Biol. Chem. Hoppe-Seyler, Vol. 377, pp. 57-65, January 1996 · Copyright © by Walter de Gruyter & Co · Berlin · New York Anisoosmotic Regulation of Hepatic Gene Expression Ulrich Warskulat, William Newsome, Birgitta Noe, Barbara Stoll and Dieter Häussinger* Medizinische Universitätsklinik, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Heinrich-Heine-Universität, Moorenstr. 5, D-40225 Düsseldorf, Germany * Corresponding author The effect of anisoosmolarity on the abundance of various mRNA species was examined in perfused rat liver and H4IIE rat hepatoma cells. Hyperosmotic ex- posure (385 mosmol/l) of isolated rat livers increased mRNA levels for tyrosine aminotransferase (TAT) by 246% and those for phosphoenolpyruvate carboxy- kinase (PEPCK) by 186%, whereas hypoosmotic expo- sure (225 mosmol/l) decreased their levels to 43% and 42%, respectively. mRNA levels for fructose-1,6-bis- phosphatase (FBP), argininosuccinate lyase (ASL), ar- gininosuccinate synthetase (ASS), glutamine synthe- tase (GS), glutaminase (GA) and glucokinase (GK) were largely unaffected. In H4IIE cells the modulation of TAT and PEPCK mRNA levels by anisoosmotic expo- sure was similar to that found in perfused rat liver. ASL and glutaminase mRNA levels were influenced in an opposite manner. The effects of anisoosmolarity on PEPCK mRNA levels in H4IIE cells were largely abolished in the presence of the protein kinase inhibi- tors H-7, H-89 and HA-1004. Other protein kinase inhib- itors such as Go-6850, KN-62, Rp-8-CPT-cAMPS, rapamycin, wortmannin, genistein or herbimycin did not prevent the osmosensitivity of PEPCK mRNA levels. Also pertussis and cholera toxin, vanadate and colchicine did not affect the osmosensitivity of PEPCK mRNA levels. The data suggest that anisoosmotic exposure acts on the levels of some but not all mRNA species and that this action may involve changes in protein phos- phorylation. They further indicate that the recently identified osmosensitive signal transduction pathway which involves a G-protein and tyrosine kinase depen- dent activation of mitogen-activated protein kinases is apparently not involved in the osmoregulation of PEPCK mRNA levels. Key words: Anisotonicity / Cell volume / Liver / mRNA / Protein kinases. Introduction Alterations of hepatocellular hydration, which reflect changes of liver cell volume on a short-term time scale, occur within minutes under the influence of hormones, cumulative substrate uptake or oxidative stress (for re- views see Lang and Häussinger, 1993, and Häussinger et al., 1994a). Such changes of cell hydration were recog- nized as an independent signal, which influences a variety of metabolic liver functions (Häussinger and Lang, 1992; Häussinger et al., 1994a), such as protein turnover and carbohydrate metabolism. Regarding the latter, hypoos- motic cell swelling stimulates glycogen synthesis (Baquet et al., 1990), flux through the pentose phosphate shunt (Saha et al., 1992), transcellular taurocholate transport (Häussinger et al., 1993) and inhibits glycolysis and glycogenolysis (Lang ef al., 1989); opposite effects are found following hyperosmotic cell shrinkage. Hepatocel- lular hydration also modifies cellular metabolism on a long-term time scale by modifying gene expression. For example, mRNA levels for tubulin (Häussinger et al., 1994b), ß-actin (Schulz ef al., 1991), ornithine decar- boxylase (Tohyama et al., 1991), and c-jun (Finkenzeller et al., 1994) are rapidly increased in response to liver cell swelling, whereas phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels (Newsome et al., 1994) and the re- plication of duck hepatitis B virus (Offensperger ef al., 1994) decreased. Regulation of PEPCK mRNA by cellular hydration probably occurs at the level of transcription and does not involve protein kinase C activation or changes in cAMP level (Newsome et al., 1994). The mechanisms by which cell volume changes affect gene expression are largely unclear, although hypoosmotic exposure was shown to induce a G-protein and tyrosine kinase depen- dent activation of MAP kinases, which could account for a swelling-induced induction of the c-jun gene due to c-jun phosphorylation (Schliess etal., 1995). The functional rele- vance of this osmosensitive signal transduction pathway (Schliess ef al., 1995) was demonstrated by the finding that both the swelling-induced alkalinization of endocyto- tic vesicles (Schreiber and Häussinger, 1995) and stimula- tion of canaliculartaurocholate excretion (Noe etal., 1996) exhibited the same inhibitor sensitivity as the swelling in- duced acivation of MAP kinases. Here, we address the question whether this osmosensitive signal transduction pathway plays a role in transducing the osmolarity effects on PEPCK mRNA levels (Newsome ef al., 1994). In addi- tion, the effect of anisoosmolarity on the levels of other mRNA species was studied in order to learn about the specificity of anisoosmolarity-effects on mRNA levels. 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