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Biomedical subjects

A Benedetti

Publications and source records attributed to A Benedetti.

At least 109 records · Page 6Linked to original sources
PubMed1994

An interleukin-1 receptor antagonist decreases fibrosis induced by dimethylnitrosamine in rat liver.

The main pathological feature of liver fibrosis is the accumulation of extracellular matrix associated with hyperplasia and activation of perisinusoidal (Ito) cells (PSC) to myofibroblast-like cells. Interleukin-1 enhances collagen synthesis by increasing the proliferative activity of cultured PSC and has been implicated in the pathogenesis of hepatic fibrosis. Interleukin-1 receptor antagonist (IL-1ra) can block the binding of IL-1 to its receptors and act as a natural inhibitor of IL-1. We have examined whether the administration of IL-1ra can interfere with the development of experimental cirrhosis induced by dimethylnitrosamine (DMN). Rats were divided in three groups and received respectively DMN, DMN + IL-1ra and IL-1ra. For each group the collagen content of the hepatic tissue and the volume density of the inflammatory infiltrate were measured. Immunostaining for laminin and alpha-smooth muscle actin were also performed. In animals given DMN + IL-1ra we observed a decreased deposition of laminin and collagen, and a decreased number of laminin-positive PSC and of alpha-smooth muscle actin reactive cells, compared with animals receiving DMN alone. The present findings suggest that the early activation of PSC in vivo is at least in part mediated by IL-1 and confirm that the administration of IL-1ra may be of interest in modifying the biological effects of IL-1.

Actins↗
PubMed1994

Alterations of hepatocyte Ca2+ homeostasis by triethylated lead (Et3Pb+): are they correlated with cytotoxicity?

Isolated rat hepatocytes were used to investigate the biochemical mechanisms of toxicity of triethyllead (Et3Pb+), a highly neurotoxic degradation product of the antiknocking petrol additive tetraethyllead. As early as 5 min from the addition of 50 microM Et3Pb+ to hepatocyte suspensions a decrease of mitochondrial membrane potential and of the capacity of mitochondria and microsomes to retain Ca2+ occurred. A dose-dependent release of mitochondrial Ca2+ as well as an inhibition of microsomal Ca(2+)-ATPase activity were also evident when Et3Pb+ (from 2.5 microM up to 50 microM) was added to, respectively, isolated liver mitochondria and microsomes. Further experiments using hepatocytes loaded with the Ca2+ indicator Fura-2AM demonstrate that 1 min from addition of Et3Pb+ the cytosolic free Ca2+ levels increased by about 3-fold. High affinity plasma membrane Ca(2+)-ATPase activity was also significantly inhibited in hepatocytes treated with Et3Pb+, suggesting that an impairement of the mechanisms controlling the efflux of extracellular Ca2+ was concomitantly involved in the rise in cytosolic Ca2+ concentration. The increase in the cytosolic Ca2+ levels caused by Et3Pb+ was followed by a rapid decline of cell viability. However, the addition of EGTA or of the intracellular Ca2+ chelator BAPTA/AM did not affect either the time-course or the extent of cytotoxicity. Conversely, fructose, a glycolytic substrate that was able to support ATP production, prevented hepatocyte death. Thus, the depletion of cellular energy stores rather than the increase in cytosolic Ca2+ appears to be the mechanism by which Et3Pb+ causes irreversible injury in isolated hepatocytes.

Animals↗
PubMed1994

Fatty acyl-CoA esters induce calcium release from terminal cisternae of skeletal muscle.

