Biomedical subjects
Publications and source records attributed to E Wöll.
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.
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(-)/ HCO3(-) 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.
1. It was postulated that swelling dependent chloride channels are involved in the proton secretion of parietal cells. Since omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are structurally related to phenol derivatives known to block swelling dependent chloride channels, we set out to test, whether these substances--which are known to block the H,K-ATPase--could also lead to an inhibition of swelling-dependent chloride channels. Swelling-dependent chloride channels--characterized in many different cell types--show highly conserved biophysical and pharmacological features, therefore we investigated the effect of omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 on swelling-dependent chloride channels elicited in fibroblasts, after the reduction of the extracellular osmolarity. 2. Omeprazole, lansoprazole and its acid activated sulphenamide form AG2000 are able to block swelling-dependent chloride channels (IClswell). 3. Lansoprazole and its protonated metabolite AG2000 act on at least two different sites of the IClswell protein: on an extracellular site which seems to be in a functional proximity to the nucleotide binding site, and on an intracellular site which allows the formation of disulfide-bridges. 4. The inhibition of the proton pump and the simultaneous blocking of chloride channels by omeprazole, lansoprazole and its acid activated sulphenamide form AG2000, as described here could be an effective mode to restrict proton secretion in parietal cells.
Described here are 2 patients who developed thrombotic microangiopathy of the kidneys after receiving high cumulative doses of the new anticancer drug gemcitabine. The first patient, who received gemcitabine for treatment of a carcinoma of the pancreas, required hemodialysis for 6 months. In the second case, a woman suffering from a cholangiocellular carcinoma, end-stage renal disease was irreversible. Clinical awareness, timely detection and discontinuation of gemcitabine are mandatory to prevent this rare but disastrous complication of gemcitabine therapy.
It is not resolved whether the anionic channel involved in volume regulation after cell swelling comprises one or more subunits. Moreover, it remains to be determined which of the different proteins cloned so far, for which an involvement in cell volume regulation has been postulated, is the ideal candidate. In this review, we consider the role of the ICln protein, cloned from MDCK cells, in cell volume regulation.
In order to evaluate the natural history, prognostic parameters and treatment modalities for metastatic uveal melanoma, a review of the clinical data from the current literature was performed based on a Medline database search. Uveal melanoma represents approximately 5% of all melanomas. It is a distinct clinico-pathological entity, differing in many aspects from cutaneous melanoma. The clinical course is unpredictable and metastatic disease can develop very late after a long disease-free interval. Uveal melanoma metastasizes haematogenously, predominantly to the liver. The most Important prognostic parameters for primary uveal melanoma are tumour diameter, the patient's age and gender, histological features and tumour location. Systemic chemotherapy that is effective in cutaneous melanoma has failed to show activity in uveal melanoma. So far only the BOLD chemotherapy regimen (dacarbazine, lomustine, vincristine and bleomycin) combined with interferon-alpha has been shown to produce an objective tumour response in approximately 20% of previously untreated patients. For metastatic disease localized to the liver, intra-arterial application of fotemustine or carboplatin or chemoembolization with cisplatin have shown useful activity, resulting in a response in up to 40% of patients. Selected patients may benefit from palliative surgery. Immunotherapy with interleukin-2 or interferon-alpha has not shown consistent activity in metastatic uveal melanoma. In conclusion, patients with uveal melanoma metastatic to the liver should undergo one of the local treatment options. Carefully selected patients with extrahepatic disease or patients failing local treatment may benefit from systemic therapy using the BOLD regimen combined with interferon.
1. 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. 2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. 3. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling. 4. 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. 5. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV. 6. 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. 7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. 8. Western analysis with a monoclonal anti gastric H+/K+ ATPase beta-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. 9. 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.
Expression of the Ha-ras oncogene in NIH 3T3 fibroblasts (+ras cells) results in growth factor-independent proliferation and marked alteration of cytoskeletal architecture including breakdown of actin stress fiber network. Compared to identical cells not expressing the oncogene (-ras cells), +ras cells exhibit a more alkaline intracellular pH (pHi) and a larger cell volume (CV), both of which are important mitogenic elements. They are due to a set point shift for activation of the Na+/H(+)-exchanger. Moreover +ras cells respond to stimuli like 0.5% fetal calf serum or bradykinin with sustained oscillation of the cell membrane potential (PD) due to stimulated Ca2+ entry which triggers pulsatile release of calcium from internal stores and subsequent activation of calcium-sensitive K+ channels. 10 mumol/l bepridil inhibit oscillations of PD and protect +ras cells against actin stress fiber depolymerization. It is shown that bepridil blocks both cellular calcium entry as measured by Mn2+ quenching of fura-2 fluorescence and activation of the Na+/H(+)-exchanger following expression of the Ha-ras oncogene. Inhibition of the Na+/H(+)-exchange with 10 mumol/l HOE 694, on the other hand, does not significantly alter Ha-ras stimulated calcium entry or cytoskeletal rearrangement. In -ras cells ionomycin (0.1 mumol/l) leads to a transient increase in Cai. This effect is paralleled by a transient depolymerization of actin stress fiber network which cannot be inhibited by HOE 694. Disruption of the actin cytoskeleton in -ras cells by cytochalasin D does not alter steady state cell volume or Na+/ H(+)-exchange activity. However, stimulation of cytochalasin-treated -ras cells with bradykinin leads to cell swelling which can be blunted by HOE 694. The results show that both cytoskeletal rearrangement and activation of the Na+/H(+)-exchanger following expression of the Ha-ras oncogene require stimulated calcium influx and Cai oscillations. The depolymerization of the actin cytoskeleton is permissive for the Na+/ H(+)-exchanger to cause cell swelling upon stimulation with bradykinin.
