Roles of the Cytoskeleton and of Protein Phosphorylation Events in the Osmotic Stress Response in EEL Intestinal Epithelium

Abstract

The eel intestinal epithelium responds to an acute hypertonic challenge by a biphasic increase of the rate of Cl^- absorption (measured as short circuit current, Isc, and creating a negative transepithelial potential, V_te, at the basolateral side of the epithelium). While the first, transient phase is bumetanide-insensitive, the second, sustained phase is bumetanide-sensitive, reflecting activation of the apically located Na⁺-K⁺-2Cl^- (NKCC) cotransporter, which correlates with the cellular RVI response.Here, we investigated the involvement of the cytoskeleton and of serine/threonine phosphorylation events in the osmotic stress-induced ion transport in the eel intestinal epithelium, focusing on the sustained RVI phase, as well as on the previously uncharacterized response to hypotonic stress.The study was carried out using confocal laser scanning microscopy, a quantitative F-actin assay, and transepithelial electrophysiological measurements (V_te and Isc) in Ussing chambers. Hypertonic stress did not detectably alter either net F-actin content or F-actin organization. In contrast, a brief exposure to hypotonic stress decreased the total cellular F-actin content in eel intestinal epithelium by about 15%, detectable morphologically mainly as a decrease in the intensity of the apical brush border F-actin labeling.The bumetanide-sensitive response of V_te and Isc to hypertonicity was potently inhibited by treatment with either cytochalasin, latrunculin A, colchicine, the protein kinase C (PKC) inhibitor chelerythrine, the myosin light chain kinase (MLCK) inhibitor ML-7, or the serine/threonine protein phosphatase inhibitor Calyculin A, but was unaffected by the PKA inhibitor H-89. The electrophysiological response of the epithelium to hypotonic stress was characterized by a sustained decrease of V_te and Isc, which was smaller and recovered faster in the presence of either cytochalasin, latrunculin A, or colchicine.It is concluded that in eel intestinal epithelium, the changes in ion transport in response to both hyper- and hypotonic stress require the integrity of both F-actin and microtubules. In addition, the shrinkage-induced activation of NKCC appears to require the activity of both PKC and MLCK. It is suggested that NKCC regulation by hypertonic stress involves an interaction between the cytoskeleton and protein phosphorylation events.

The cloning of eel osmotic stress transcription factor and the regulation of its expression in primary gill cell culture

“…Therefore, our observation supports the notion that Ostf induction is dependent on the integrity of the cytoskeleton, which is modulated in the process of cell volume regulation. Using eel intestinal epithelia, Lionetto et al reported that colchicine treatment inhibited the hypertonicity-induced shortcircuit current by about 50-80% (Lionetto et al, 2002). This observation supports the important role of the cytoskeleton in cellular osmo-responses.…”

“…Consistently, the incubation abolished the induction of Ostf mRNA expression. Since the change in intracellular ion strength would affect cell volume and cytoskeleton architecture (Di Ciano et al, 2002;Lionetto et al, 2002;Tse et al, 2006), we decided to test the effect of colchicine (an inhibitor of microtubule polymerization) to hypertonicityinduced Ostf expression. Obviously, colchicine treatment reduced Ostf gene expression.…”

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

“…Our data show that inhibition of the MLCK pathway altered the expression levels of Na + /K + -ATPase and TauT mRNA. Intriguingly, the osmotic stress response in eel intestinal epithelia was also found to be modulated by inhibitors of MLCK (Lionetto et al, 2002). In the mammalian system, hyperosmotic stress stimulated MLC2 phosphorylation and NKCC activity to facilitate cell volume restoration (Hoffmann and Pedersen, 2007).…”

Hypotonic shock mediation by p38 MAPK, JNK, PKC, FAK, OSR1 and SPAK in osmosensing chloride secreting cells of killifish opercular epithelium

“…In eel intestine, chelerythrine and calyculin A were potent inhibitors of the activation of NKCC-mediated ion uptake in an RVI response to hypertonic shock (Lionetto et al, 2002). In human cervical cancer cells, hypotonic shock evokes activation of extracellular signal activated kinase (ERK1 and ERK2) and p38 MAPK through a PKC-dependent pathway that also involves intracellular calcium (Shen et al, 2001).…”