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Biomedical subjects

F Piccinini

Publications and source records attributed to F Piccinini.

At least 19 recordsLinked to original sources
PubMed1999

PKCdelta acts as a growth and tumor suppressor in rat colonic epithelial cells.

We have analysed the expression of three calcium-independent isoforms of protein kinase C (PKC), PKCdelta, PKCepsilon and PKCzeta, in an in vitro model of colon carcinogenesis consisting of the nontumorigenic rat colonic epithelial cell line D/WT, and a derivative src-transformed line D/src. While PKCzeta and PKCepsilon showed similar protein levels, PKCdelta was markedly decreased in D/src cells when compared to the D/WT line. To assess whether down-regulation of PKCdelta was causally involved in the neoplastic phenotype in D/src cells, we prepared a kinase-defective mutant of PKCdelta. Stable transfection of this sequence caused morphological and growth changes characteristic of partial transformation in D/WT cells. Moreover, to test whether PKCdelta was involved in growth control and transformation in this model, we overexpressed PKCdelta in D/src cells. Transfected cells underwent marked growth and morphological modifications toward the D/WT phenotype. In a late stage in culture, transfected cells ceased to proliferate, rounded up and degenerated into multinucleated, giant-like cells. We conclude that PKCdelta can reverse the transformed phenotype and act as a suppressor of cell growth in D/src cells. Moreover, our data show that downregulation of this isoenzyme of PKC may cooperate in the neoplastic transformation induced by the src oncogene in D/WT cells.

Animals↗
PubMed1999

Effect of L-arginine load on platelet aggregation: a comparison between normotensive and preeclamptic pregnant women.

BACKGROUND: In the present study we hypothesized that a derangement of the L-arginine-nitric oxide system could be involved in the development of the hypercoagulative status found during preeclampsia. In order to verify such hypothesis we have compared the effects of nitric oxide substrate, L-arginine on platelet aggregation. Moreover, we have also measured the L-citrulline plasma levels as a stochiometric metabolite resulting from the conversion L-arginine to nitric oxide. METHODS: Nine preeclamptic women and 11 normotensive pregnant women were enrolled for the study. Subjects were infused with saline and with 30gr of L-arginine. Blood samples were drawn during the saline infusion (30 min), during L-arginine administration (30 min) and 30 min thereafter. ADP and collagen-induced platelet aggregation was studied as per Born with a dual-channel aggregometer (Chrono-Log, Mascia Brunelli, Italy) and L-citrulline was measured by HPLC. RESULTS: In normotensive women the infusion significantly decreased ADP and collagen-induced aggregation after 15 minutes of L-arginine load; whereas no effects were observed in preeclamptic women. Similarly in normotensive but not in preeclamptic women L-arginine load was able to increase L-citrulline plasma levels. CONCLUSIONS: In normotensive women the in vivo L-arginine administration decreases platelet aggregation with an increase of L-citrulline plasma levels. On the contrary, no effects were observed in preeclamptic women. These findings confirm that a hypercoagulative status characterizes preeclampsia and that such phenomenon could be explained by a derangement of the platelet L-arginine-nitric oxide pathway.

Adenosine Diphosphate↗
PubMed1998

Protein kinase Cepsilon is oncogenic in colon epithelial cells by interaction with the ras signal transduction pathway.

We have shown previously that overexpression of the epsilon isoform of protein kinase C (PKCepsilon) in rat colonic epithelial cells causes malignant transformation, possibly by interacting with the ras signal transduction pathway (Oncogene 12: 847, 1996). We have now performed experiments to examine certain early steps in the ras signaling pathway. A marked increase of Raf-1 phosphorylation was detected in tumorigenic ras-transformed D/ras as well as in D/epsilon cells (overexpressing PKCepsilon), compared to the nontumorigenic D/WT parental line. Moreover, in the PKCepsilon-transformed D/epsilon cell line, stable transfection with a dominant-negative raf-1 (DNraf) sequence caused complete regression of the neoplastic phenotype. These results suggested that PKCepsilon-induced transformation was associated with increased Raf-1 activation, and that DNraf could block the oncogenic effect of PKCepsilon. Furthermore, transfection of D/WT cells with dominant-negative ras induced arrest of cell growth, and subsequent transfection with PKCepsilon cDNA enhanced cell proliferation and induced neoplastic transformation. These results suggest that ras acts upstream of PKCepsilon, and that overexpression of PKCepsilon circumvents the block in cell proliferation caused by dominant-negative ras. We conclude that PKCepsilon exerts its oncogenic activity in rat colonic cells by affecting the ras signaling cascade at the level of Raf-1 activation.