The sensory neuropeptide secretoneurin (SN) triggers chemotactic migration of monocytes. We have investigated the possibility that SN, like other chemoattractants such as formyl-Met-Leu-Phe and chemokines, might stimulate migration of monocytes by G protein and protein kinase C (PKC) activation and induce Ca2+i release. We report that preincubation of monocytes with pertussis toxin inhibited SN chemotaxis. Staurosporine, an inhibitor of PKC, significantly decreased SN-induced chemotaxis of monocytes, suggesting that PKC may be involved in the signaling. Tyrphostin-23, which inhibits tyrosin kinase, did not affect SN-induced chemotaxis of monocytes. This suggests that SN uses a signaling mechanism that is coupled to pertussis toxin-sensitive G proteins. Involvement of phospholipase C beta as a result of PKC activation is suggested by a SN-induced increase of intracellular Ca2+ concentration in monocytes.
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. Moreover, 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.
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 x G x G, a glycine repeat located at the predicted outer mouth of the ICln channel protein.
Cell swelling in Madin-Darby canine kidney (MDCK) cells by reduction of extracellular osmolarity (omission of 70 ad 150 mmol/l mannitol, respectively) leads to the activation of anion of channels and Ca2+ sensitive K+ channels. The K+ channel activation leads to an initial transient hyperpolarization of the cell membrane potential (PD) followed by a sustained depolarization due to activation of anion channels. The present study elucidates the role of intracellular calcium (Ca2+i) in regulatory cell volume decrease (RVD) of MDCK cells. While reduction of extracellular osmolarity by omitting 70 mmol/l mannitol did not lead to a detectable change in Ca2+i, severe cell swelling by omitting 150 mmol/l mannitol led to a transient rise in Ca2+i. PD changes, on the other hand, were not different under either condition. In addition, the response of PD to cell swelling was not altered by treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate diester, pertussis toxin or cholera toxin. In the nominal absence of extracellular Ca2+, reduction of extracellular osmolarity did not lead to an increase in Ca2+i and no initial transient hyperpolarization was observed, whereas addition of 10 mumol/l ATP still led to a significant hyperpolarization. Omission of extracellular Ca2+ was followed by a strong decrease in cell membrane resistance (Rm) due to activation of a depolarizing cation conductance. Subsequent readdition of Ca2+ caused a marked increase in Ca2+i due to Ca2+ influx. This Ca2+ entry was further stimulated by cell swelling. RVD was significantly blunted in the absence of extracellular Ca2+. The results suggest that cell swelling stimulates a Ca2+-permeable pathway in the cell membrane favoring Ca2+ entry into the cell with subsequent activation of Ca2+-sensitive K+ 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.
BACKGROUND: The antiviral drugs AZT and acyclovir are generally used in the treatment of infections with human immunodeficiency virus (HIV) and herpes simplex virus (HSV). These substances are known to impede virus replication by premature nucleic acid chain termination. It is not yet clear, however, if this is the sole mechanism responsible for the antiviral and/or the numerous side effects observed in patients treated with these agents. We investigated the swelling-induced chloride current in fibroblasts, which we demonstrated is closely related or identical to a cloned epithelial chloride channel, ICln: This chloride channel can be blocked by nucleotides. MATERIALS AND METHODS: Electrophysiological, fluorescence optical, and volume measurements were made to determine the effect of nucleoside analogs on the swelling-dependent chloride current (ICl) in NIH 3T3 fibroblasts and in human T cell lymphoma (H9) cells and the cAMP-dependent chloride current in CaCo cells. RESULTS: AZT and acyclovir block the swelling-dependent chloride current and the chloride flux in fibroblasts, and the regulatory volume decrease (RVD) and ICl in H9 cells. This immediate effect can be substantially reduced by the simultaneous incubation of the cells with thymidine-5'-diphosphate (TDP) or uridine, both of which are by themselves unable to affect ICl. CONCLUSIONS: We show here a novel molecular mechanism by which antiviral drugs of the nucleoside analog family could lead to impairments of the kidney, bone marrow, gastrointestinal, and neuronal functions, and how these side effects could possibly be restricted by the presence of TDP or uridine.
