Most mammalian cells have developed compensatory mechanisms to respond to the variable osmotic stress caused by changes in the concentrations of intracellular osmo-active substances (e.g. glucose, amino acids, lactate) or by variations in the osmolarity of the surrounding medium. In response to osmotic cell swelling, the Regulatory Volume Decrease (RVD) is triggered and directs a reduction in the tonicity of the cell by the concerted opening of cation and anion selective ion channels. To date, the K⁺ and Cl^- conductances activated upon hypo-osmotic stimulation have been characterised electrophysiologically in many different cell systems. The molecular identity of the channels however, as well as the mechanism(s) involved in their activation have not yet been fully clarified and may differ between cell types. In this review, we will evaluate the different signalling pathways activated by osmotic cell swelling and discuss their putative role(s) in ion channel regulation, in maintaining cellular volume homeostasis, and in auto- and paracrinic signal transduction, with emphasis on intestinal epithelial cells.

Human Intestine 407 cells respond to osmotic cell swelling by the activation of Cl−- and K+-selective ionic channels, as well as by stimulating an organic osmolyte release pathway readily permeable to taurine and phosphocholine. Unlike the activation of volume-regulated anion channels (VRAC), activation of the organic osmolyte release pathway shows a lag time of ∼30–60 s, and its activity persists for at least 8–12 min. In contrast to VRAC activation, stimulation of organic osmolyte release did not require protein tyrosine phosphorylation, active p21rho, or phosphatidylinositol 3-kinase activity and was insensitive to Cl− channel blockers. Treatment of the cells with putative organic anion transporter inhibitors reduced the release of taurine only partially or was found to be ineffective. The efflux was blocked by a subclass of organic cation transporter (OCT) inhibitors (cyanine-863 and decynium-22) but not by other OCT inhibitors (cimetidine, quinine, and verapamil). Brief treatment of the cells with phorbol esters potentiated the cell swelling-induced taurine efflux, whereas addition of the protein kinase C (PKC) inhibitor GF109203X largely inhibited the response, suggesting that PKC is involved. Increasing the level of intracellular Ca2+ by using A-23187- or Ca2+-mobilizing hormones, however, did not affect the magnitude of the response. Taken together, the results indicate that the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves a PKC-dependent step.

Osmotic swelling of Intestine 407 cells leads to an immediate increase in cell surface membrane area as determined using the fluorescent membrane dye FM 1-43. In addition, as measured by tetramethylrhodamine isothiocyanate (TRITC)-dextran uptake, a robust (>100-fold) increase in the rate of endocytosis was observed, starting after a discrete lag time of 2-3 min and lasting for ϳ10 In mammalian cells, hypotonic cell swelling leads to the activation of cell volume regulatory processes, which in general involves a transient increase in the K ϩ and Cl Ϫ conductances (for reviews see Refs. 1-3). As a result, KCl leaves the cell, and cellular volume is rapidly restored (regulatory volume decrease). In addition to ion channel activation, osmotic swelling, like many other forms of mechanical stress, is known to promote the release of ATP, a potentially auto-or paracrinic factor acting through plasma membrane purinoceptors (4 -7). Extracellular ATP has been shown to regulate to the regulatory volume decrease response in a number of different cell types (4,5,8,9), either through the stimulation of a Ca 2ϩ -dependent K ϩ efflux (8) or by the activation of the volume-sensitive Cl Ϫ channels (4, 9). In Intestine 407 cells, extracellular ATP is not required for the direct activation of volume-sensitive Cl Ϫ channels (7). However, (sub)micromolar concentrations of extracellular ATP were able to potentiate the hypotonicity-provoked Cl Ϫ efflux in a Ca 2ϩ -dependent manner (7). In addition, osmotically induced ATP release was found to be critically involved in the activation of extracellular signal-regulated protein kinase (Erk) 1 -1/2 in Intestine 407 cells (7). Although the role of Erk-1/2 activation in the regulatory volume decrease response remains to be elucidated, activation of these MAP kinases by cell swelling has been observed in all cell models studied so far (10 -20).To date, several potential mechanisms have been proposed to explain cellular release of ATP. These include the following: 1) leakage due to (local) membrane damage; 2) activation of specific channel(s) or transporter(s); and 3) exocytotic events. Previously, members of the ABC-superfamily of transporters were suggested to permeate ATP (for reviews see Refs. 21 and 22). Intestine 407 cells, however, lack CFTR expression, and in the subclone we use, P-glycoprotein expression was not detected (7, 10) arguing against a role for ABC transporters in the ATP release. Because the cell swelling-induced ATP release differs from the activation of osmo-sensitive Cl Ϫ channels in both the time scale of activation/inactivation and in its sensitivity to inhibitors (6, 7), it was concluded that ATP does not permeate through volume-sensitive anion channels. In contrast, in Intestine 407 cells, the ATP release was found to depend largely on [Ca 2ϩ ] i as well as on an intact cytoskeleton (7). In this study, we pursued the hypothesis that regulated exocytosis might be involved in the release of ATP. In a number of cell models it was found that disruption of soluble N-ethy...

In response to osmotic cell swelling, Intestine 407 cells react with a rapid and transient activation of phospholipase D (PLD). To investigate the role of PLD during the regulatory volume decrease, cells were treated with 1-butanol resulting in a depletion of PLD substrates. Activation of volume-regulated anion channels, but not the cell swelling-induced release of taurine, was largely inhibited in the presence of low concentrations of 1-butanol. In addition, hypotonicity-induced exocytosis, ATP release and subsequent endocytosis were found to be largely abrogated. The results support a model of cell volume regulation in which PLD plays an essential role in the cell swelling-induced vesicle cycling and in the activation of volume-sensitive anion channels.

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