M. Paulmichl

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.

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Ficks law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

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.

Effect of inhibitors of Na+/H+‐exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes

Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N‐formyl‐Met‐Leu‐Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP‐induced cell swelling. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP‐induced increase in CV. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP‐stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. Western analysis with a monoclonal anti gastric H+/K+ATPase β‐subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+‐dependent H+‐extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.

The effect of hypoosmolarity on the electrical properties of Madin Darby canine kidney cells

The present study has been performed to test for the effect of hypotonic extracellular fluid on the electrical properties of Madin Darby canine kidney (MDCK)-cells. The volume of suspended MDCK-cell is 1,892±16 fl (n=8) in isotonic (298.7 mosmol/l) extracellular fluid. Exposure of the cells to hypotonic (230.7 mosmol/l) extracellular fluid is followed by cellular swelling to 2,269±18 fl (n=4) and subsequent volume regulatory decrease to 2,052±22 fl (n=4) within 512 s. Volume regulatory decrease is abolished by quinidine (1 mmol/l) and by lipoxygenase inhibitor nordihydroguaiaretic acid (50 μmol/l). The potential difference across the cell membrane averages −53.6±0.9 mV (n=49) in isotonic extracellular perfusates. Reduction of extracellular osmolarity depolarizes the cell membrane by +25.7±0.8 mV (n=67), reduces the apparent potassium selectivity of the cell membrane, from 0.55±0.07 (n=9) to 0.09±0.01 (n=26), and increases the apparent chloride selectivity from close to zero to 0.34±0.02 (n=21). Potassium channel blocker barium (1 mmol/l) depolarizes the cell membrane by +15.2±1.1 mV (n=13). In the presence of barium, reduction of extracellular osmolarity leads to a further depolarization by +14.0±1.4 mV (n=12). Addition of chloride channel blocker anthracene-9-COOH (1 mmol/l) leads to a hyperpolarization of the cell membrane by −6.7±2.2 mV (n=11). In the presence of anthracene-9-COOH, reduction of the extracellular osmolarity leads to a depolarization by +22.4±1.7 mV (n=11). Application of 1 mmol/l quinidine depolarizes the cell membrane to −6.6±0.5 mV (n=8) and virtually abolishes the effect of reduced extracellular osmolarity on cell membrane potential. Nordihydroguaiaretic acid (50 μmol/l), a substance known to inhibit lipoxygenase, increases steady state cell membrane potential in isotonic extracellular fluid to −58.8±1.8 mV (n=10) and blunts the depolarizing effect of hypotonic extracellular fluid (+5.4±1.5 mV,n=10). In conclusion, exposure of MDCK-cells to hypotonic media depolarizes the cell membrane by activation of a conductive pathway, which is insensitive to both barium and anthracene-9-COOH. The conductive pathway is possibly activated by leucotrienes.

Apparent chloride conductance of subconfluent Madin Darby canine kidney cells

In incompletely confluent Madin Darby canine kidney (MDCK)-cells continuous measurements of the potential difference across the cell membrane (PD) were made with conventional microelectrodes during rapid changes of extracellular chloride concentration. During control conditions mimicking in vivo situation, PD averages −50.3 ±0.7 mV. Reduction of extracellular chloride concentration from 122 mmol/l to 64.5 mmol/l depolarizes the cell membrane by +1.8±0.2 mV while reduction to 16 mmol/l leads to a transient, variable depolarization followed by a hyperpolarization of the cell membrane by −11.8 ±1.4 mV. 1 mmol/l anthracene-9-COOH hyperpolarizes the cell membrane by −10.7±1.0 mV, and abolishes the effect of altered extracellular chloride concentration (−0.6±0.5 mV), 1 μmol/l diphenylamine-2-carboxylate hyperpolarizes the cell membrane by −11.7±1.4 mV. 10 μmol/l furosemide hyperpolarize the cell membrane by −11.4±1.4 mV. Step increases of extracellular potassium concentration from 5.4 to 20 mmol/l depolarize the cell membrane by +14.9±1.0 mV in the absence of inhibitors, by +24.2±1.3 mV in the presence of anthracene-9-COOH and by +28.8±0.7 mV in the presence of furosemide. 10 μmol/l isoproterenol depolarize the cell membrane by +2.4±0.3 mV and increase the depolarizing effect of reducing extracellular chloride concentration to 64.5 mmol/l (+2.9±0.4 mV). 1 μmol/l forskolin depolarizes the cell membrane by +5.8±1.0 mV. In conclusion, chloride conductance of subconfluent MDCK-cells may be small during control conditions, is apparently decreased by anthracene-9-COOH and reduction of extracellular chloride concentration but is enhanced by isoproterenol.

