Activation of the MAPKs ERK1/2 by cell swelling in turbot hepatocytes.

BACKGROUND INFORMATION: Activation of MAPKs (mitogen-activated protein kinases), in particular ERK1/2 (extracellular-signal-regulated kinase 1/2), has been reported to take place in a large variety of cell types after hypo-osmotic cell swelling. Depending on cell type, ERK1/2 phosphorylation can then serve or not the RVD (regulatory volume decrease) process. The present study investigates ERK1/2 activation after aniso-osmotic stimulations in turbot hepatocytes and the potential link between phosphorylation of these proteins and RVD. RESULTS: In turbot hepatocytes, Western-blot analysis shows that a hypo-osmotic shock from 320 to 240 mOsm kg(-1) induced a rapid increase in ERK1/2 phosphorylation, whereas a hyper-osmotic shock from 320 to 400 mOsm kg(-1) induced no significant change in the phosphorylation of these proteins. The hypo-osmotic-induced ERK1/2 phosphorylation was significantly prevented when hypo-osmotic shock was performed in the presence of the specific MEK (MAPK/ERK kinase) inhibitor PD98059 (100 microM). In these conditions, the RVD process was not altered, suggesting that ERK1/2 did not participate in this process in turbot hepatocytes. Moreover, the hypo-osmotic-induced activation of ERK1/2 was significantly prevented by breakdown of extracellular ATP by apyrase (10 units ml(-1)), by inhibition of purinergic P2 receptors by suramin (100 microM) or by calcium depletion using EGTA (1 mM) and thapsigargin (1 microM). CONCLUSIONS: In turbot hepatocytes, hypo-osmotic swelling but not hyper-osmotic shrinkage induced the activation of ERK1/2. However, these proteins do not seem to be involved in the RVD process. Their hypo-osmotic-induced activation is partially due to cascades of signalling events triggered by the binding of released ATP on purinergic P2 receptors and requires the presence of calcium.

Involvement of respiratory chain in the regulatory volume decrease process in turbot hepatocytes.

Regulatory volume decrease (RVD) constitutes a fundamental process that turbot (Scophthalmus maximus) hepatocytes are able to perform when exposed to hypo-osmotic stress. RVD is an integrative mechanism that involves various elements of the cellular machinery. Among others, ATP is an essential protagonist: released following hypo-osmotic shock, it acts as an auto/paracrine factor to trigger other signalling events. The origin of this ATP remains unclear and, to the best of our knowledge, no information exists about the role of mitochondrial respiration in RVD. Therefore, we propose to analyse the potential link between RVD and the respiratory chain, with a focus on ATP release and exocytosis. Using inhibitors of mitochondrial respiration, RVD was shown to be dependent on respiratory chain activity. However, we demonstrated an indirect role of mitochondrial respiration: ATP could be synthesized and then stored in intracellular vesicles until the moment cells release it to face hypo-osmotic swelling. However, the involvement of exocytosis in this process needs to be further investigated.

Volume regulation following hyposmotic shock in isolated turbot (Scophthalmus maximus) hepatocytes.

Regulatory volume decrease (RVD) following hyposmotic stimulation was studied in isolated turbot, Scophthalmus maximus, hepatocytes. Exposed to a reduced osmolality (from 320 to 240 mosm kg(-1)), cells first swelled and then exhibited a RVD. Volume regulation was significantly inhibited in presence of NPPB, 9-AC, acetazolamide, DIDS and barium. Taken together, these results could suggest that RVD operated via separate K+ and Cl- channels and probably Cl-/HCO3(-) exchanger in turbot hepatocytes. The K+/Cl- cotransporter could also be involved as furosemide and DIOA strongly inhibited the process whereas NEM, a K+/Cl- cotransporter activator, added under isosmotic conditions, led to cell shrinkage. RVD in turbot hepatocytes appeared also to depend on proteins p38 MAP kinase and tyrosine kinase but not on proteins ERK 1/2. Arachidonic acid and leukotrienes could also be involved since inhibition of synthesis of both these compounds by quinacrine and NDGA, respectively, inhibited the volume regulation. Likewise, Ca2+ has been proved to be an essential messenger as RVD was prevented in absence of Ca2+. Finally, this work provides bases for novel studies on cell volume regulation in marine teleosteans.