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Hepatocytes Isolated (hepatocyte + isolated)
Selected AbstractsCytosolic calcium regulates liver regeneration in the rat,HEPATOLOGY, Issue 2 2010Laura Lagoudakis Liver regeneration is regulated by growth factors, cytokines, and other endocrine and metabolic factors. Calcium is important for cell division, but its role in liver regeneration is not known. The purpose of this study was to understand the effects of cytosolic calcium signals in liver growth after partial hepatectomy (PH). The gene encoding the calcium-binding protein parvalbumin (PV) targeted to the cytosol using a nuclear export sequence (NES), and using a discosoma red fluorescent protein (DsR) marker, was transfected into rat livers by injecting it, in recombinant adenovirus (Ad), into the portal vein. We performed two-thirds PH 4 days after Ad-PV-NES-DsR or Ad-DsR injection, and liver regeneration was analyzed. Calcium signals were analyzed with fura-2-acetoxymethyl ester in hepatocytes isolated from Ad-infected rats and in Ad-infected Hela cells. Also, isolated hepatocytes were infected with Ad-DsR or Ad-PV-NES-DsR and assayed for bromodeoxyuridine incorporation. Ad-PV-NES-DsR injection resulted in PV expression in the hepatocyte cytosol. Agonist-induced cytosolic calcium oscillations were attenuated in both PV-NES,expressing Hela cells and hepatocytes, as compared to DsR-expressing cells. Bromodeoxyuridine incorporation (S phase), phosphorylated histone 3 immunostaining (mitosis), and liver mass restoration after PH were all significantly delayed in PV-NES rats. Reduced cyclin expression and retinoblastoma protein phosphorylation confirmed this observation. PV-NES rats exhibited reduced c-fos induction and delayed extracellular signal-regulated kinase 1/2 phosphorylation after PH. Finally, primary PV-NES,expressing hepatocytes exhibited less proliferation and agonist-induced cyclic adenosine monophosphate responsive element binding and extracellular signal-regulated kinase 1/2 phosphorylation, as compared with control cells. Conclusion: Cytosolic calcium signals promote liver regeneration by enhancing progression of hepatocytes through the cell cycle. (HEPATOLOGY 2010;) [source] Gap junction-mediated intercellular communication in a long-term primary mouse hepatocyte culture systemHEPATOLOGY, Issue 5 2003Stephanie A. Stoehr Gap junction-mediated intercellular communication (GJIC) is critical for maintaining integral cellular processes including differentiation and growth control. The disruption of GJIC has been correlated with aberrant function in many cell types, including hepatocytes in vivo; therefore it is imperative that cellular model systems support intercellular communication to simulate normal cellular functions. Functional GJIC has been shown in long-term primary rat hepatocyte cultures, which have been implemented widely to study various aspects of hepatocellular function; however, the onset of transgenic technology in murine species has necessitated the development of a primary mouse hepatocyte system. In this report, we analyze GJIC in a dimethylsulfoxide (DMSO)-containing long-term primary mouse hepatocyte culture system. The cells retain morphologic and biochemical characteristics of differentiated hepatocytes through day 30 post plating, including liver-specific gene expression. We further show that connexin32 and connexin26 expression and gap junction plaque formation increase over time in culture concomitant with an increase in GJIC between adjoining primary mouse hepatocytes. In conclusion, the findings described in this study make it possible to maintain differentiated primary mouse hepatocytes that also show GJIC in long-term culture for 30 days. In addition, this system has the potential to be extended to study primary mouse hepatocytes isolated from genetically engineered mice. [source] Fasting modulates metabolic responses to cortisol, GH and IGF-I in Arctic charr hepatocytesJOURNAL OF FISH BIOLOGY, Issue 6 2005Ø. Aas-Hansen Hepatocytes in primary culture from fed and 2 month fasted Arctic charr Salvelinus alpinus were exposed to physiological doses of either cortisol, salmon growth hormone (GH), salmon insulin-like growth factor-I (IGF-I) or a combination of salmon GH and salmon IGF-I. Fasting significantly lowered medium glucose levels compared to the fed fish, but had no significant effects on hepatocyte glycogen content or on the activities of enzymes involved in the intermediary metabolism. Cortisol