ad libitum mice) and in both old (27-month-old) slightly and severely restricted groups. Very old (45-month-old) severely restricted animals had intermediate lipid peroxidation potentials. Fatty acid composition of liver homogenates was also determined. Significant differences between groups were found for only three fatty acids. Linoleic acid (18:2(n-6)) decreased in aged slightly restricted animals while it remained stable in severely restricted animals during aging. Dihomo-gamma-linolenic acid (20:3(n-6)) was higher in very old restricted animals than in old slightly restricted animals. Docosahexaenoic acid (22:6(n-3)) decreased in old slightly restricted animals. These results indicated that the effect of diets on hepatic fatty acid composition and the potential for microsomal lipid peroxidation in mice was dependent on the degree of caloric restriction and age.

Fe3+ binding to liposomes of different phospholipid composition.

The possibility that phospholipid polar heads may influence lipid peroxidation by affecting the binding and location of the metal catalyst was investigated. The multilamellar liposomes containing dimyristoyl phosphatidylcholine (DMPC) and an equal amount of either dipalmitoyl phosphatidylcholine (DPPC) or dipalmitoyl phosphatidic acid (DPPA) were utilized to study Fe3+ location. Two simple colorimetric methods were utilized, which were developed to evaluate: a) Fe3+ both sequestered within and bound in a non reducible form on the surface of the liposomes; Fe3+ chelated to strong complexing agents present both on the surface and in the inner compartment of the liposomes. The results obtained clearly show that the two types of polar heads differently bind the metal. Whereas DMPC/DPPC liposomes do not affect Fe3+ detection by both methods, suggesting that the metal is not internalized and strongly bound, DMPC/DPPA liposomes greatly interfere with Fe3+ detection. Analysis of the binding data indicates that a large amount of the metal is sequestered by the liposomes both in a complexed and free form.

Phospholipid polar heads affect the generation of oxygen active species by Fe2+ autoxidation.

The possibility that phospholipid polar heads may influence Fe2+ reaction with molecular oxygen and, thus, the generation of oxygen active species was investigated. Dipalmitoyl phosphatidylcholine (DPPC) and DPPC/dipalmitoyl phosphatidic acid (DPPA) were utilized as model liposomes. Fe2+ oxidation, oxygen consumption, nitro blue tetrazolium reduction and 2-deoxyribose degradation were the parameters evaluated. Comparison of the results obtained clearly shows that the two types of polar heads differently affect iron chemistry. DPPC liposomes are ineffective. By contrast, Fe2+ oxidation by oxygen occurs in the presence of DPPC/DPPA liposomes. During this reaction, species able to reduce nitro blue tetrazolium and to degrade 2-deoxyribose are generated. The results obtained indicate that the polar heads of phospholipids, by influencing Fe2+ autoxidation, generate dangerous oxygen species which may exert an active role in the oxidation of the associated hydrophobic components of the phospholipids.

The influence of phospholipid polar head on the lipid hydroperoxide dependent initiation of lipid peroxidation.

In a buffer (Mes) and at a pH (6.5) where Fe2+ is very stable, we have studied the peroxidation of liposomes catalyzed by FeCl2. The liposomes studied, prepared by sonolysis, contained either phosphatidylcholine or 1:1 molar ratio of phosphatidylcholine and phosphatidic acid. The presence of the negatively charged phospholipid causes: 1) rapid Fe2+ oxidation and oxygen consumption; 2) increased generation of lipid hydroperoxides; 3) decreased generation of thiobarbituric acid-reactive materials; 4) very low inhibition of Fe2+ oxidation and lipid hydroperoxide generation by BHT; 5) inhibition of the termination phase of lipid peroxidation at high FeCl2 concentrations. A hypothesis is proposed to explain the results obtained.

The influence of pH on OH. scavenger inhibition of damage to deoxyribose by Fenton reaction.

Hydroxyl radicals (OH.) can be formed in aqueous solution by direct reaction of hydrogen peroxide (H2O2) with ferrous salt (Fenton reaction). OH. damage to deoxyribose, measured as formation of thiobarbituric acid-reactive material, was evaluated at different pHs to study the mechanism of action of classical OH. scavengers. OH. scavenger effect on Fe2+ oxidation was also evaluated in the same experimental conditions. In the absence of OH. scavengers, OH. damage to deoxyribose is higher at acidic compared to neutral and moderately basic pH. At acidic pH deoxiribose is per se able to inhibit Fe2+ oxidation by H2O2. Most of OH. scavengers tested inhibit deoxyribose damage and Fe2+ oxidation in a similar manner: both inhibitions are most relevant at acidic pH and decrease by increasing the pH. These results are not due to OH. scavenger inhibition of Fenton reaction. The influence of pH on the parameters studied appears to be due to the competition of deoxyribose and OH. scavengers for iron. These results suggest the prominent role of iron binding in the degradation of deoxyribose and in the OH. scavenging ability of different compounds. Results obtained with triethylenetetramine, a iron chelator with a low rate constant with OH., confirm that both deoxyribose and the OH. scavengers interact with iron bringing about a site specific Fenton reaction; that the OH. formed at these sites oxidize these molecules to their radical forms which in turn reduce the Fe3+ produced by Fenton reaction. The results presented indicate that most of classical OH. scavengers exert their effect predominantly by preventing the site specific reaction between Fe2+ and H2O2 on the deoxyribose molecule.

Effect of spermine on association of protein kinase C with phospholipid vesicles.

The in vitro mechanism by which polyamines affect protein kinase C (PK C) activation process was investigated in a reconstituted system consisting of purified enzyme and phospholipid vesicles of various phosphatidylserine content. It was found that the addition of spermine greatly interferes with the association of PK C to liposomes. This tetramine, at micromolar concentrations, was most potently effective while other polyamines such as spermidine and putrescine were almost ineffective; therefore the modulatory action appeared to be structure specific. The spermine effect is dramatically influenced by the density of the phosphatidylserine present on the liposome, suggesting the complex formation with the acidic component on phospholipid vesicles to be the mechanism by which this polyamine exerts its modulatory action.