Martin Gschwentner

Mechanisms Sensing and Modulating Signals Arising From Cell Swelling

Cell volume alterations are involved in numerous cellular events like epithelial transport, metabolic processes, hormone secretion, cell migration, proliferation and apoptosis. Above all it is a need for every cell to counteract osmotic cell swelling in order to avoid cell damage. The defence against excess cell swelling is accomplished by a reduction of the intracellular osmolarity by release of organic- or inorganic osmolytes from the cell or by synthesis of osmotically less active macromolecules from their specific subunits. De-spite the large amount of experimental data that has accumulated, the intracellular mechanisms underlying the sensing of cell volume perturbations and the activation of volume compensatory processes, commonly summarized as regulatory volume decrease (RVD), are still only partly revealed. Moving into this field opens a complex scenario of molecular rearrangements and interactions involving intracellular messengers such as calcium, phosphoinositides and inositolphosphates as well as phosphoryla-tion/dephosphorylation processes and cytoskeletal reorganization with marked cell type- and tissue specific variations. Even in one and the same cell type significant differences regarding the activated pathways during RVD may be evident. This makes it virtually im-possible to unambigously define common sensing- and sinaling pathways used by differ-ent cells to readjust their celll volume, even if all these pathways converge to the activa-tion of comparatively few sets of effectors serving for osmolyte extrusion, including ion channels and transporters. This review is aimed at providing an insight into the manifold cellular mechanisms and alterations occuring during cell swelling and RVD.

Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells

Abstract. The ability of cells to readjust their volume after swelling, a phenomenon known as regulatory volume decrease (RVD), is a fundamental biological achievement guaranteeing survival and function of cells under osmotic stress. This article reviews the mechanisms of RVD in mammalian cells with special emphasis on the activation of ion channels during RVD.

Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels

Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

Na+/H+Exchangers: Linking Osmotic Dysequilibrium to Modified Cell Function

The Na+/H+ exchangers (NHEs) are among the major ion transporters involved in cell volume regulation. NHE activation leads to a cellular influx of Na+ ions and extrusion of H+ ions, which are readily replenished from intracellular buffers. This will result in a net import of Na+. In many systems NHE operates in parallel to Cl–-/ HCO33– exchange, resulting in cellular uptake of NaCl. The influx of osmotically obliged water will consequently lead to cell swelling. This makes NHEs suitable to serve as powerful mechanisms for increasing cell volume (CV). The low volume threshold for NHE activation enables the cells to respond to very minute reductions of the CV. By the coupling to the export of H+ ions cell volume regulatory NHE activation may lead to changes in intracellular pH. On the other hand NHEs are activated by a broad variety of ligands and by intracellular acidosis, which, in turn, may consequently lead to cell swelling. In addition, NHEs are linked to other intracellular proteins and structures, like e.g. the cytoskeleton, which themelves are involved in the regulation of numerous cellular processes. Therefore NHEs link CV regulation to a diversity of cellular functions, both in physiological and pathophysiological conditions. Six isoforms of the Na+/H+ exchanger, termed NHE1 - 6, have been cloned so far. NHE 1 - 5 are located in the plasma membrane, whereas NHE6 is sorted to the mitochondrial membrane. NHE1 and NHE6 are the ubiquitously expressed isoforms. The expression of the isoforms NHE2 to NHE5 is restricted to specific tissues and the pattern of their expression, as well as their subcellular localization indicate that they fulfill specialized functions. Cell shrinkage induced activation has been shown for NHE1,2 and 4. In contrast, NHE3 is inhibited by cell shrinkage. In many cells several isoforms are present and assigned to specific membrane domains where they may serve a functional crosstalk between the different ion transporters.

Cell swelling stimulates cytosol to membrane transposition of ICln.

ICln is a multifunctional protein that is essential for cell volume regulation. It can be found in the cytosol and is associated with the cell membrane. Besides its role in the splicing process, ICln is critically involved in the generation of ion currents activated during regulatory volume decrease after cell swelling (RVDC). If reconstituted in artificial bilayers, ICln can form ion channels with biophysical properties related to RVDC. We investigated (i) the cytosol versus cell membrane distribution of ICln in rat kidney tubules, NIH 3T3 fibroblasts, Madin-Darby canine kidney (MDCK) cells, and LLC-PK1 epithelial cells, (ii) fluorescence resonance energy transfer (FRET) in living fibroblasts between fluorescently tagged ICln and fluorochromes in the cell membrane, and (iii) possible functional consequences of an enhanced ICln presence at the cell membrane. We demonstrate that ICln distribution in rat kidneys depends on the parenchymal localization and functional state of the tubules and that cell swelling causes ICln redistribution from the cytosol to the cell membrane in NIH 3T3 fibroblasts and LLC-PK1 cells. The addition of purified ICln protein to the extracellular solution or overexpression of farnesylated ICln leads to an increased anion permeability in NIH 3T3 fibroblasts. The swelling-induced redistribution of ICln correlates to altered kinetics of RVDC in NIH 3T3 fibroblasts, LLC-PK1 cells, and MDCK cells. In these cells, RVDC develops more rapidly, and in MDCK cells the rate of swelling-induced depolarization is accelerated if cells are swollen for a second time. This coincides with an enhanced ICln association with the cell membrane.

