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.     

γ<Subscript>1</Subscript>-Dependent Down-regulation of Recombinant Voltage-gated Ca<Superscript>2+</Superscript> Channels

(1) Voltage-gated Ca²⁺ (Ca_V) channels are multi-subunit membrane complexes that allow depolarization-induced Ca²⁺ influx into cells. The skeletal muscle L-type Ca_V channels consist of an ion-conducting Ca_V1.1 subunit and auxiliary α₂δ−1, β₁ and γ₁ subunits. This complex serves both as a Ca_V channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α₂δ−1 and β₁ subunits regulate Ca_V channel function, there is far less information on the γ₁ subunit. Previously, we characterized the interaction of γ₁ with the other components of the skeletal Ca_V channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca²⁺ current density in myotubes from γ₁ null mice. (3) In the current report, using Western blotting we show that the expression of the Ca_V1.1 protein is significantly lower when it is heterologously co-expressed with γ₁. Consistent with this, patch-clamp recordings showed that transient transfection of γ₁ drastically inhibited macroscopic currents through recombinant N-type (Ca_V2.2/α₂δ−1/β₃) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ₁ subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca²⁺ current density following γ₁ transfection.     

PA1b,a plant peptide,induces intracellular [Ca<Superscript>2+</Superscript>] increase via Ca<Superscript>2+</Superscript> influx through the L-type Ca<Superscript>2+</Superscript> channel and triggers secretion in pancreatic β cells

Using alginic acid to adsorb polypeptides at pH 2.7, we isolated a peptide pea albumin 1b (PA1b) from pea seeds. The PA1b is a single chain peptide consisting of 37 amino acid residues with 6 cysteines which constitutes the cystine-knot structure. Using microfluorometry and patch clamp techniques, we found that PA1b significantly elevated the intracellular calcium level ([Ca²⁺]_i) and elicited membrane capacitance increase in the primary rat pancreatic β cells. The PA1b effect on [Ca²⁺]_i elevation was abolished in the absence of extracellular Ca²⁺ or in the presence of L-type Ca²⁺ channel blocker, nimodipine. Interestingly, we found that PA1b significantly depolarized membrane potential, which could lead to the opening of voltage-dependent L-type Ca²⁺ channels and influx of extracellular Ca²⁺, and then evoke robust secretion. In this study we identified the plant peptide PA1b which is capable of affecting the excitability and function of mammalian pancreatic β cell.     

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.     

Investigation of the encapsulation of metal cations (Cu<Superscript>2+</Superscript>, Zn<Superscript>2+</Superscript>, Ca<Superscript>2+</Superscript> and Ba<Superscript>2+</Superscript>) by the dipeptide Phe–Phe using natural bond orbital theory and molecular dynamics simulation

Complexes of the dipeptide phenylalanine–phenylalanine (Phe–Phe) with divalent metal cations (Cu²⁺, Zn²⁺, Ca²⁺ and Ba²⁺) were studied at the B3LYP and MP2 levels of theory with the basis sets 6-311++G(d,p) and 6-31 + G(d) in the gas phase. The relative energies of these complexes indicated that cation–π bidentate/tridentate conformations are more favourable than other conformations with uncoordinated rings. These findings were confirmed by the calculated values of thermodynamic parameters such as the Gibbs free energy. Natural bond orbital (NBO) analysis was carried out to explore the metal–ligand coordination in Phe–Phe–Cu²⁺/Zn²⁺ complexes. Possible orbital transitions, types of orbitals and their occupancies were determined for a range of Phe–Phe–Cu²⁺/Zn²⁺ complexes. The charge transfer involved in various orbital transitions was explored by considering the second-order perturbation energy. NBO analysis revealed that the change transfer is stronger when the metal cation uses both the 4s + 4p subshells rather than just its 4p subshell. We also performed molecular dynamics (MD) simulations to check the stability and consistency of the most favourable binding motifs of Cu²⁺, Zn²⁺, Ca²⁺ and Ba²⁺ with Phe–Phe over time. The structures of the Phe–Phe–Cu²⁺/Zn²⁺/Ca²⁺/Ba²⁺ complexes obtained using MD simulation were found to be in good agreement with those obtained in the DFT-based calculations.

