Roles of Volume-sensitive Cl<Superscript>−</Superscript> Channel in Cisplatin-induced Apoptosis in Human Epidermoid Cancer Cells

The anti-cancer drug cisplatin induces apoptosis by damaging DNA. Since a stilbene-derivative blocker of Cl⁻/HCO₃⁻ exchangers and Cl⁻ channels, SITS, is known to induce cisplatin resistance in a manner independent of intracellular pH and extracellular HCO₃⁻, we investigated the relation between cisplatin-induced apoptosis and Cl⁻ channel activity in human adenocarcinoma KB cells. A stilbene derivative, DIDS, reduced cisplatin-induced caspase-3 activation and cell death, which were detected over 18 h after treatment with cisplatin. DIDS was also found to reduce sensitivity of KB cells to 5-day exposure to cisplatin. Whole-cell patch-clamp recordings showed that KB cells functionally express volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels which are activated by osmotic cell swelling and sensitive to DIDS. Pretreatment of the cells with cisplatin for 12 h augmented the magnitude of VSOR Cl⁻ current. Thus, it is concluded that cisplatin-induced cytotoxicity in KB cells is associated with augmented activity of a DIDS-sensitive VSOR Cl⁻ channel and that blockade of this channel is, at least in part, responsible for cisplatin resistance induced by a stilbene derivative.     

Volume-sensitive Cl<Superscript>−</Superscript> Current in Bovine Adrenocortical Cells: Comparison with the ACTH-induced Cl<Superscript>−</Superscript> Current

In a previous study performed on zona fasciculata (ZF) cells isolated from calf adrenal glands, we identified an ACTH-induced Cl⁻ current involved in cell membrane depolarization. In the present work, we describe a volume-sensitive Cl⁻ current and compare it with the ACTH-activated Cl⁻ current. Experiments were performed using the whole-cell patch-clamp recording method, video microscopy and cortisol-secretion measurements. In current-clamp experiments, hypotonic solutions induced a membrane depolarization to −22 mV. This depolarization, correlated with an increase in the membrane conductance, was sensitive to different Cl⁻ channel inhibitors. In voltage-clamp experiments, hypotonic solution induced a membrane current that slowly decayed and reversed at −21 mV. This ionic current displayed no time dependence and showed a slight outward rectification. It was blocked to variable extent by different conventional Cl⁻-channel inhibitors. Under hypotonic conditions, membrane depolarizations were preceded by an increase in cell volume that was not detected under ACTH stimulation. It was concluded that hypotonic solution induced cell swelling, which activated a Cl⁻ current involved in membrane depolarization. Although cell volume change was not observed in the presence of ACTH, biophysical properties and pharmacological profile of the volume-sensitive Cl⁻ current present obvious similarities with the ACTH-activated Cl⁻ current. As compared to ACTH, hypotonic solutions failed to trigger cortisol production that was weakly stimulated in the presence of high-K⁺ solution. This shows that in ZF cells, membrane depolarization is not a sufficient condition to fully activate secretory activities.This revised version was published online in August 2005 with a corrected cover date.     

The role of Cl<Superscript>−</Superscript> in pollen germination and tube growth

The involvement of Cl^? in cytoplasm polarization in the pollen tube and membrane potential control during pollen germination in vitro was studied by fluorescence techniques in Nicotiana tabacum. Cl^? release from cells was blocked by the anion channel inhibitor nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or by the addition of Cl^? to the incubation medium. The concentrations of the inhibitor (40 μM) and extracellular Cl^? completely inhibiting pollen germination (200 mM) and pollen tube growth (100 mM) were used. The release of anions from the pollen grain has been revealed in the first minutes of hydration also in the presence of 200 mM Cl^?. The inhibitor blocked this process completely, which points to the significance of the NPPB-sensitive anion channels in the transmembrane Cl^? transport at the early activation stage. The pollen tube membrane was hyperpolarized in the presence of 100 mM Cl^?; however, exogenous Cl^? had no effect on the compartmentalization and organelle movement in the tube. The inhibitor depolarized the plasma membrane in the pollen grain and tube and affected the polar organization of the cytoplasm and organelle movement. Thus, activity of NPPB-sensitive chloride channels was required to regulate the potential on the plasma membrane and to maintain the functional compartmentalization of the cytoplasm, which provides for the polar growth.     