The effect of palmitoyl-CoA (PCoA) on Ca2+ fluxes in unfractionated SR, longitudinal tubules (LSR) and terminal cisternae (TC) subfractions, obtained from rabbit fast-twitch skeletal muscles, was investigated. After MgATP-dependent Ca2+ preloading, PCoA released Ca2+ from unfractionated SR and TC, but not from LSR. Both the extent and the rate of PCoA-induced Ca2+ release from TC were increased in a dose-dependent manner, the half-maximal effect being attained at [PCoA] of approximately 6 microM. Ruthenium red, a Ca2+ release channel blocker, completely inhibited PCoA-induced Ca2+ release, whereas caffeine, a Ca2+ release channel agonist, depleted TC of Ca2+ and prevented the PCoA action. Scatchard plot analysis of [3H]-ryanodine binding showed that PCoA increased the affinity without affecting Bmax. The action of PCoA was mimicked by a nonhydrolysable analog. The present results indicate that PCoA interacts and opens the Ca2+ release channel (ryanodine receptor) of TC and that the mechanism of action involves binding rather than hydrolysis.

Acyl Coenzyme A↗
PubMed1994

Chronic ethanol feeding increases apoptosis and cell proliferation in rat liver.

The present study was conducted to evaluate if the increased rate of apoptosis previously reported in the liver of ethanol-treated rats was accompanied by increased cell renewal. A quantitative analysis of apoptosis was performed in rats fed an ethanol-containing liquid diet for 5 weeks. S-phase cells were demonstrated by immunohistochemistry, using the Bromodeoxyuridine/anti-Bromodeoxyuridine method. In ethanol-fed rats apoptosis was five times greater than in pair-fed controls. Bromodeoxyuridine-labelled hepatocytes increased from 0.07 +/- 0.009% in controls to 0.17 +/- 0.013% (p < 0.001) and Bromodeoxyuridine-labelled lipocytes (desmin-positive sinusoidal cells) increased from 3.43 +/- 0.28% to 6.60 +/- 1.04% (p < 0.001). The lobular distribution of labelled cells was modified with a shift towards the perivenular areas. The results of this study suggest that the replacement of liver cells lost by ethanol-induced apoptosis is not impaired in intact (non-operated) animals. The impaired regeneration following partial hepatectomy reported in ethanol-fed rats is possibly due to differences in the extent of parenchymal loss, to altered relationships between hepatocytes and blood supply and to the modalities of regeneration involved.

Alcoholism↗
PubMed1994

Immunohistochemical analysis of S-phase cells in normal human and rat liver by PC10 monoclonal antibody.

The expression of proliferating cell nuclear antigen (PCNA) was examined in normal human and rat liver fixed in either formaldehyde or methanol, and was compared with the incorporation of bromodeoxyuridine (BrdU) in S-phase cells. Codistribution of PCNA and BrdU was assessed in rat liver by double immunohistochemical staining using PC10 and anti-BrdU monoclonal antibodies to identify labelled nuclei of parenchymal and sinusoidal cells. In formaldehyde-fixed human biopsies (n = 13) PCNA-labelling index (PCNA LI) was 0.43 +/- 0.24% (mean +/- SEM) for hepatocytes and 0.09 +/- 0.03% for sinusoidal cells. A great interspecimen variability was observed and a preferential lobular distribution was not evident. In methanol-fixed human liver (n = 8) the immunostaining was strong. PCNA LI was 0.05 +/- 0.01% for hepatocytes and 0.14 +/- 0.01% for sinusoidal cells. 75% of labelled hepatocytes and 60% of labelled sinusoidal cells were found in acinar zone 1. In formaldehyde-fixed rat liver (n = 10) a weak nuclear staining and a great interspecimen variability were evident. LI was 0.13 +/- 0.07% for hepatocytes and 0.40 +/- 0.21% for sinusoidal cells without preferential acinar distribution. In methanol-fixed rat liver (n = 10), PCNA LI was 0.14 +/- 0.02% for hepatocytes and 0.40 +/- 0.04% for sinusoidal cells. 64% of labelled hepatocytes and 50% of labelled sinusoidal cells were found in zone 1. Only on methanol-fixed material did double immunohistochemistry show an almost complete overlap of BrdU and PCNA labelling. The PCNA LIs and the zonal distribution of labelled nuclei as obtained in methanol-fixed material are in keeping with previous reports using 3H-thymidine (3H-Thy) incorporation, suggesting that PCNA immunostaining represents a valid alternative to 3H-Thy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Plasma membrane order parameter in periportal and perivenular hepatocytes isolated from ethanol-treated rats.