Animals↗
PubMed1997

L-arginine infusion decreases platelet aggregation through an intraplatelet nitric oxide release.

Nitric Oxide (NO) inhibits platelet aggregation via activation of an intraplatelet soluble guanylate cyclase which induces an increase in cyclic GMP (1). It has been also demonstrated that platelets contain a constitutive, calcium-dependent, NO synthase which is activated by collagen-induced platelet aggregation. This leads to a NO synthesis from L-Arginine (L-Arg), which in turn increases cyclic GMP and down-regulates platelet aggregation (2). In vitro administration of supraphysiological concentrations of L-Arg enhances platelet cyclic GMP levels by increasing NO production and reduces platelet aggregation. This effect is reversed by pre-incubation with NO-synthase inhibitors (3). These results indicate that the L-Arg: NO pathway plays an important role in the modulation of human platelet aggregation (4). In vivo L-Arg, when administered i.v., induces hypotension (5) and vasodilatation (6,7) in humans, and when orally supplemented reduces platelet aggregability both in hypercholesterolemic rabbits and healthy men (8,9).

Adenosine Diphosphate↗
PubMed1996

Epsilon PKC acts like a marker of progressive malignancy in rat liver, but fails to enhance tumorigenesis in rat hepatoma cells in culture.

We have analyzed the expression pattern of epsilon protein kinase C (PKC) in normal liver tissue, in hyperplastic liver nodules and in hepatocellular carcinomas generated in the rat with the Solt-Farber protocol. A progressive increase in PKC epsilon expression was observed in nodules and carcinomas compared to normal liver tissue, suggesting that the expression level of this PKC isoenzyme could be associated with increased malignancy. To test this hypothesis, the well differentiated, poorly tumorigenic MH1C1 rat hepatoma cell line was stably transfected with a full length epsilon PKC cDNA. No increase in growth rate, saturation density, soft agar growth or in vivo tumorigenicity was observed in transfected cells, compared to parental or mock-transfected cells. These results indicate that epsilon PKC does not seem to participate in signaling pathways involved in neoplastic transformation or malignant progression in our liver cell model. The fact that epsilon PKC overexpression is tumorigenic in several other cell types suggests that this effect might be strictly cell- and tissue-specific.

Animals↗
PubMed1996

Overexpression of protein kinase C epsilon is oncogenic in rat colonic epithelial cells.

We have analysed the expression of five protein kinase C [PKC] isoforms in an in vitro model using nontumorigenic rat colonic epithelial cells FRC/TEX CL D [D/WT] and in the related tumorigenic Ha-ras-transformed FRC/TEX CL D/H-ras line [D/ras]. The PKC subspecies alpha, delta, epsilon and xi were expressed at the protein level in both D/WT and D/ras cells, while beta PKC was undetectable in both lines. The levels of expression of the delta and xi isoforms were similar in D/WT and D/ras cells. Alpha PKC expression was decreased and epsilon PKC was increased in D/ras cells compared to the D/WT line. To assess whether overexpression of epsilon PKC was linked to the transformed phenotype, we have generated from D/WT cells two clones (D/epsilon-5 and D/epsilon-9) which stably overexpress epsilon PKC about fivefold. Overexpression of epsilon PKC caused marked morphological changes in both transfected clones, which were accompanied by increased saturation densities and anchorage-independent colony formation in semisolid agar. These growth effects were attenuated or reversed by chronic incubation with phorbol 12-myristate 13-acetate. Furthermore, D/epsilon-5 and D/epsilon-9 cells formed tumors in athymic nude mice with 100% incidence while the parental D/WT or vector alone (D/MV12) controls produced no tumors. We conclude that epsilon PKC can act as an oncoprotein when modestly overproduced in nontumorigenic D/WT colonic cells, and that this isoform of PKC may be linked to ras-modulated signal transduction leading to neoplastic transformation in colonic epithelium.

Animals↗
PubMed1995

Protein kinase C isozymes during liver regeneration.