Transmembrane transport of K<Superscript>+</Superscript> and Cl<Superscript>−</Superscript> during pollen grain activation in vivo and in vitro

We studied the possibility of K⁺ and Cl⁻ efflux from tobacco pollen grains during their activation in vitro or on the stigma of a pistil. For this purpose the X-ray microanalysis and spectrofluorometry were applied. We found that the relative content of potassium and chlorine in the microvolume of pollen grain decreases during its hydration and activation on stigma. Efflux of these ions was found both in vivo and in vitro. In model in vitro experiments anion channel inhibitor NPPB ((5-nitro-2-(3-phenylpropylamino) benzoic acid) in the concentration that was blocking pollen germination, reduced Cl⁻ efflux; potassium channel inhibitor TEA (tetraethylammonium chloride) partially reduced K⁺ efflux and lowered the percent of activated cells. Another blocker of potassium channels Ba²⁺ caused severe decrease in cell volume and blocked the activation. In general, the obtained data demonstrates that the initiation of pollen germination both in vivo and in vitro involves the activation of K⁺ and Cl⁻ release. An important role in these processes is played by NPPB-, TEA- and Ba²⁺-sensitive plasmalemma ion channels.     

A Voltage-Dependent Ca<Superscript>2+</Superscript> Influx Pathway Regulates the Ca<Superscript>2+</Superscript>-Dependent Cl<Superscript>−</Superscript> Conductance of Renal IMCD-3 Cells

We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca²⁺-dependent Cl⁻ current (I_CLCA). Here we report that I_CLCA activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al³⁺-sensitive, voltage-dependent, Ca²⁺ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (−60 mV), I_CLCA was found to be inactive and resting currents were predominantly K⁺ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of I_CLCA (T _0.5 ~ 500 s), while repolarization in turn resulted in a monoexponential decay in I_CLCA (T _0.5 ~ 100 s). The activation of I_CLCA by depolarization was reduced by lowering extracellular Ca²⁺ and completely inhibited by buffering cytosolic Ca²⁺ with EGTA, suggesting a role for Ca²⁺ influx in the activation of I_CLCA. However, raising bulk cytosolic Ca²⁺ at −60 mV did not produce sustained I_CLCA activity. Therefore I_CLCA is dependent on both an increase in intracellular Ca²⁺ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca²⁺ influx pathway that displays equal permeability to Ca²⁺ and Ba²⁺ ions and that is blocked by extracellular Al³⁺ and La³⁺. Furthermore, Al³⁺ completely and reversibly inhibited depolarization-induced activation of I_CLCA, thereby directly linking Ca²⁺ influx to activation of I_CLCA. We speculate that during sustained membrane depolarization, calcium influx activates I_CLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.     

Changes in K<Superscript>+</Superscript>, Na<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> balance and K<Superscript>+</Superscript>, Cl<Superscript>−</Superscript> fluxes in U937 cells induced to apoptosis by staurosporine: On cell dehydration in apoptosis

The K⁺, Na⁺, and Cl⁻ balance and K⁺ (Rb⁺) and ³⁶Cl⁻ fluxes in U937 cells induced to apoptosis by 0.2 or 1 μM staurosporine were studied using flame emission and radioisotope techniques. It is found that two-thirds of the total decrease in the amount of intracellular osmolytes in apoptotic cells is accounted for by monovalent ions and one-third consists of other intracellular osmolytes. A decrease in the amount of monovalent ions results from a decrease in the amount of K⁺ and Cl⁻ and an increase in the Na⁺ content. The rate of ³⁶Cl⁻, Rb⁺ (K⁺), and ²²Na⁺ equilibration between cells and the medium was found to significantly exceed the rate of apoptotic change in the cellular ion content, which indicates that unidirectional influxes and effluxes during apoptosis may be considered as being in near balance. The drift of the ion flux balance in apoptosis caused by 0.2 μM staurosporine was found to be associated with the increased ouabain-resistant Rb⁺ (K⁺) channel influx and insignificantly altered the ouabain-sensitive pump influx. Severe apoptosis induced by 1 μM staurosporine is associated with reduced pump fluxes and slightly changed channel Rb⁺ (K⁺) fluxes. In apoptotic cells, the 1.4–1.8-fold decreased Cl⁻ level is accompanied by a 1.2–1.6-fold decreased flux.     