Hepatic ethanol (EtOH) metabolism has been assumed to involve hepatocytes differently, according to their location in the hepatic acinus. This study's aim was to gain information on plasma membrane (PM) order parameter in periportal (PP) and perivenular (PV) hepatocyte-enriched fractions isolated by a digitonin-collagenase perfusion technique from rats pair-fed for 6-8 wk liquid diets containing either EtOH or isocaloric carbohydrates. Fluorescence polarization (P) studies have been performed to measure PM order parameter of PP and PV hepatocytes cultured for 2-6 h on glass cover slips and labeled with 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific probe for PM of living cells. Fluorescence polarization and microscopy indicated that TMA-DPH is a suitable probe to study PM order parameter in subconfluent rat hepatocyte monolayers where it labeled, after a rapid incorporation, PM of cells. In pair-fed control rats, PM order parameter was lower in PP hepatocytes than in PV cells (P = 0.366 +/- 0.013 vs. 0.381 +/- 0.021, respectively, P < 0.02; n = 7). In EtOH-treated rats, these zonal differences tended to disappear (P = 0.419 +/- 0.012 in PP cells vs. 0.417 +/- 0.007 in PV cells; n = 7). In addition, the order parameter was significantly higher either in PP or PV hepatocytes compared with pair-fed control animals (P < 0.002 and 0.003, respectively). A 30-min culture of cells in the presence of 40-200 mM EtOH significantly decreased the PM order parameter of hepatocytes isolated from pair-fed control rats with respect to EtOH-treated animals both in PP and PV cells (P < 0.01 and 0.02, respectively; n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A rapid immunostaining method for frozen sections.

A simple and rapid one-step method for demonstrating immunohistochemical markers (leukocyte common antigen, cytokeratin, etc.) is described, which can help define the nature of poorly differentiated neoplasms for diagnosis using frozen section. Microwave irradiation was used to speed immunohistochemical analysis using "Enhanced Polymer One-step Staining" (EPOS) reagents on cryostat sections from a variety of pathologic samples. Reproducible results were obtained using EPOS reagents for leukocyte common antigen and cytokeratin. The overall procedure takes less than 10 min and can be completed during surgery.

Frozen Sections↗
PubMed1993

CoA and fatty acyl-CoA derivatives mobilize calcium from a liver reticular pool.

The effect of CoA and fatty acyl-CoA esters on Ca2+ fluxes has been studied in isolated liver microsomes and in digitonin-permeabilized hepatocytes. When microsomes were loaded with increasing concentrations of Ca2+ (6-29 nmol/mg of protein), the extent to which CoA and palmitoyl-CoA released Ca2+ increased. At 23 nmol of Ca2+/mg of protein, half-maximal [CoA] and [palmitoyl-CoA] were 35 and 50 microM respectively. Under conditions of minimal Ca2+ loading, net release of Ca2+ was absent, but Ca2+ translocation from a CoA-sensitive to a CoA-insensitive pool took place. The effect of CoA required the presence of fatty acids, probably to form fatty acyl esters. In permeabilized hepatocytes, the pool(s) mobilized by CoA (or by palmitoyl-CoA) appeared to be different from that mobilized by Ins(1,4,5)P3.

Acyl Coenzyme A↗
PubMed1993

Latency is the major determinant of UDP-glucuronosyltransferase activity in isolated hepatocytes.

The glucuronidation of p-nitrophenol was measured in intact, saponin- and alamethicin-treated isolated mouse hepatocytes. In saponin-permeabilized cells the elevation of extrareticular UDP-glucuronic acid concentration enhanced the rate of glucuronidation threefold. When intracellular membranes were also permeabilized by alamethicin, a further tenfold increase in the glucuronidation of p-nitrophenol was present. Parallel measurements of the ER mannose 6-phosphatase activity revealed that saponin selectively permeabilized the plasma membrane, whereas alamethicin permeabilized both plasma membrane and ER membranes. The inhibition of p-nitrophenol glucuronidation by dbcAMP in intact hepatocytes was still present in saponin-treated cells and disappeared in alamethicin-permeabilized hepatocytes. It is suggested that the permeability of the endoplasmic reticulum membrane is a major determinant of glucuronidation not only in microsomes but in isolated hepatocytes as well.