Protein kinase C isozyme (alpha, beta, delta and zeta) activities have been studied in compensatory cell proliferation at 20 and 24 hours after partial hepatectomy. Chromatographic analysis of PKC isozymes in male rats showed an activation of delta at 20 h after partial hepatectomy and of alpha, beta and zeta at 24 in correspondence with S phase peak. A peculiar sexual dimorphism, which matched sexual differences in DNA synthesis, has been observed. The administration of an excess of choline was able to modulate the protein kinase C isozyme pattern in females in relation to DNA synthesis and c-myc expression. Our data suggest that alpha, beta and zeta PKC positively regulated, while delta negatively controlled the liver compensatory growth after partial hepatectomy.

Animals↗
PubMed1995

NMR evaluation of changes in myocardial high energy metabolism produced by repeated short periods of ischemia.

Following our previous results which demonstrated that repeated short periods (2 min) of ischemia are capable of protecting the isolated rat heart from a subsequent global ischemia (30 min), in the present study we have concentrated on the metabolic changes occurring in rat hearts during six 2 min ischemia/3 min reperfusion cycles. Cardiac high-energy phosphates were monitored using 31P-NMR. Phosphocreatine levels fell (50-60%) during each ischemic period, and recovered to 70-80% of their initial values during reperfusion. P(i) rose by 59% during the first ischemic period, but increased less during subsequent ischemias (30% during the 6th occlusion, P < 0.05 vs. the first ischemic period) returning to baseline levels after each reperfusion. [ATP], pH, and [Mg2+] remained almost unaffected, but there was a decrease in HPLC-determined effluent ATP catabolites. The first occlusion led to a 95% drop in contractile function (P < 0.001 vs. baseline), but this recovered to 73% upon reperfusion (P < 0.02 vs. baseline), and was 65% at the end of the protocol. Phosphorylation potential (PP = [ATP]/([ADP].[P(i)]) correlated exponentially with total purine (r = 0.90) and with adenosine + inosine release (r = 0.81), and by the 6th ischemia/reperfusion cycle, exceeded that observed in controls by 21% (P < 0.05). We conclude that repeated short periods of ischemia do not lead to any significant alteration in the absolute myocardial ATP, but are associated with an enhanced cytosolic energy state in the heart, that enables the myocardium to reach a steady albeit lower functional state. Adenosine (+inosine) release may be involved in the regulation of the energy supply-demand balance.

Adaptation, Physiological↗
PubMed1995

Pharmacological action of a new spin trapping compound, 2-phenyl DMPO, in the adriamycin-induced cardiotoxicity.

Adriamycin (ADR)-induced cardiotoxicity was adopted in this investigation as a reliable model of radical-dependent myocardial pathology allowing both quantitative studies of drug activity in the isolated organ and in vivo comparison of the cardio-protection vs. general toxicity. Since commercially available lipophilic spin trapping compounds were shown to develop significant protective activity, in this investigation a newly synthesized spin trap (2-phenyl-DMPO) was studied. In Langendorff rat heart, 200 microM ADR induced a significant impairment of contractile performance, while 2-phenyl-DMPO was not cardiotoxic up to the 5 mM concentration. By this dose, 2-phenyl-DMPO induced a significant protection against the ADR-induced contractile impairment. In in vivo experiments, ADR (9 mg/kg i.v.) produced a significant impairment of ECG, coronary flow and contractility. The continuous administration of 2-phenyl-DMPO i.p. by osmotic pump delivering 0.3 mumol/hr was unable to protect the animals against the cardiotoxic signs. Seven days after ADR administration, severe general toxicity (arrest of body weight increase) and myelotoxicity were also observed. 2-phenyl-DMPO was unable to protect the animals from these toxic signs. The present results confirm that lipophilic spin traps can be a new class of antiradical drugs, as confirmed by the experiments performed in the isolated heart with the 2-phenyl-DMPO; however, this last compound is probably metabolized in vivo to inactivate derivatives.

Analysis of Variance↗
PubMed1994

Involvement of the alpha isoenzyme of protein kinase C in the growth inhibition induced by phorbol esters in MH1C1 hepatoma cells.

