Nitric oxide increases oxygen free radicals injury in isolated rat hepatocytes

Nitric oxide increases oxygen free radicals injury in isolated rat hepatocytes

Cell biology." molecular biology"/imction [ P2 C6/189 ] NITRIC OXIDE INCREASES OXYGEN FREE RADICALS INJURY IN ISOLATEDRAT HEPATOCYTES. P. Caraceni, ...

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Cell biology." molecular biology"/imction

[ P2 C6/189 ] NITRIC OXIDE INCREASES OXYGEN FREE RADICALS INJURY IN ISOLATEDRAT HEPATOCYTES.

P. Caraceni, *YM. Kim, A. Oasbarrini, #AB. Bode, *JR. Lancaster Jr, DH Van Thiel. Oklahoma Transplant Insitute, Oklahoma City, USA and Depts. of *Surgery and #Physiology, University of Pittsburgh, Pittsburgh, USA. The effect of the interaction between oxygen free radicals (OFR) and nitric oxide (NO) on the pathogenesis of the hepatic damage observed during inflammation, immune activation, and ischemia-reperfusion injury is unclear. The aim of this study was to determine the effect of endogenous NO production superimposed upon OFR-induced cell injury in isolated rat hepatocytes. Two experimental groups were used: 1) cytokines pre-treated hepatocytes, which produce great amounts of NO; and 2) non-treated hepatocytes, which do not produce NO. The cells were exposed to the effect of 02- and H202 generated by txanthine oxidase/hypoxanthine (XO). Cell injury was assessed by trypan blue uptake and lipid peroxidation (LP) by measuring TBARS and conjugated diene. Endogenous free radicals formation was detected with luminol chemiluminescence (LC). Treatment with XO for 4 h produced a significant greater cell injury and LP in stimulated hepatecytes than in control untreated hepatocytes. The addition of NMMA, a NO inhibitor, decreased cell injury and LP to a level similar to that observed in the untreated hepatocytes. LC was substantially greater in stimulated than in unstimulated hepatocytes. Morever, LC was significantly enhanced by L-arginine, precursor of the NO synthesis, and competitively inhibited by NMMA ordy in treated hepatocytes. These results suggest that in rat hepatocytes: 1) endogenous production of NO potentiates cell injury and LP caused by exogenous OFR, and 2) the damaging effect of NO may be related to the formation of more reactive free radicals, such as peroxynitrite that can be detected by the increase in LC.

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P2C6/191 ] SWELLING OF RAT HEPATOCYTES STIMULATES LYSOSOMAL ALKALINIZATION: MEDIATION BY CELL CYTOSKELETON G.L. Busch. H. VOlkl. P. Dartsch. D. H/iussin~er. F. Lan~ Physiologisches Institut I, Universit/it Tfibingeh, Ttibingen, Germany. In view of the importance of cell volume for proteolysis and the pH-dependence of autophagic hepatic proteolysis, the effect of cell swelling on vesicular pH was investigated. Using two different fluorescent dyes, acridine orange (AO) and FITCdextran, the effect of cell swelling on intravesicular pH was determined in both rat and human hepatocytes. Hypoosmotic solution (-40raM NaC1) increased AO fluorescence intensity in human and rat hepatocytes by 69 + 3% (n=9) and 39 + 1% (n=36), respectively, indicating an alkalinization of the AOloaded vesicular compartments. Pretreatment (1 h) of those cells with 5 ~ I colcemid, a substance which depolymerizes microtubules, reduced the response to 11 + 1% (12) and 17 + 1% (35), respectively. Pretreatment (lh) of rat hepatocytes with 5 laM colchicine, an additional microtubule inhibitor, reduced the response to 13 + 1% (n=26) while lumicolchicine, an inactive stereoisomer of colchicine, had no effect. Using F1TCdextran, the mean pH of acidic intracellular compartments in rat hepatocytes was found to be 5.92 + 0.16 (n=7). Following exposure to hypoosmotic solution, there was an alkalinization of those compartments to pH 6.35 + 0.11. Isoosmotic swelling of rat hepatecytes with 40 nM insulin, 2 mM BaC12, and 2 mM L-glutamine also induced vesicular alkalinization as indicated by the increase in AO fluorescence intensity. In conclusion, cell swelling by various mechanisms appears to induce an alkalinization of acidic intracellular compartments, including lysosomes. Hypoosmotic-induced cell swelling may exert this alkalinizing effect via a microtubule-dependent mechanism.