treatment had no effect on hepatocyte glycogen content or on the enzyme activities investigated, but resulted in a significant increase in medium glucose concentration in hepatocytes isolated from fasted, but not fed fish. GH and IGF-I treatments, both singly and in combination, significantly increased the glycogen content of hepatocytes isolated from fed fish, with less pronounced effects on hepatocytes isolated from fasted fish. The combination of GH and IGF-I significantly increased lactate dehydrogenase activity regardless of the feeding state and significantly reduced the phosphenolpyruvate carboxykinase activity and medium glucose concentration in hepatocytes isolated from fed fish. Further, GH and IGF-I significantly increased the activities of alanine aminotransferase and aspartate aminotransferase in hepatocytes isolated from fasted fish, but not fed fish. There were no effects of GH, IGF-I, or their combination, on glucose 6-phosphate dehydrogenase or 3-hydroxyacyl-CoA dehydrogenase activities. The results demonstrated that nutritional status of the animal modulates hepatocyte responsiveness to metabolic hormones, and suggested a role for GH and IGF-I in hepatic glycogen conservation. [source] Metabolism of isometamidium in hepatocytes isolated from control and inducer-treated ratsJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2006I. BOIBESSOT Little is known about the metabolism and mechanism of action of the trypanocide, isometamidium (ISM), the major drug used for prophylaxis of trypanosomiasis. We have investigated its metabolism and distribution in isolated rat hepatocytes using liquid chromatography-mass spectrometry and confocal laser scanning microscopy (CLSM). Two putative metabolites were formed, which were proposed to be a mono-acetyl derivative and an oxidized metabolite (SII). This is the first demonstration of the hepatic metabolism of ISM, as previous in vivo studies were hampered by dose-limiting toxicity and insensitive analytical methods. The intrinsic fluorescence of the drug enabled its intracellular uptake to be followed by CLSM. It is taken up rapidly into the nucleolus, nuclear membrane and endoplasmic reticulum within 5 min, and retained in the nucleus for at least 24 h. Persistent binding of ISM to cellular macromolecules may contribute to its prophylactic effect in vivo. Pretreatment of rats with 3-methylcholanthrene, phenobarbitone (PB) or the widely used pyrethroid pesticide, deltamethrin, resulted in an increase in metabolism of ISM to the proposed SII after 1 h incubation with hepatocytes. 3-methylcholanthrene was the most potent inducer, causing a maximal 19.5-fold induction of SII formation after exposure of hepatocytes to ISM for 1 h compared with formation by control hepatocytes. In comparison, at the 1 h timepoint deltamethrin pre-treatment caused a 10.2-fold induction, and PB only 8.2 fold. [source] Monoamine oxidase A and B activities in embryonic chick hepatocytes: differential regulation by retinoic acidCELL BIOCHEMISTRY AND FUNCTION, Issue 2 2002Antonietta Nicotra Abstract Monoamine oxidases (MAOs) A and B are two isoenzymes involved in the degradation of many biological amines in the nervous system and in peripheral organs. In the present work hepatocytes isolated from 14-day-old chick embryos were used as a model system to determine whether retinoic acid (RA) is capable of modulating the activity of the two MAO forms. RA is a retinoid that, by binding with nuclear receptors, interferes with the expression of specific genes in many differentiation processes. Enzymic activity was measured with a radiochemical method using serotonin and ,-phenylethylamine as preferential substrates for MAO A and MAO B, respectively. The specific activity of the two forms was measured in hepatocytes cultured for 24, 48 and 72,h in the presence and the absence of serum. RA stimulated MAO B but not MAO A activity, in a dose- and time-dependent way, and only in the presence of serum. Maximum stimulation (about 3.5-fold) was obtained after treatment with 5,,M RA for 72,h. Kinetic analysis of MAO B activity showed an increase in Vmax in treated hepatocytes (5,,M RA for 72,h) with no change in Km. In conclusion, the present work shows that RA selectively elicits MAO B activity in cultured chick embryonic hepatocytes, this stimulation requires the presence of some factors present in the serum and is probably due to an increase in the number of enzyme molecules. Copyright © 2001 John Wiley & Sons, Ltd. [source] | ||||||||||