Fluorescence-optical measurements of chloride movements in cells using the membrane-permeable dye diH-MEQ.

Fluorescence-optical measurements of the intracellular chloride concentration facilitate identification of chloride movements across the cell membrane of living cells. The two main dyes used for this purpose are 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) and 6-methoxy-quinolyl acetoethyl ester (MQAE). The use of both substances is impaired by their poor membrane permeability and therefore limited loading of the cells to be studied. Here we report the use of 6-methoxy-N-ethylquinolinium iodide (MEQ), a chloride-sensitive dye for which a membrane-permeable form is easily prepared. This makes the loading procedure as easy as with the acetoxymethyl (AM) forms of other dyes for sensing intracellular ions. In addition, the original method, which described absolute concentration measurements of chloride in the cytosol, was modified in so far as only relative measurements were made. This avoids the known limitations of single wavelength excitation and emission dyes with respect to exact concentration measurements. More-over, to enhance the signal-to-noise ratio the driving force for chloride was considerably increased by changing the original direction of the anion flux in the cells under investigation. We verified the method by using fibroblasts and activating ICln, a putative chloride channel cloned from epithelial cells and of paramount importance in the regulatory volume decrease in these cells. In the presence of SCN− the MEQ quench measured in NIH 3T3 fibroblasts is dramatically enhanced in hypotonically challenged cells compared with cells under isotonic conditions. Antisense oligodeoxynucleotides sensing ICln considerably impeded the swelling-induced chloride current (ICl) in NIH 3T3 fibroblasts. Accordingly, the chloride movement measured by the SCN− quench of the MEQ signal was significantly reduced. Similar results can be obtained in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), two known blockers of chloride transport in the plasma membrane of a variety of cells. In conclusion, fluroscence-optical measurements using MEQ as the chloride-sensitive dye provide a reliable and easy-to-use method for measuring changes of the chloride flux across the cell membrane of living cells.

Insight into the Structure-Function Relation of Chloride Channels

Chloride channels are highly selective transport proteins ubiquitously expressed in eukaryotic cells. Biophysical methods allow discrimination between several different types of chloride channels with

ICln: a chloride channel paramount for cell volume regulation.

Cell volume regulation is a ubiquitous cell regulatory mechanism based on meticulously controlled ion transport mechanisms. Keeping the absolute volume constant seems to be of the highest priority for most cells and is achieved at the expense of altered intracellular ion concentrations. We have been able to demonstrate that ICln, a chloride channel cloned from epithelial cells, is paramount for the ability of swollen cells to regulate their volume back to that under resting conditions. A unique feature of ICln is the distinct sensitivity of these channels for nucleotides and nucleoside analogues added to the extracellular fluid. In addition, cromolyn sodium and nedocromil sodium, drugs used by patients with asthma, are able to impede the function of these channels.

Structure and function of the ion channel ICln.

Normal function of organs and cells is tightly linked to the cytoarchitecture. Control of the cell volume is therefore vital for the organism. A widely established strategy of cells to counteract swelling is the activation of chloride and potassium channels, which leads to a net efflux of salt followed by water – a process termed regulatory volume decrease. Since there is evidence for swelling-dependent chloride channels (IClswell) being activated also during pathological processes, the identification of the molecular entity underlying IClswell is of utmost importance. Several proteins are discussed as the channel forming IClswell, i.e. phospholemman, p-glycoprotein, CLC-3 and ICln. In this review we would like to focus on the properties of ICln, a protein cloned from a m̲adin d̲arby c̲anine K̲idney (MDCK) cell library whose expression in Xenopus laevis oocytes resulted in a nucleotide sensitive outwardly rectifying chloride current closely resembling the biophysical properties of IClswell.

Blockade of swelling-induced chloride channels by phenol derivatives

1 . In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as ICln, a protein isolated from a cDNA library derived from Madin Darby canine kidney (MDCK) cells. ICln expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid) and NPPB (5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2 . RVD in NIH3T3 fibroblasts and MDCK cells is temperature‐dependent. 3 . RVD, the swelling‐dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4 . The cyclic AMP‐dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium‐activated chloride current in fibroblasts is insensitive. 5 . The binding site for the two phenol derivatives onto ICln seems to be distinct but closely related to the nucleotide binding site identified as G × G × G, a glycine repeat located at the predicted outer mouth of the Icln channel protein.