[ P2 C6/190 ]

[ P2C6/192 I

A PUTATIVE NUCLEOTIDE RECEPTOR OF T H E HEPATOCYTE PLASMA MEMBRANE. PURIFICATION AND PARTIAL CHARACTERIZATION.

PEPTIDERGIC INNERVATION OF HUMAN FETAL LIVER

W. Kreisel. C. Spamer, W. M/Sssner, Ch. Dietz, W. Gerok. C. Heilmann, Dept. of Gastroenterology, Medizinische Universit~itsklinik, D-79106, Freiburg, Germany. We report on the isolation and characterization of a 230 kDa-protein (SDS-PAGE) of hepatocyte plasma membranes that fulfils several criteria to be identified with a nucleotide receptor. The protein has been purified from Triton X-100 extracts of rat liver plasma membranes by sequential affinity chromatography on two lectins and 5'AMP-Sepharose. The 230 kDa-protein is composed of two S-S linked 120 kDaand 110 kDa-proteins of large structural homology, each containing 5 to 6 hybrid-type glycanes. It is covalently modified by Ca 2*dependent (Ko~[c~2+1= 3.5x 10"TM)autophosphorylation and Mg2+-dependent dephosphorylation, and can be photoaffinity-radiolabeled with 8-N3-[~,-32P]ATP, demonstrating one, or more, binding site(s) for ATP and probably for other nucleotides. It exhibits nucleotidase activity of broad specificity. The P2-purinoceptor antagonist Suramin effectively inhibits autophosphorylation at/zM concentrations. Conclusion~: From hepatocyte plasma membranes a 230 kDa hybrid-type glycoprotein has been purified with structural and catalytic properties to serve a nucleotide receptor function that may be feed-back regulated by [Ca2+]i via a cytosolically exposed regulatory Ca 2√∑binding site in co-operation with a phosphorylation site.

aim of our study was to define the ontogeny of hunan intrahepatic innervation and to assess the distribution and the nature of peptidergic nerves in humanfetal liver using imTunohistoct~mical methods. Our material consisted of Bouin~ or for= malin-fixed, p a r a f f i n ~ normal liver tissue frcm 14 human fetuses (20-40 weeks of gestation) and 10 adults. Weused the peroxidase-antiperoxidasetechnique (PAP) and polyclonal anti: bodies to pan-neural markers (NSE, neurofilam~, S-I00 protein, P~ 9.5) and neuropeptides (scmatostatin, ~PY, galanin, calcito: nin, CC~P). A rich neural supply was observed in the portal tracts and acini of humanadult liver. Intrasinusoidal fibers were noted throughout the acini, although they were most at~mdant in acinar zone I. The peptidergic innervation was characterised by the presence of nerve fibres containing I~Y. In humanfetal liver, a rich neural network appearedto be established in the portal tracts, mainly in close proximity to hepatic artery branches, at 20 weeks of gestation. The density of the nerve fibres reached adult levels betweenthe 32rid and 33rd week. Intra acinar innervation appeared at 40 weeksof gestation and i t was restricted in acinar zones I. Nerve fibres containing galanin,

D. Tiniakos, G. Tiniakos, A.D. Burt* Dept. of Pathology, NIMTS Hospital, Athens, Greece *Un~e. Dept. of Pathology, Newcastle upon Tyne, U.IC

sonatostatin, calcitonin and CC~RPappearedat 22, 26, 29 and 32 weeks respectively and they were localised in the portal tracts ~til birth, with the exception of scmatostatinergic nerves which disappeared before the 39th gestational week. ~Y-czntaining nerves were not demonstrated in humanfetal liver. It is suggested that the intrahepatic peptidergic neural network observed in the fetus may play an important role in liver functions necessary only during life in utero.