Alamethicin↗
PubMed1993

Evidence that stimulation of plasma-membrane Ca2+ inflow is an early action of glucagon and dibutyryl cyclic AMP in rat hepatocytes.

The ability of glucagon (1 nM) and of dibutyryl cyclic AMP (50 microM) to increase cytosolic free Ca2+ concentration ([Ca2+]i) in Fura-loaded rat hepatocytes was examined in a system wherein Ca2+ inflow was induced by the re-admission of excess Ca2+ to a nominally Ca(2+)-free medium. An increase in [Ca2+]i did not occur in the absence of either agonist, but did so after co-addition of either agonist with Ca2+. Increasing the time between addition of dibutyryl cyclic AMP (or of glucagon) and Ca2+ led to increases in [Ca2+]i; half-maximal and maximal increases were observed at 0 s (i.e. at co-addition) and 5-7 s respectively. Dibutyryl cyclic AMP and Ca2+ each exhibited a concentration-dependence when their respective concentrations were changed for a fixed time interval between additions. Half-maximal and maximal effects were obtained with 30 microM and 50 microM dibutyryl cyclic AMP and with 0.5 mM and approx. 1 mM Ca2+ respectively. The data demonstrate an early action of glucagon and dibutyryl cyclic AMP on [Ca2+]i. It is argued that the agonist-induced rise in [Ca2+]i results from an increase in plasma-membrane Ca2+ inflow, an effect that appears to occur much earlier than that on mobilization of internal stores of Ca2+.

Animals↗
PubMed1993

Intraluminal calcium of the liver endoplasmic reticulum stimulates the glucuronidation of p-nitrophenol.

The relationship between the intraluminal Ca2+ content of endoplasmic reticulum and the rate of the glucuronidation of p-nitrophenol was investigated in isolated rat hepatocytes. Different agents which decrease the Ca2+ level in the endoplasmic reticulum [calcium ionophores (A23187, ionomycin) or Ca(2+)-ATPase inhibitors(thapsigargin,2,5-di-(t-butyl)-1,4-benzohydroquinone+ ++)] inhibited the conjugation of p-nitrophenol. Depletion of intracellular Ca2+ stores by preincubation of hepatocytes in the absence of free Ca2+ (in the presence of excess EGTA) also decreased the rate of glucuronidation; Ca2+ re-admission to EGTA-treated hepatocytes restored glucuronidation. In intact liver microsomes the p-nitrophenol UDP-glucuronosyl-transferase activity was not modified by varying the external free Ca2+ concentrations within a cytosol-like range. Emptying of the Ca2+ from the lumen of microsomal vesicles by A23187, after MgATP-stimulated Ca2+ sequestration, decreased the glucuronidation of p-nitrophenol. A similar effect was observed in filipin-permeabilized hepatocytes. In native and in detergent-treated microsomes, Ca2+ (1-10 mM) increased the p-nitrophenol UDP-glucuronosyltransferase activity. It is suggested that the physiological concentration of Ca2+ in the lumen of the endoplasmic reticulum is necessary for the optimal activity of p-nitrophenol UDP-glucuronosyltransferase; the depletion of Ca2+ decreases the activity of the enzyme.

Animals↗
PubMed1993

Membrane heterogeneity in isolated rat hepatocytes and liver plasma membrane subfractions: a comparative study using DPH and its cationic derivative TMA-DPH.