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Graphical Abstract Conformational search on encapsulation of divalent metal cations (Ca²⁺, Zn²⁺, Ca²⁺, Ba²⁺) by the Phe-Phe dipeptide

     

β<Subscript>2</Subscript>-Integrin-Mediated Adhesion and Intracellular Ca<Superscript>2+</Superscript> Release in Human Eosinophils

Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO₃-buffered HybriCare medium maintained in a humidified 5% CO₂ incubator at 37°C. Impedance increased by more than 1 kΩ within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca²⁺] that precedes adhesion with BAPTA-AM (10 μM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca²⁺] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with β₂-integrins, or jasplakinolide (2 μM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 μM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca²⁺] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.     

Effect of different methods of Ca<Superscript>2+</Superscript> extraction from PSII oxygen-evolving complex on the Q<Subscript>A</Subscript><Superscript>−</Superscript> oxidation kinetics

Lumenal extrinsic proteins PsbO, PsbP, and PsbQ of photosystem II (PSII) protect the catalytic cluster Mn₄CaO₅ of oxygen-evolving complex (OEC) from the bulk solution and from soluble compounds in the surrounding medium. Extraction of PsbP and PsbQ proteins by NaCl-washing together with chelator EGTA is followed also by the depletion of Ca²⁺ cation from OEC. In this study, the effects of PsbP and PsbQ proteins, as well as Ca²⁺ extraction from OEC on the kinetics of the reduced primary electron acceptor (Q_A ^?) oxidation, have been studied by fluorescence decay kinetics measurements in PSII membrane fragments. We found that in addition to the impairment of OEC, removal of PsbP and PsbQ significantly slows the rate of electron transfer from Q_A ^? to the secondary quinone acceptor Q_B. Electron transfer from Q_A ^? to Q_B in photosystem II membranes with an occupied Q_B site was slowed down by a factor of 8. However, addition of EGTA or CaCl₂ to NaCl-washed PSII did not change the kinetics of fluorescence decay. Moreover, the kinetics of Q_A ^? oxidation by Q_B in Ca-depleted PSII membranes obtained by treatment with citrate buffer at pH 3.0 (such treatment keeps all extrinsic proteins in PSII but extracts Ca²⁺ from OEC) was not changed. The results obtained indicate that the effect of NaCl-washing on the Q_A ^? to Q_B electron transport is due to PsbP and PsbQ extrinsic proteins extraction, but not due to Ca²⁺ depletion.     

Ca<Superscript><Emphasis Type="Italic">2+</Emphasis></Superscript>-Induced Cl<Superscript>−</Superscript> Efflux at Rat Distal Colonic Epithelium

With the aid of the halide-sensitive dye 6-methoxy-N-ethylquinolinium iodide (MEQ), changes in intracellular Cl^- concentration were measured to characterize the role of Ca²⁺-dependent Cl^- channels at the rat distal colon. In order to avoid indirect effects of secretagogues mediated by changes in the driving force for Cl^- exit (i.e., mediated by opening of Ca²⁺-dependent K⁺ channels), all experiments were performed under depolarized conditions, i.e., in the presence of high extracellular K⁺ concentrations. The Ca²⁺-dependent secretagogue carbachol induced a stilbene-sensitive Cl^- efflux, which was mimicked by the Ca²⁺ ionophore ionomycin. Surprisingly, the activation of Ca²⁺-dependent Cl^- efflux was resistant against blockers of classical Ca²⁺ signaling pathways such as phospholipase C, protein kinase C and calmodulin. Hence, alternative pathways must be involved in the signaling cascade. One possible signaling molecule seems to be nitric oxide (NO) as the NO donor sodium nitroprusside could induce Cl^- efflux. Vice versa, the NO synthase inhibitor N-ω-monomethyl-arginine (l-NMMA) reduced the carbachol-induced Cl^- efflux. This indicates that NO may be involved in part of the signaling cascade. In order to test the ability of the epithelium to produce NO, the expression of different isoforms of NO synthase was verified by immunohistochemistry. In addition, the cytoskeleton seems to play a role in the activation of Ca²⁺-dependent Cl^- channels. Inhibitors of microtubule association such as nocodazole and colchicine as well as jasplakinolide, a drug that enhances actin polymerization, inhibited the carbachol-induced Cl^- efflux. Consequently, the activation of apical Cl^- channels by muscarinic receptor stimulation differs in signal transduction from the classical phospholipase C/protein kinase C way.     

