To date, very little is known about the molecular identity of the receptor involved in osmosensing in vertebrates. A specific class of osmoreceptors belonging to the family of histidine kinases has been identified in fungi, yeast, and plants cells (Grefen and Harter, 2004; Hohmann, 2002; Urao et al., 2000). Histidine kinases, however, as a part of the so-called two-component systems, are rarely expressed in mammalian cells. Several alternative mechanisms for osmosensing have been proposed, including macromolecular crowding and signaling via the extracellular matrix and integrin receptors, as well as activation of mechanosensitive ion channels (Haussinger et al., 2006; Mongin and Orlov, 2001; Okada, 1997; Pasantes-Morales et al., 2006b). For intestinal epithelial cells, the role of integrins and their effects on cytoskeletal rearrangements are of a particular interest because of the sensitivity of VRAC in these cells for agents that affect the integrity of the cytoskeleton and by the observation of several groups that osmotic cell swelling is accompanied by a cytoskeletal reorganization (Carton et al., 2003; Cornet et al., 1993; Foskett and Spring, 1985; Pedersen et al., 1999; Tilly et al., 1996).
Although differences may exist between cell types, several general features of cell signaling in response to hypoosmotic stress have emerged. Especially the requirement of protein tyrosine phosphorylation for the activation of VRAC has been well documented. In the presence of tyrosine kinase inhibitors such as herbimycin A, genistein, or tyrphostins, the cell swelling-activated anion conductance was found to be largely reduced, whereas (per) vanadate inhibition of phosphotyrosine phosphatases, to reduce phosphotyrosine phosphatase activity, potentiated the anion efflux triggered by nonsaturating hypoosmotic stimulation (Shi et al., 2002; Sorota, 1995; Tilly et al., 1993; Voets et al., 1998). Many hypotonicity-activated (targets of) tyrosine kinases have been reported, including the focal adhesion kinase, receptor tyrosine kinases such as the epidermal growth factor receptor, phosphatidylinositol 3-kinase, and members of the families of src and MAP kinases (for review, see Pasantes-Morales et al., 2006a). A direct activation of VRAC, however, has only been established for the src-like p56lck tyrosine kinase in Jurkat T lymphocytes. For these cells, strong evidence exists that p56lck is both essential and adequate for channel activation (Lepple-Wienhues et al., 1998). This notion is supported by our observation that the hypotonicity-provoked anion efflux in
Intestine 407 cells is largely reduced after treating the cells with damna-canthal, an inhibitor ofp56lck (B. C. Tilly, unpublished results). In Caco-2 colonocytes, however, targeting c-src to caveolae was found to inhibit VRAC (Trouet et al., 200l).
In addition to tyrosine kinases and/or phosphatases, the involvement of G proteins in the activation of the RVD has been reported. Indeed, activation of G proteins by the intracellular administration of GTPgS rapidly induced an anion-selective current in several cell types, including human HT29cl19A colonocytes (Tilly et al., 1991; Voets et al, 1998). Furthermore, a regulatory role for the Ras-related G-protein p21Rho in the activation of VRAC has been reported for Intestine 407 cells and various other cell types (Carton et al., 2002; Estevez et al., 2001; Pedersen et al., 2002; Tilly et al., 1996; Voets et al., 1998). Most plausibly, p21Rho exerts its function through the induction of cytoskeletal remodeling. In Intestine 407 cells, the related p12Ras and its downstream target Raf-1, which are also activated during osmotic cell swelling, did not affect VRAC regulation but were found to be essential for activation of the Erk-1/2 map kinases (Van der Wijk et al., 1998). Notably, although p21Rho is essential for VRAC regulation, studies in bovine endothelial cells have shown that Rho activation alone is not sufficient to induce opening of the channel (Carton et al., 2002). The hypotonicity-induced release of organic osmolytes was not sensitive to tyrosine kinase or phosphatase inhibition and did not require p21Rho or PtdIns-3-kinase activity, indicating that the efflux is regulated independently of VRAC (Tomassen et al., 2004).
Osmotic cell swelling is often accompanied by an increase in vesicle (re-)cycling and exocytosis (Bruck et al., 1992; Okada et al., 1992; Strbak and Greer, 2002; Van der Wijk et al., 2003). In Intestine 407 cells and several other cell types, an increase in volume leads to the extracellular release of ATP, an almost universal cellular response to mechanical stress, through a mechanism involving exocytosis (Hazama et al., 1999; Van der Wijk etal., 1998, 2003). Although extracellular ATP is not directly involved in the regulation of VRAC, purinergic receptor activation was found to trigger the hypotonicity-provoked stimulation of Erk-1/2 in Intestine 407 cells (Van der Wijk, 1998, 1999). It has been reported that exocytosis also contributes to the development of the compensatory anion conductance by the recruitment of additional anion channels to the plasma membrane (Lim et al., 2006).
Osmotic cell swelling activates several distinct signaling pathways that are often also associated with (neuro-)hormone and/or growth factor-associated signal transduction. Whereas some of these cascades are coupled to the regulation ofVRAC, others, such as the ATP-provoked activation of Erk-1/2, are apparently not involved in channel regulation and, although speculative, may have a function in restoring cellular homeostasis and in maintaining cell viability. Importantly, with the notable exception of p56lck in Jurkat T cells, activation of these signaling molecules by itself is not sufficient to activate VRAC. Linking these widely utilized signaling modules to an independent, but yet unidentified, "volume sensor'' not only preserves the specificity of the response, but also couples the RVD to pathways activated by hormonal stimulation, thereby facilitating the correction of small changes in cell volume that may occur during hormone-induced changes in cellular metabolism.
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