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) has been studied in hepatocytes isolated from rat liver and in isolated plasma membrane subfractions (cLPM, canalicular membranes and bLPM, basolateral membranes) using frequency domain fluorometry. The decay has been analyzed either by using a model of discrete exponential components or a model that assumes a continuous distribution of lifetime values in order to study different aspects of membrane heterogeneity. The results obtained by the two analyses are practically superimposable but the distributional approach allows an evaluation of membrane heterogeneity through the width of the distribution that has shown particularly significant differences when freshly hepatocytes are compared with in vitro aged hepatocytes. Moreover, the comparison of the distributional analysis of the two probes has shown in cLPM a tendency to higher values of the main lifetime component and a narrower distribution width with respect to bLPM. These results indicate changes of membrane domain organization that have been discussed in relation with the specific lipid composition that characterizes the two membrane subfractions. Our results indicate that frequency domain fluorometry may be used to study membrane heterogeneity in intact cells and isolated membranes.

Animals↗
PubMed1993

Physiological concentrations of inorganic phosphate affect MgATP-dependent Ca2+ storage and inositol trisphosphate-induced Ca2+ efflux in microsomal vesicles from non-hepatic cells.

1. MgATP-dependent 45Ca2+ uptake by microsomes obtained from various non-hepatic tissues, namely rat brain, rat solid Morris hepatoma 3924A and human platelets, was measured in the presence of P(i) at low, cytosol-like, concentrations. 2. Increasing P(i) concentrations (0.5-3 mM) caused a progressive enlargement of the 45Ca(2+)-storage capacity of all the microsomal fractions. 3. As a result of P(i) stimulation of Ca2+ uptake, 45Ca2+ and [32P]P(i) were co-accumulated by the three microsomal fractions. 4. The time course for 45Ca2+ and [32P]P(i) accumulation in brain microsomes revealed a biphasic 45Ca2+ uptake: a rapid phase was followed by a second, slower, phase, which depended on the presence of P(i). During the P(i)-dependent phase, the uptake of 45Ca2+ was paralleled by the uptake of [32P]Pi. 5. The passive efflux of Ca2+ was paralleled by the efflux of P(i) and vice versa. In fact, the inhibition of active Ca2+ uptake by excess EGTA, or lowering the P(i) concentration of the incubation system by dilution, caused the release of 45Ca2+ and [32P]P(i) from 45Ca2+ or [32P]P(i) pre-loaded brain microsomes. The Ca2+ ionophore A23187 also released 45Ca2+ and [32P]P(i). 6. Ca2+ efflux by A23187 was rapid (t 1/2 approx. 2 s) and independent of the extent of intravesicular Ca2+ loading, which indicates that Ca2+ and P(i) do not form intravesicular insoluble complexes. 7. The progressive increase in Ca2+ accumulation, depending on P(i) stimulation, resulted in a proportional increase in the amount of Ca2+ releasable by InsP3 in the three non-hepatic microsomal fractions and in digitonin-permeabilized platelets. 8. Concomitantly to Ca2+, microsomal P(i) was also released by InsP3.

Adenosine Triphosphate↗
PubMed1993

Regulation of intracellular pH in isolated periportal and perivenular rat hepatocytes.

BACKGROUND: Liver acinus shows a well-known metabolic zonation. The aim of this study was to investigate intracellular pH (pHi) regulation in isolated periportal (PP) and perivenular (PV) rat hepatocytes. METHODS: 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein was used as pH-sensitive dye. Hepatocyte subconfluent monolayers were acid-loaded by exposure to 20 mmol/L NH4Cl and alkali-loaded by reducing external CO2 and HCO3- at an external pH of 7.4. RESULTS: In the presence of HCO3-/CO2, (1) baseline pHi was higher in PP (7.25 +/- 0.018) than in PV hepatocytes (7.20 +/- 0.013) (P < 0.05); (2) pHi recovery from an acid load was 40% higher in PP than in PV hepatocytes (P < 0.02) and was inhibited by amiloride by 36% in PV and 7% in PP hepatocytes; (3) DIDS inhibited amiloride-independent pHi recovery from an acid load by 65% in PP and 52% in PV cells. In the absence of HCO3-/CO2, baseline pHi and pHi recovery from an acid load were not significantly different in PP and PV hepatocytes. pHi recovery from an alkali load was 30% higher in PV than in PP cells (P < 0.02). CONCLUSIONS: Our data suggest that isolated PP rat hepatocytes show higher activity for Na(+)-HCO3- cotransport, whereas PV cells show greater activity for Cl-/HCO3- exchanger.