The involvement of Ca<Superscript>2+</Superscript> gradients,Ca<Superscript>2+</Superscript> fluxes,and CaM kinase II in polarization and germination of<Emphasis Type="Italic"> Silvetia compressa</Emphasis> zygotes

Previous work has shown that distinct Ca²⁺ gradients precede and predict the loci of germination of the zygotes of the brown alga, Silvetia compressa (J. Agardh) E. Serrão, T.O. Cho, S.M. Boo et Brawley, that are polarized by unilateral blue light. We show here that dark-grown S. compressa zygotes also form cytosolic Ca²⁺ gradients prior to germination and then germinate from the site of elevated Ca²⁺. In no case did germination occur without a prior formation of a Ca²⁺ gradient. Using the self-referencing Ca²⁺-selective probe, we measured highly localized influx of Ca²⁺ during photopolarization, indicating that extracellular stores supply at least some of the Ca²⁺ needed to construct a gradient. Finally, we find that germination was inhibited by a bath-applied inhibitor of calcium/calmodulin-dependent kinase II (CaM kinase II), KN-93 (but not by its inactive analog, KN-92), and by an injected inhibitory peptide for the kinase. KN-93 did not interfere with the photopolarization of the zygotes, consistent with the view that calmodulin is not involved in the initial response to light. The KN-93 results indicate that the requirement for active CaM kinase II for germination ends about 2 h before overt germination. We conclude that Ca²⁺ gradients, generated in part by localized calcium entry from the seawater, are an essential part of the process of polarity development and expression in these cells, regardless of the nature of the external cue that directs the orientation of the axis. Calmodulin and CaM kinase II are involved in interpreting (but not in establishing) the calcium gradient, allowing germination to occur at the site of elevated calcium, but CaM kinase II appears not to be involved in the initiation of germination.     

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.     

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.     

The Protective Effect of Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channel Opener,Nicorandil, Combined With Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> Exchange Blocker KB-R7943 on Myocardial Ischemia–Reperfusion Injury in Rat

To study the protective effect of mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP channel) opener, nicorandil, combined with Na⁺/Ca²⁺ exchange blocker KB-R7943 on myocardial ischemia–reperfusion injury in isolated rat hearts; the isolated rat heart was perfused by modified Langendorff device, after 15-min balanced perfusion, 45-min ischemia (about left and right coronary perfusion flow reduced to 5% of the original irrigation flow), and 2-h reperfusion were performed. Forty Wistar rats were randomly divided into four groups: control group, nicorandil group, KB-R7943 group, and the combination of nicorandil and KB-R7943 group. After 45-min ischemia and then 2-h reperfusion, the myocardial infarct size was 34.31% in control group, 26.35% in nicorandil group, 28.74% in KB-R7943 group, and 19.23% in combination of nicorandil and KB-R7943 group. SOD activity in coronary perfusion fluid was the highest in the combination of nicorandil and KB-R7943 group, and MDA content was the lowest. In the combination drug group compared with the control group, myocardial ultrastructural injury was significantly reduced. The combination of nicorandil and KB-R7943 significantly reduced myocardial infarct size, significantly reduced myocardial ultrastructural damage, could increase coronary perfusion fluid SOD activity, and reduced MDA levels.     

The 1.3 isoform of Na<Superscript>+</Superscript>–Ca<Superscript>2+</Superscript> exchanger expressed in guinea pig tracheal smooth muscle is less sensitive to KB-R7943

The sodium–calcium exchanger (NCX) plays a major role in the regulation of cytosolic Ca²⁺ in muscle cells. In this work, we performed force experiments to explore the role of NCX during contraction and relaxation of Cch-stimulated guinea pig tracheal smooth muscle strips. This tissue showed low sensitivity to NCX inhibitor KB-R7943 (IC50, 57 ± 2 μM), although a complete relaxation was obtained by NCX inhibition at 100 μM. Interestingly, relaxation after washing the agonist was prolonged in the absence of external Na⁺, whereas washing without Na⁺ and in the presence of KB-R7943 resembled control conditions with physiological solution. Altogether, this suggests the reversal of NCX to a Ca²⁺ influx mode by the manipulation on the Na⁺ gradient, which can be inhibited by KB-R7943. In order to understand the low sensitivity to KB-R7943, we studied the molecular aspects of the NCX expressed in this tissue and found that the isoform of NCX expressed is 1.3, similar to that described in human tracheal smooth muscle. Sequencing revealed that amino acid 19 in exon B is phenylalanine, whereas in its human counterpart is leucine, and that the first amino acid after exon D is aspartate instead of glutamate in humans. Results herein presented are discussed in term of their possible functional implications in the exchanger activity and thus in airway physiology.     

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.     