Conventional electrophysiology and 2', 7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein fluorescence have been applied to elucidate the effects of metabolic acidosis on straight proximal tubules of the mouse kidney. Reduction of extracellular bicarbonate concentration from 20 to 10 mmol/l leads to a decline of intracellular pH from 7.00 +/- 0.06 to 6.85 +/- 0.05, a depolarization of the cell membrane (PDbl) from -72 +/- 1 to -59 +/- 2 mV, a decrease of the basolateral transference number for potassium (tK) from 0.80 +/- 0.01 to 0.54 +/- 0.03, an increase of the basolateral transference number for bicarbonate (tb) from 0.16 +/- 0.02 to 0.42 +/- 0.03 and an increase of the fractional resistance of the basolateral over the luminal cell membrane (Rb/Ra) by 64 +/- 8%. Upon return to 20 mmol/l bicarbonate after a 5-min exposure to 10 mmol/l bicarbonate, the intracellular pH approached a more alkaline value (7.28 +/- 0.08) than before exposure to acidosis. Despite the intracellular alkalosis, PDbl (-67 +/- 1 mV) and tK (0.73 +/- 0.02) remained significantly below, and tb (0.26 +/- 0.02) and Rb/Ra (32 +/- 8%) significantly above the respective values before induction of acidosis. Even transient exposure of the tubules to 40 mmol/l extracellular bicarbonate did not restore the original electrophysiological properties of the tubule cells. It is concluded that both a rapidly reversible and a long-lasting decrease of proximal tubular K+ conductance follows cellular acidosis.
Among the sequelae of ras oncogene expression are intracellular alkalinization and increase of cell volume, both phenomena attributed at least in part to activation of the Na+/H+ exchanger. The present study was performed to elucidate the effects of HOE 694--a novel inhibitor of the Na+/H+ exchanger--on intracellular pH, cell volume, cytoarchitecture and cell proliferation of ras oncogene expressing NIH 3T3 fibroblasts. Following transient exposure of the cells to 20 mmol/l NH3/NH4+, intracellular pH decreases sharply. The following slow realkalinization is completely blocked by 10 mumol/1 HOE 694. Half-maximal inhibition is achieved by 100 nmol/l HOE 694. Cell proliferation is inhibited by HOE 694 with similar potency, whereas the increase in cell volume and cytoskeletal transformation are not prevented, even by 10 mumol/l HOE 694.
In ras oncogene expressing cells, bradykinin leads to intracellular alkalinization by activation of the Na+/H+ exchanger. This effect is paralleled by oscillatory increase of intracellular calcium activity and cell shrinkage. Staurosporine (1 mumol/l) is not sufficient to prevent bradykinin induced intracellular alkalinization, thus pointing to a protein kinase C independent pathway for the activation of Na+/H+ exchange. The present study has been performed to elucidate, whether the increase of intracellular calcium contributes to cell shrinkage and activation of the Na+/H+ exchanger. To this end, the effects of the calcium ionophore ionomycin have been tested. Ionomycin leads to a dose dependent increase of intracellular calcium activity. At 100 nmol/l ionomycin intracellular calcium is increased from 114 +/- 17 nmol/l to 342 +/- 24 nmol/l (n = 9), a value within the range of intracellular calcium concentrations following application of bradykinin. The calcium increase is paralleled by a decrease of cell volume by 12 +/- 2% (n = 5) and an increase of intracellular pH from 6.78 +/- 0.02 to 6.90 +/- 0.03 (n = 11), values similar to those following application of bradykinin. The alkalinizing effect of ionomycin is completely abolished in the presence of the novel Na+/H+ exchange inhibitor HOE 694 (10 mumol/l), but is not inhibited by 1 mumol/l staurosporine. Inhibition of K+ and Cl- channels by barium (5 mmol/l) and ochratoxin-A (5 mumol/l) prevents both ionomycin induced cell shrinkage and protein kinase C independent intracellular alkalinization. It is concluded that bradykinin leads to intracellular alkalinization mainly by increasing intracellular calcium concentration. Calcium triggers calcium sensitive K+ channels, and presumably Cl- channels, the subsequent loss of cellular KCl leads to cell shrinkage which, in turn, activates Na+/H+ exchange.
Pteridines are heterocyclic compounds which are synthesized and released by human monocytes/macrophages following stimulation by interferon-gamma. Their concentration in various body fluids proved to be indicative for the stimulation of the cellular immune system, and determination of pteridines has become an important diagnostic tool. We show that pteridine derivatives, namely neopterin (N), 7,8-dihydroneopterin (NH2), and 5,6,7,8-tetrahydrobiopterin (BH4) increase intracellular calcium (Cai) in human monocytic cells. Significant increases of Cai are observed at 10 nmol/l NH2, at 100 nmol/l BH4 and at 1 mol/l N, i.e. at concentrations encountered in vivo. At a concentration of 1 mumol/l, Cai is increased (from a control value of 145 +/- 7 nmol/l) to 464 +/- 62 nmol/l (NH2), 340 +/- 41 nmol/l (BH4) and 344 +/- 46 nmol/l (N), respectively. The increase of Cai depends on the presence of extracellular calcium and is likely to be due to activation of a calcium channel. We show that the absence of extracellular calcium or the addition of lanthanum ions to the extracellular fluid fully reverses the pteridine-induced increase of Cai. According to these observations, pteridines may mimic the effects of other inflammatory mediators on monocytic cells and seem to be involved in the crosstalk of immunocompetent cells.