Animals↗
PubMed1993

Cell proliferation following extrahepatic biliary obstruction. Evaluation by immunohistochemical methods.

The aim of the present investigation was to conduct an immunohistochemical study using bromodeoxyuridine (BrdU) incorporation as a marker of S-phase cells and cytokeratins as markers of biliary epithelial cells, in bile-duct-ligated rats at intervals of 1, 3, 7, 14 and 21 days after total biliary obstruction. Data obtained using only BrdU incorporation by S-phase nuclei were compared with those obtained by the simultaneous demonstration of S-phase nuclei and cytoplasmic cytokeratins. The labelling index of parenchymal liver cells and of biliary epithelial cells was evaluated as an index of the cellular growth pattern after total biliary obstruction. Our data show that following total biliary obstruction: (a) cell proliferation follows a similar pattern for biliary epithelial cells hepatocytes with a peak on the 3rd day; (b) the labelling index is significantly higher in biliary epithelial cells than in hepatocytes; and (c) sequential immunohistochemical staining using cytokeratin as a marker allows better identification of biliary epithelial cells, especially when the ductular lumen is not clearly outlined, or in isolated biliary cells which appear as components of the wall of the ducts of Hering.

Animals↗
PubMed1993

Primary amenorrhoea and infertility due to a mutation in the beta-subunit of follicle-stimulating hormone.

We report a woman with primary amenorrhoea and infertility associated with an isolated deficiency of pituitary follicle-stimulating hormone (FSH), but normal luteinizing hormone (LH) secretion. Ovulation was induced by administration of exogenous FSH and resulted in a successful pregnancy. Sequence analysis of the FSH beta-subunit gene indicated that she is homozygous for a two nucleotide frameshift deletion in the coding sequence. Her mother and son are heterozygous for this mutation. This deletion results in an alteration of amino acid codons 61-86 followed by a premature termination codon. The predicted truncated beta-subunit peptide lacks regions which are important for association with the alpha subunit and for binding to and activation of the FSH receptor. Abnormalities of FSH structure or function might be an under recognised but treatable cause of infertility.

Adult↗
PubMed1993

Intracellular alkalinization stimulates bile flow and vesicular-mediated exocytosis in IPRL.

Intracellular pH recovery from an acute alkaline load in rat hepatocytes is mediated by a Cl(-)-HCO3- exchanger, which is electroneutral, Na+ independent, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) sensitive. Stimulation of this Cl(-)-HCO3- exchanger requires intact microtubules, suggesting that vesicular transport may be required to activate this exchanger. To determine if intracellular alkalinization stimulates biliary HCO3- excretion and bile flow in the intact liver by vesicle-mediated exocytosis, isolated perfused rat livers (IPRL) were alkalinized by two protocols. Isohydric changes in CO2 and HCO3- concentrations induced transient increases in bile flow by 36% (P < 0.01), which were abolished by DIDS (0.01 mM), inhibited by pretreatment with colchicine (P = 0.01), but not affected by membrane depolarization with the K(+)-channel blocker BaCl2 (1 mM). Similarly, perfusion with 20 mM NH4Cl produced a 42% increase in bile flow (P < 0.01) and a 26% increase in biliary HCO3- excretion. Both the increases in bile flow and HCO3- excretion were almost completely blocked by DIDS and inhibited by pretreatment with colchicine (P < 0.01). Biliary excretion of horseradish peroxidase was also increased during intracellular alkalinization with either protocol (P < 0.01). These findings suggest that intracellular alkalinization stimulates bile flow and biliary HCO3- excretion. Microtubule-dependent vesicular-mediated exocytosis is involved in this response.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