The role of Ca<Superscript>2+</Superscript>, H<Superscript>+</Superscript>, and Cl<Superscript>−</Superscript> ions in generation of variation potential in pumpkin plants

The contributions of Ca²⁺, H⁺, and Cl⁻ in generation of variation potentials (VP) in 3- to 4-week-old pumpkin (Cucurbita pepo L., cv. Mozoleevskaya) plants were assessed. During VP generation, transient alkalinization of the medium around the stem was recorded with a potentiometric method. The pH changes were kinetically similar to the electric potential changes and were apparently due to temporal suppression of the plasma-membrane electrogenic H⁺ pump. These data and the observed inhibition of VP in the stem zone treated locally with a metabolic inhibitor (NaN₃) indicate that the VP generation is related to the reversible suppression of the H⁺-pump. The anion channel blocker (ethacrynic acid) decelerated significantly the front slope of VP and reduced the VP amplitude. A short-term increase in external Cl⁻ concentration around the stem was observed during potential transients representing the VP front slope and the pulses integrated into VP. The removal of Ca²⁺ from extracellular medium inhibited the VP generation. It is proposed that Ca²⁺ plays a role in activation of anion channels and in the H⁺-pump inactivation. The VP generation is probably determined by a complex mechanism, with contributions from passive ion fluxes (Ca²⁺, Cl⁻) moving along the electrochemical gradients and from changes in the electrogenic pump activity.     

Monitoring Cl<Superscript>−</Superscript> Movement in Single Cells Exposed to Hypotonic Solution

Self-referencing ion - selective electrodes (ISEs), made with Chloride Ionophore I-Cocktail A (Fluka), were positioned 1–3 μm from human embryonic kidney cells (tsA201a) and used to record chloride flux during a sustained hyposmotic challenge. The ISE response was close to Nernstian when comparing potentials (V_N) measured in 100 and 10 mM NaCl (ΔV_N = 57 ± 2 mV), but was slightly greater than ideal when comparing 1 and 10 mm NaCl (ΔV_N = 70 ± 3 mV). The response was also linear in the presence of 1 mm glutamate, gluconate, or acetate, 10 μm tamoxifen, or 0.1, 1, or 10 mm HEPES at pH 7.0. The ISE was ∼3 orders of magnitude more selective for Cl⁻ over glutamate or gluconate but less than 2 orders of magnitude move selective for Cl⁻ over bicarbonate, acetate, citrate or thiosulfate. As a result this ISE is best described as an anion sensor. The ISE was ‘poisoned’ by 50 μm 5−nitro-2-(3phenylpropyl-amino)-benzoic acid (NPPB), but not by tamoxifen. An outward anion efflux was recorded from cells challenged with hypotonic (250 ± 5 mOsm) solution. The increase in efflux peaked 7–8 min before decreasing, consistent with regulatory volume decreases observed in separate experiments using a similar osmotic protocol. This anion efflux was blocked by 10 μm tamoxifen. These results establish the feasibility of using the modulation of electrochemical, anion-selective, electrodes to monitor anions and, in this case, chloride movement during volume regulatory events. The approach provides a real-time measure of anion movement during regulated volume decrease at the single-cell level.     

Control of Epithelial Ion Transport by Cl<Superscript>−</Superscript> and PDZ Proteins

Inhibition of epithelial Na⁺ channels (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated previously. Recent studies suggested a role of cytosolic Cl⁻ for the interaction of CFTR with ENaC, when studied in Xenopus oocytes. In the present study we demonstrate that the Na⁺/H⁺-exchanger regulator factor (NHERF) controls expression of CFTR in mouse collecting duct cells. Inhibition of NHERF largely attenuates CFTR expression, which is paralleled by enhanced Ca²⁺-dependent Cl⁻ secretion and augmented Na⁺ absorption by the ENaC. It is further demonstrated that epithelial Na⁺ absorption and ENaC are inhibited by cytosolic Cl⁻ and that stimulation by secretagogues enhances the intracellular Cl⁻ concentration. Thus, the data provide a clue to the question, how epithelial cells can operate as both absorptive and secretory units: Increase in intracellular Cl⁻ during activation of secretion will inhibit ENaC and switch epithelial transport from salt absorption to Cl⁻ secretion.This revised version was published online in August 2005 with a corrected cover date.     

Modulation of Na<Superscript>+</Superscript>/Mg<Superscript>2+</Superscript> exchanger stoichiometry ratio by Cl<Superscript>−</Superscript> ions in basolateral rat liver plasma membrane vesicles