MH1C1 rat hepatoma cells express the alpha isoenzyme as the only phorbol-ester sensitive isoform of protein kinase C (PKC). In this cell line, phorbol 12-myristate 13-acetate (PMA) induced a marked, dose-dependent growth inhibition. The administration of the PKC inhibitor staurosporine was able to mimic the effect of the phorbol ester on cell growth in a dose-dependent fashion, whereas the PKC activator arachidonic acid stimulated cell proliferation. Exposure of cells to an antisense oligonucleotide specific for alpha PKC caused a significant impairment of cell growth. These data suggest that the alpha PKC activity is required for proliferation of MH1C1 cells.

Alkaloids↗
PubMed1994

Protein kinase C isozyme pattern in liver hyperplasia.

Lead nitrate, a potent activator of protein kinase C, is able to induce reversible rat liver hyperplasia. This phenomenon shows sex-related growth differences: liver hyperplasia as well as its regression by apoptosis occurred earlier and was more pronounced in male than in female rats. Dietary choline administration to females causes a shift of growth pattern towards the male values. Analysis of protein kinase C isoenzymes with hydroxylapatite column chromatography at time points crucial for lead-induced liver proliferation in male, female and choline-treated female rats showed a significant down-regulation of beta and alpha PKC activities and a marked activation of epsilon PKC. The fluctuation of these activities could be related to the rates of DNA synthesis. These data suggest that the observed PKC isoenzymes could be involved in the signal transduction pathway leading to lead-induced liver proliferation.

Animals↗
PubMed1994

Protective effect of dietary selenium supplementation on delayed cardiotoxicity of adriamycin in rat: is PHGPX but not GPX involved?

The involvement of Se enzymes in the protection against the oxidative stress induced by adriamycin (ADR) in rat heart has been studied in animals fed for 10 weeks at three different levels of Se content (low = 0.02 ppm; normal = 0.5 ppm; high = 1.0 ppm) and receiving a weekly injection of 3 mg/kg ADR for 4 weeks. ECG (QaT duration) and contractility of isolated atria were measured. The high-Se diet showed a significant protection on both parameters. To assess the hypothesis that an increase of specific activity of antioxidant Se enzymes may account for the cardioprotective effect of selenium, glutathione peroxidase (GPX), and phospholipid hydroperoxide glutathione peroxidase (PHGPX) were tested. The assays were performed on ventricles isolated from treated rats. At the end of the experimental period, GPX (cytosolic enzyme) did not show any significant difference between controls and ADR-treated at any level of Se content, thus excluding its involvement in the cardioprotection observed in high-Se ADR-treated animals. PHGPX, which is present both in cytosol and in the cell membrane, showed a trend to increase its activity in the presence of ADR treatment only in the membrane fraction; however, the statistical significance was reached only in the low-Se group (+100%). This observation suggests that membrane PHGPX might be involved in the cellular mechanism of adaptation of the heart to the toxic effects of ADR; however, the behavior of these enzymes does not seem to account for the significant protection of selenium supplementation both on ECG and on contractile indices of ADR cardiotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Intracellular magnesium homeostasis is involved in the functional recovery of preconditioned rat heart.

The role of ionic derangements in preconditioned hearts (PC) was assessed by measuring pHi, high-energy phosphates (HEP) and [Mg2+] by 31P NMR. Control (C) Langendorff rat hearts were subjected to 30' ischemia and 30' reflow. PC underwent 4x(2' ischemia + 3' reperfusion) before prolonged ischemia and reflow. In this model, the contractile recovery of PC hearts at the end of reflow (rate-pressure product RPP: 50 +/- 12% vs. 5 +/- 5% of C, p = 0.004) is not related to higher HEP or pHi levels than in C. [Mg2+]i increased significantly during ischemia both in C and PC organs; upon reperfusion its level remained significantly high in C (p < 0.001), while it regained the normal value in PC hearts. This behavior might improve in turn the SR calcium handling in PC organs, eventually contributing to the contractile recovery.

Adenosine Triphosphate↗
PubMed1993

Analysis of calcium-dependent protein kinase C isoforms in the early stages of diethylnitrosamine-induced rat hepatocarcinogenesis.