<Superscript>1</Superscript>H–<Superscript>15</Superscript>N correlation spectroscopy of nanocrystalline proteins

The limits of resolution that can be obtained in ¹H–¹⁵N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide ¹H resonances. Heteronuclear decoupling of ¹⁵N from the ¹H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly ²H and ¹⁵N enriched protein where the amides have been exchanged in normal water, MAS at 20 kHz, and WALTZ-16 decoupling of the ¹⁵N nuclei. The combination of these techniques results in average ¹H lines of only 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and ¹⁵N decoupling are described for achieving the best possible performance in these experiments.     

Flow of Deposited Inorganic N in Two Gleysol-dominated Mountain Catchments Traced with <Superscript>15</Superscript>NO<Subscript>3</Subscript><Superscript>−</Superscript> and <Superscript>15</Superscript>NH<Subscript>4</Subscript><Superscript>+</Superscript>

In two mountain ecosystems at the Alptal research site in central Switzerland, pulses of ¹⁵NO₃ and ¹⁵NH₄ were separately applied to trace deposited inorganic N. One forested and one litter meadow catchment, each approximately 1600 m², were delimited by trenches in the Gleysols. K¹⁵NO₃ was applied weekly or fortnightly over one year with a backpack sprayer, thus labelling the atmospheric nitrate deposition. After the sampling and a one-year break, ¹⁵NH₄Cl was applied as a second one-year pulse, followed by a second sampling campaign. Trees (needles, branches and bole wood), ground vegetation, litter layer and soil (LF, A and B horizon) were sampled at the end of each labelling period. Extractable inorganic N, microbial N, and immobilised soil N were analysed in the LF and A horizons. During the whole labelling period, the runoff water was sampled as well. Most of the added tracer remained in both ecosystems. More NO₃⁻ than NH₄⁺ tracer was retained, especially in the forest. The highest recovery was in the soil, mainly in the organic horizon, and in the ground vegetation, especially in the mosses. Event-based runoff analyses showed an immediate response of ¹⁵NO₃⁻ in runoff, with sharp ¹⁵N peaks corresponding to discharge peaks. NO₃⁻ leaching showed a clear seasonal pattern, being highest in spring during snowmelt. The high capacity of N retention in these ecosystems leads to the assumption that deposited N accumulates in the soil organic matter, causing a progressive decline of its C:N ratio.     

Ca<Superscript>2+</Superscript>-CaMKKβ pathway is required for adiponectin-induced secretion in rat submandibular gland

Adiponectin functions as a promoter of saliva secretion in rat submandibular gland via activation of adenosine monophosphate-activated protein kinase (AMPK) and increased paracellular permeability. Ca²⁺ mobilization is the primary signal for fluid secretion in salivary acinar cells. However, whether intracellular Ca²⁺ mobilization is involved in adiponectin-induced salivary secretion is unknown. Here, we found that full-length adiponectin (fAd) increased intracellular Ca²⁺ and saliva secretion in submandibular glands. Pre-perfusion with ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) combined with thapsigargin (TG), an endoplasmic reticulum Ca²⁺-ATPase inhibitor, abolished fAd-induced salivary secretion, AMPK phosphorylation, and enlarged tight junction (TJ) width. Furthermore, in cultured SMG-C6 cells, co-pretreatment with EGTA and TG suppressed fAd-decreased transepithelial electrical resistance and increased 4-kDa FITC-dextran flux responses. Moreover, fAd increased phosphorylation of calcium/calmodulin-dependent protein kinase (CaMKKβ), a major kinase that is activated by elevated levels of intracellular Ca²⁺, but not liver kinase B1 phosphorylation. Pre-perfusion of the isolated gland with STO-609, an inhibitor of CaMKKβ, abolished fAd-induced salivary secretion, AMPK activation, and enlarged TJ width. CaMKKβ shRNA suppressed, whereas CaMKKβ re-expression rescued fAd-increased paracellular permeability. Taken together, these results indicate that adiponectin induced Ca²⁺ modulation in rat submandibular gland acinar cells. Ca²⁺-CaMKKβ pathway is required for adiponectin-induced secretion through mediating AMPK activation and increase in paracellular permeability in rat submandibular glands.     

Methyl-β-cyclodextrin inhibits Ca<Superscript>2+</Superscript>-responses induced by glutoxim and molixan in macrophages

Using Fura-2AM microfluorimetry, we have shown for the first time that methyl-β-cyclodextrin, inducing cholesterol extraction from membranes and raft disruption, significantly inhibits glutoxim- and molixan-induced Ca²⁺-responses in rat peritoneal macrophages. The results suggest that intact rafts are necessary for signaling cascade induced by glutoxim or molixan and leading to intracellular Ca²⁺ concentration increase in macrophages.