Calcium-dependent activation of Erk-1 and Erk-2 after hypo-osmotic astrocyte swelling

Abstract

The influence of hypo-osmotic cell swelling on the activity of the mitogen-activated protein (MAP) kinases Erk-1 and Erk-2 (where Erk stands for extracellular signal-regulated protein kinase) was studied in cultured rat astrocytes. Hypo-osmotic treatment led within 10 min to an increased activity of Erk-1 and Erk-2, which became maximal at 20 min and returned to the basal level within 60 min. Moreover, exposure to hypo-osmotic conditions induced a biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i): a rapid peak-like increase was followed by a sustained plateau. The absence of extracellular Ca2+ completely abolished Erk activation as well as the plateau of the [Ca2+]i response after hypo-osmotic stimulation. Application of wortmannin and agents to elevate intracellular cAMP levels also completely blocked Erk activation but were without effect on the biphasic [Ca2+]i response to hypo-osmotic treatment of the cells, suggesting a role of PtdIns 3-kinase and the Ras/Raf pathway downstream of the calcium signal. Protein kinase C (PKC) and Ca2+/calmodulin (CaM)-dependent kinases are unlikely to play a role in the hypo-osmolarity-induced signalling towards MAP kinases, as revealed by the blockage of PKC and CaM kinases. Inhibition of tyrosine kinases, pertussis-toxin- or cholera-toxin-sensitive G-proteins and phospholipase C had no effect on the [Ca2+]i response; the Erk response to hypo-osmolarity was also largely unaltered. This is different from the swelling-induced MAP kinase activation in hepatocytes, which was shown to occur via a calcium-independent but G-protein- and tyrosine kinase-dependent mechanism. Thus osmo-signalling towards MAP kinases might exhibit cell-type-specific features.

Ammonia induces RNA oxidation in cultured astrocytes and brainin vivo

“…Primary astrocytes were prepared from the cortices of cerebral hemispheres of newborn Wistar rats and cultured for 2 to 3 weeks as described. 15 Purity of the cell culture as determined by GFAP and S-100 immunohistochemical staining was greater than 95%. Immunoreactivity of the neuron-specific nuclear NeuN was not detectable in the astrocytes culture.…”

Regulatory function of hyperosmotic stress-induced signaling cascades in the expression of transcription factors and osmolyte transporters in freshwater Japanese eel primary gill cell culture

“…In mammalian studies, hypertonicity induced phosphorylation of MAPKs, including ERK1/2, p38 MAPK and JNK (Wehner et al, 2003). In Madin-Darby canine kidney (MDCK) cells, H4IIE rat hepatoma cells and bovine aortic endothelial cells, activation of ERK1/2 was detectable within 10min of treatment and reached a maximum level in 20-30min (Itoh et al, 1994;Duzgun et al, 2000;Schliess et al, 1996). Hypertonic stress-induced activation of p38 MAPK was reported in the human leukemic monocyte lymphoma cell line U937 and in murine inner medullary collecting duct (IMCD) cells (Shrode et al, 1995;Zhang and Cohen, 1996).…”

Functional and molecular characterization of multiple K–Cl cotransporter isoforms in corneal epithelial cells

“…MAPK pathways are activated by tyrosine phosphorylation induced by diverse extracellular stimuli, e.g., anisosmotic challenge, mechanical stress, and growth factors. Hypotonicity-induced swelling is accompanied by a rapid and transient increase of tyrosine phosphorylation of multiple proteins, including p44/42 (i.e., Erk1/2) and p38MAPK (Noe et al, 1996;Sadoshima et al, 1996;Schliess et al, 1995;Schliess et al, 1996;Sinning et al, 1997;Tilly et al, 1996a;Tilly et al, 1996b;Zhang et al, 1998). Moreover, increased tyrosine phosphorylation was found to be a critical step in activating the RVD process (Tilly et al, 1996a;Tilly et al, 1993).…”