The profiles of the calcium-dependent protein kinase C (PKC) isozymes alpha, beta, and gamma were examined in subcellular fractions from Fischer 344 rat liver during the early stages (48 h, 96 h, 7 d, and 60 d) of diethylnitrosamine (DEN)-induced carcinogenesis, using the Solt-Farber "resistant hepatocyte" model (DEN-2-acetylaminofluorene-partial hepatectomy; DEN-AAF-PH), and then related to the presence of focal or nodular gamma-glutamyl transpeptidase (GGT)-positive morphologic changes in the liver. After DEAE and hydroxyapatite column chromatography, two peaks, immunologically identified as PKC-alpha and -beta isoforms, were detected in the liver of normal (alpha/beta ratio = 4.0) and treated rats. In DEN-AAF-PH hepatocarcinogenesis an increase in PKC-alpha expression was found after PH (+43 +/- 19% at 48 h, alpha/beta ratio = 5.1; +125 +/- 25% at 96 h, alpha/beta ratio = 4.8), whereas the PKC-beta isoform appeared less significantly modified (+11 +/- 3% at 48 h and +89 +/- 17% at 96 h). Seven and 60 days after PH, a marked increase in the PKC-alpha (+96 +/- 20% and +150 +/- 48%, respectively) and PKC-beta isoforms (+158 +/- 41%, alpha/beta ratio = 3.1 and +130 +/- 26%, alpha/beta ratio = 4.4, respectively), occurred along with the appearance of GGT-positive altered hepatic foci and nodules in the liver sections. Sham hepatectomy caused PKC-alpha and -beta isoform activities similar to those of normal controls. In contrast, saline-AAF-PH-treated rats had downregulation of PKC-alpha after PH (alpha/beta ratio = 1.8 at 96 h), possibly due to the mitoinhibitory effect of the carcinogen AAF on normal uninitiated hepatocytes. Immunohistochemical analysis with monoclonal antibodies to PKC-alpha and -beta revealed diffuse positive cytoplasmic signals in GGT-positive foci and nodules in rat liver. Taken together, these preliminary results, using the Solt-Farber model of liver carcinogenesis, suggest a role for PKC in tumor promotion. They also suggest that the PKC-alpha isoform may play a specific role in clonal expansion of DEN-initiated hepatocytes after PH.

2-Acetylaminofluorene↗
PubMed1993

Effects of the spin trap alpha-phenyl N-tert-butyl nitrone on myocardial function and flow: a dose-response study in the open-chest dog and in the isolated rat heart.

Alpha-phenyl N-tert-butyl nitrone (PBN) is widely used in spin-trapping experiments, but its possible toxicity has not been systematically evaluated. The purpose of this study was to investigate the effects of different doses of PBN on cardiac function in vivo (open-chest dogs) and in vitro (isolated rat hearts). In open-chest dogs, PBN was infused intracoronarily to achieve coronary arterial concentrations ranging from 1.6 mM to 10.0 mM. At coronary arterial concentrations of 1.6 mM and 2.5 mM, PBN had no appreciable effect on regional myocardial function (assessed as systolic wall thickening). However, coronary arterial concentrations of PBN of 5.0 mM and 10.0 mM produced a marked reduction and, eventually, a complete loss of systolic wall thickening (53% of baseline values after 30 min at 5.0 mM and 14% after 30 min at 10.0 mM). Furthermore, PBN increased coronary blood flow by approximately 25% at 2.5 mM and by > 100% at 10.0 mM. In isolated rat hearts, perfusion with 2.5 and 5.0 mM PBN for 60 min did not significantly affect global myocardial function, assessed as developed pressure, rate-pressure product, and positive and negative dP/dt. At the 10.0 mM concentration, however, these variables were significantly decreased after 30 min (developed pressure: -77% vs. controls; rate-pressure product: -84%; +dP/dt: -60%; -dP/dt: -70%); two out of five hearts stopped beating within 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Intestinal absorption of manganese: an in vitro study.

Our work on isolated rat intestine aimed at studying the hypothesis that the intestinal transport of manganese was carrier-mediated and, consequently, subjected to saturation. Our results confirm this hypothesis, assessing the concentration of carrier saturation for manganese at 0.5 mM. As this concentration, which may determine self-limitation in the intestinal absorption of this metal, is much higher than the maximal allowed concentration of the EEC Standard (50 mg/l), food and water should be carefully monitored for their manganese content in order to avoid reaching toxic concentrations in blood and tissues.

Animals↗