Short term regulation of hepatocyte glutathione content by hepatic sinusoidal cells in co-culture.
An inadequate balance between oxidant species and antioxidant mechanisms may constitute the primary mechanisms of a number of pathologies. The liver plays a central role in this balance: parenchymal hepatic cells contain and export especially high levels of the antioxidant glutathione and activated Kupffer cells release inflammation mediators and reactive oxygen species. There is growing evidence of a paracrine regulation of hepatic function by means of a fluent intercellular communication which must still be fully elucidated, especially in basal conditions. In vivo models provide often too complex results but, in vitro, tissue interactions are left aside; therefore it is important to find new experimental models to address cell communication studies. Here we propose the complementary use of three models to study liver glutathione system regulation in basal conditions: pure parenchymal cells primary cultures, addition of sinusoidal cell conditioned media to parenchymal cells and co-culture of sinusoidal cells using porous membranes. We have also developed a high specifity immunofluorescent method for the complete characterization of sinusoidal cell populations by flow cytometry and confocal microscopy. Our results show that Kupffer cells possess higher levels of reactive oxygen species than sinusoidal endothelial cells even in basal conditions. We also report that the glutathione content of hepatic parenchymal cells in basal conditions is regulated by a sinusoidal-parenchymal cells cross-talk and suggest the existence of a paracrine circuit in the management of liver oxidative stress.
Catalá
M
M
Department of Natural Sciences and Applied Mathematics, Higher School of Experimental Science and Technology, Universidad Rey Juan Carlos, Spain. myriam.catala@urjc.es
Pagani
R
R
Portolés
M T
MT
D000818:Animals; D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000378:metabolism / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007728:Kupffer Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D017077:Culture Media, Conditioned; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D007770:L-Lactate Dehydrogenase; D005978:Glutathione