The Na⁺/Mg²⁺ exchanger represents the main Mg²⁺ extrusion mechanism operating in mammalian cells including hepatocytes. We have previously reported that this exchanger, located in the basolateral domain of the hepatocyte, promotes the extrusion of intravesicular trapped Mg²⁺ for extravesicular Na⁺ with ratio 1. This electrogenic exchange is supported by the accumulation of tetraphenyl-phosphonium within the vesicles at the time when Mg²⁺ efflux occurs. In this present study, the role of extra- and intra-vesicular Cl^? on the Na⁺/Mg²⁺ exchange ratio was investigated. The results reported here suggest that Cl^? ions are not required for the Na⁺ to Mg²⁺ exchange to occur, but the stoichiometry ratio of the exchanger switches from electrogenic (1Na _in ⁺ :1 Mg _out ²⁺ ) in the presence of intravesicular Cl^? to electroneutral (2Na _in ⁺ :1 Mg _out ²⁺ ) in their absence. In basolateral liver plasma membrane vesicles loaded with MgCl₂ labeled with ³⁶Cl^?, a small but significant Cl^? efflux (~30 nmol Cl^?/mg protein/1 min) is observed following addition of NaCl or Na-isethionate to the extravesicular medium. Both Cl^? and Mg²⁺ effluxes are inhibited by imipramine but not by amiloride, DIDS, niflumic acid, bumetanide, or furosemide. In vesicles loaded with Mg-gluconate and stimulated by Na-isethionate, an electroneutral Mg²⁺ extrusion is observed. Taken together, these results suggest that the Na⁺/Mg²⁺ exchanger can operate irrespective of the absence or the presence of Cl^? in the extracellular or intracellular environment. Changes in trans-cellular Cl^? content, however, can affect the modus operandi of the Na⁺/Mg²⁺ exchanger, and consequently impact "cellular" Na⁺ and Mg²⁺ homeostasis as well as the hepatocyte membrane potential.     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.     

Molecular Weight and Subunit Composition of the Sensitive to GABA-Ergic Ligands Cl<Superscript>−</Superscript>/HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>-Stimulated Mg<Superscript>2+</Superscript>-ATPase from Plasma Membrane of Rat Brain

The molecular weight and subunit composition of Cl-,HCO3(-)- and picrotoxin-stimulated Mg2+-ATPase from rat brain plasma membrane solubilized in sodium deoxycholate were studied by gel filtration chromatography. The enzyme activity eluted from a Sephacryl S-300 column in a single peak associated with a protein of molecular weight approximately 300 kD and a Stokes radius of 5.4 nm. The enzyme-enriched fraction, concentrated and denatured by SDS, migrated through a Sephacryl S-200 column as three peaks with molecular weights of approximately 57, 53, and 45 kD. SDS-PAGE also showed three major protein bands with molecular weights of about 57, 53, and 48 kD. The molecular weight and subunit composition of the Cl- and HCO3(-)-stimulated Mg2+-ATPase from neuronal membrane of rat brain are similar with the molecular properties of GABA(A)-benzodiazepine receptor complex from mammalian brain but are different from those of P-type transport ATPases.     

Anionic Selectivity Sequence of the Cl<Superscript>−</Superscript>–H<Superscript>+</Superscript> Symporter in the Synaptosomal Preparation from Rat Brain Cortex

The Na⁺/H⁺ exchanger has been the only unequivocally demonstrated H⁺-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl⁻–H⁺ symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl⁻–H⁺ symporter is NO₃⁻ > Br⁻ > Cl⁻ >> I⁻ = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 μM inhibits the gluconate-dependent alkalinization by 30 ± 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization. Special issue article in honor of Dr. Ricardo Tapia.     

The Role of K<Superscript>+</Superscript>-Cl<Superscript>−</Superscript>-Cotransporter-2 in Neuropathic Pain

The pain sensory system normally functions under a fine balance between excitation and inhibition. When this balance is perturbed for some reason, it leads to neuropathic pain. There is accumulating evidence that attributes this pain generation to specific dysfunctions of the inhibitory system in the spinal cord. One possible mechanism leading to the induction of these dysfunctions is the down-regulation of K⁺-Cl^?-cotransporter-2 (KCC2) expression. In fact, various neuropathic pain models indicate a decrease of KCC2 expression in the spinal cord. The alteration of KCC2 expression affects GABAergic and glycinergic neurotransmissions, because KCC2 is a potassium-chloride exporter and serves to maintain intracellular chloride concentration. When there is a low level of KCC2 expression, GABAergic and glycinergic neurotransmissions transform from inhibitory signals to excitatory signals. In this review, the hypothesis that an alteration of KCC2 expression has a crucial influence on the initiation/development or maintenance of neuropathic pain is discussed. In addition, it is suggested that the alteration of inhibitory signals is dependent on the time after peripheral nerve injury.     