Effects of epinephrine on electrical properties of Madin-Darby canine kidney cells

The present study has been performed, to test for the influence of epinephrine on the potential difference across the cell membrane (PD) of Madin-Darby canine kidney (MDCK) cells. Under control conditions, mimicking the in vivo situation, PD averages −53.3±0.9 mV (n=37). Increasing extracellular potassium concentration from 5.4 to 10 and 20 mmol/l depolarizes the cell membrane by +4.3±0.4 mV (n=5) and +15.8±1.2 mV (n=5), respectively. The application of 1 μmol/l epinephrine leads to sustained hyperpolarization of the cell membrane to −71.5±0.7 mV (n=37). In the presence of epinephrine, increasing extracellular potassium concentration from 5.4 to 20 mmol/l depolarizes the cell membrane by +30.6 ±0.2 mV (n=5); 1 mmol/l barium depolarizes the cell membrane by +14.8±0.7 mV (n=20) and abolishes the effect of step increases of extracellular potassium concentration from 5.4 to 10 mmol/l. In the presence of barium, epinephrine leads to a transient hyperpolarization by −31.2 ±1.2 mV (n=18). During this transient hyperpolarization, the cell membrane is sensitive to extracellular potassium concentration despite the continued presence of barium; 10 μmol/l verapamil depolarizes the cell membrane to −41.0±2.6 mV (n=11). In the presence of verapamil, the hyperpolarizing effect of epinephrine is only transient; 10 μmol/l phentolamine depolarizes the cell membrane by +3.0±0.6 mV (n=8). In the presence of phentolamine, the effect of epinephrine is virtually abolished (+0.4±0.6 mV,n=8); 1 μmol/l isoproterenol depolarizes the cell membrane by +2.8±0.8 mV (n=8). In the norminal absence of extracellular calcium, epinephrine leads to a transient hyperpolarization, which can only be elicited once. In conclusion, cpinephrine hyperpolarizes MDCK cells by increasing the apparent potassium conductance. This effect is transmitted by α-receptors and may be mediated by increases of intracellular calcium activity.

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.

Cell Volume Regulation in Renal Cortical Cells

Both proximal renal tubule cells and cultured Madin-Darby canine kidney (MDCK) cells are capable of regulating their volume in hypotonic media. Regulatory cell volume decrease in proximal straight tubules is impaired by barium, amiloride and acetazolamide and depends on the presence of bicarbonate and of sodium, whereas it is unaffected by complete removal of extracellular chloride. The observations may point to parallel loss of potassium through potassium channels as well as of bicarbonate and sodium via a bicarbonate-sodium cotransport. Alternatively, potassium/hydrogen ion exchange or potassium bicarbonate cotransport could be involved. In MDCK cells, exposure to hypotonic media apparently leads to the activation of an anion channel, while potassium conductance is rather decreased. In both proximal tubules and MDCK cells, volume regulatory decrease is possibly triggered by leucotrienes, which may be released during cell swelling. Cell volume is altered in a variety of conditions even at isotonic extracellular fluid and cell volume-regulatory mechanisms are likely to participate in regulation of renal transepithelial transport.