Calculation of Na<Superscript>+</Superscript>/Cl<Superscript>−</Superscript> Selectivity in Tight Junctions between Epithelial Cells by Methods of Nonlocal Electrostatics with Pole Models of Dielectric Function without an Overscreening Effect

A mathematical model of Na⁺/Cl^? selectivity in tight junctions (TJs) between epithelial cells was developed. It was demonstrated that Na⁺/Cl^? selectivity in TJs depends on the total charge of amino-acid residues of claudin macromolecules within TJs, as well as on the ion-distribution coefficients between TJs and free solution. It was demonstrated that the obtained formulas predict a change of Na⁺/Cl^? selectivity in TJs for Cl^?/Na⁺ selectivity if the sign of the total charge of amino-acid residues in TJ changes. The calculated Na⁺/Cl^? selectivity value for MDCK cells coincides with the experimental data of (Colegio et al., 2002). To calculate a change in ion-solvation energy during their transition to TJs, formulas for nonlocal electrostatics with one-, two-, and three-pole models of dielectric function without taking into account the overscreening effect were used.     

PsbP-induced protein conformational changes around Cl<Superscript>−</Superscript> ions in the water oxidizing center of photosystem II

PsbP is an extrinsic protein of PSII having a function of Ca²⁺ and Cl^? retention in the water-oxidizing center (WOC). In order to understand the mechanism how PsbP regulates the Cl^? binding in WOC, we examined the effect of PsbP depletion on the protein structures around the Cl^? sites using Fourier transform infrared (FTIR) spectroscopy. Light-induced FTIR difference spectra upon the S¹→S₂ transition were obtained using Cl^?-bound and NO₃^?-substituted PSII membranes in the presence and absence of PsbP. A clear difference in the amide I band changes by PsbP depletion was observed between Cl^?-bound and NO₃^?-substituted PSII samples, indicating that PsbP binding perturbed the protein conformations around the Cl^?ion(s) in WOC. It is suggested that PsbP stabilizes the Cl^? binding by regulating the dissociation constant of Cl^? and/or an energy barrier of Cl^? dissociation through protein conformational changes around the Cl^? ion(s).     

Comparative properties of sensitive to GABA<Subscript>A</Subscript>-ergic ligands Cl<Superscript>−</Superscript>, HCO<Subscript>3</Subscript><Superscript>−</Superscript>-activated Mg<Superscript>2+</Superscript>-ATPase from brain plasma membranes of fish and rats

Action of Cl^? + HCO₃ ^?1 ions on Mg²⁺-ATPase from brain plasma membranes of fish and rats has been studied. Maximal effect of the anions on the “basal” Mg²⁺-ATPase activity is revealed in the presence of 10 mM Cl^? and 3 mM HCO₃ ^?1 at physiological values of pH of incubation medium. The studied Cl^?, HCO₃ ^?-activated Mg²⁺-ATPases of both animal species, by their sensitivity to SH-reagents (5,5-dithio-bis-nitrobenzoic acid, N-ethylmaleimide), oligomycin, and orthovanadate, are similar to transport ATPase of the P-type, but differ from them by molecular properties and by sensitivity to ligands of GABA_A-receptors. It has been established that the sensitive to GABA_A-ergic ligands, Cl^?, HCO₃ ^?-activated Mg²⁺-ATPase from brain of the both animal species is protein of molecular mass around 300 kDa and of Stock’s radius 5.4 nm. In fish the enzyme is composed of one major unit of molecular mass approximately 56 kDa, while in rats-of three subunits of molecular masses about 57, 53, and 45 kDa. A functional and structural coupling of the ATP-hydrolyzing areas of the studied enzyme to sites of binding of GABA_A-receptor ligands is suggested.     

Role of unusual CD4<Superscript>+</Superscript> CD28<Superscript>−</Superscript> T cells in acute coronary syndrome

Acute coronary syndrome (ACS) is a group of clinical symptoms that results from complete or partial occlusive thrombus, which is caused by coronary an atherosclerotic plaque rupture or erosion. According to a recent study, CD4⁺ CD28⁻ T cells are found in atherosclerotic plaques and the peripheral circulation blood in patients with ACS, these cells play an important role in plaque ruptures. CD4⁺ CD28⁻ T cells are an unusual subset of helper cells, which expand and have harmful effects in ACS. In this review, we discuss the current issues on the generation of CD4⁺ CD28⁻ T cells and focus on their phenotypic and functional characteristics relevant to the development of cardiovascular events. Targeting the CD4⁺ CD28⁻ T cells subset in ACS could provide novel therapeutic means to prevent acute life-threatening coronary events.     

Estimation of denitrification potential in a karst aquifer using the <Superscript>15</Superscript>N and <Superscript>18</Superscript>O isotopes of NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>

A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr²⁺ and Cl^– and concentrations of stable isotope ¹⁸O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of ¹⁵N and ¹⁸O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in ¹⁵N and ¹⁸O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems.