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. 相似文献
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. 相似文献
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 (1Nain+:1 Mgout2+) in the presence of intravesicular Cl? to electroneutral (2Nain+:1 Mgout2+) 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. 相似文献
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 (ICLCA). Here we report that ICLCA 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), ICLCA 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 ICLCA (T0.5 ~ 500 s), while repolarization in turn resulted in a monoexponential decay in ICLCA (T0.5 ~ 100 s). The activation of ICLCA 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 ICLCA. However, raising bulk cytosolic Ca2+ at −60 mV did not produce sustained ICLCA activity. Therefore ICLCA 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 ICLCA, thereby directly linking Ca2+ influx to activation of ICLCA. We speculate that during sustained membrane depolarization, calcium influx activates ICLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells. 相似文献
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. 相似文献
Using vesicles of symbiosome membrane (SM), it was shown that the Ca2+-ATPase can function as an ATP-energized Ca2+/H+ antiporter. The initial rate of the acidic shift inside the vesicles, as well as the rate of the ITP-dependent alkalization of the medium inside them markedly increased in the presence of valinomycin. This process was rapidly stopped by eosin Y, a known inhibitor of the type IIB Ca2+-ATPase. ITP-dependent uptake of Ca2+ was blocked after the addition to the reaction mixture of nigericin in the presence of K+. Under these conditions, the alkaline shift of pH inside the vesicles occurred, leading to the inhibition of operation of the calcium pump in SM. Evaluation of the pH shifts inside the vesicles by using pH-indicator pyranine confirmed the ion-exchange mechanism of the Ca2+-ATPase functioning in the SM. 相似文献
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. 相似文献
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. 相似文献
A model of the HK2a subunit of the rabbit colonic H+, K+
ATPase has been generated using the crystal structure of the Ca+2 ATPase as a template. A pairwise sequence alignment of the deduced primary sequences of the two proteins demonstrated that they share 29% amino acid sequence identity and 47% similarity. Using O (version 7) the model of HK2a was constructed by interactively mutating, deleting, and inserting the amino acids that differed between the pairwise sequence alignment of the Ca+2 ATPase and HK2a. Insertions and deletions in the HK2a sequence occur in apparent extra-membraneous loop regions. The HK2a model was energy minimized and globally refined to a level comparable to that of the Ca+2 ATPase structure using CNS. The charge distribution over the surface of HK2a was evaluated in GRASP and possible secondary structure elements of HK2a were visualized in BOBSCRIPT. Conservation and placement of residues that may be involved in ouabain binding by the H+, K+
ATPase were considered and a putative location for the subunit was postulated within the structure.Figure Possible architecture of the HK2a subunit. The residue in green is the lysine (position 517, Fig. 1) that lies in the nucleotide binding pocket and the residue in red is the aspartic acid at the phosphorylation site (position 385). Based on an alignment with the Ca+2 ATPase, ten transmembrane helices were modeled into HK2a. The ten transmembrane helices are drawn as rods and shown in different colors for clarity. From left to right, the transmembrane helix designations are M10 (blue), M7 (gray), M8 (purple), M9 (orange), M5 (pink), M6 (green), M3 (brown), M4 (cyan), M2 (teal), and M1 (almond). 相似文献
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. 相似文献
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. 相似文献
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.
We investigate the electrophysiological salt stress response of the salt-sensitive charophyte Chara australis as a function of time in saline artificial pond water (saline APW) containing 50 mM NaCl and 0.1 mM CaCl2. The effects are due to an increase in Na+ concentration rather than an increase in Cl− concentration or medium osmolarity. A previous paper (Shepherd et al. Plant Cell Environ 31:1575–1591, 2008) described the
rise in the background conductance and inhibition of proton pumping in saline APW in the first 60 min. Here we investigate
the shift of membrane potential difference (PD) to levels above −100 mV and the change of shape of the current–voltage (I/V)
profiles to upwardly concave. Arguing from thermodynamics, the I/V characteristics can be modeled by channels that conduct
H+ or OH−. OH− was chosen, as H+ required an unrealistic increase in the number/permeability of the channels at higher pH levels. Prolonged exposure to saline
APW stimulated opening of more OH− channels. Recovery was still possible even at a PD near −50 mV, with partial return of proton pumping and a decrease in OH− current following APW wash. Upon change of pH from 7 to 9, the response was consistent with previously observed I/V characteristics
of OH− channels. For a pH change to 6, the response was transient before channel closure but could still be modeled. The consequences
of opening of H+ or OH− channels while the cell is under salt stress are discussed. 相似文献
Leptin, a 16-kDa cytokine produced mainly by the adipose tissue, is known to increase energy expenditure while at the same
time lowering food intake by acting directly on the hypothalamus. ObRb, the leptin receptor mostly involved in intracellular
signaling, is expressed in a wide range of tissues, thus allowing leptin to affect a much broader diversity of biological
processes. High concentrations of leptin are encountered in patients with hyperleptinemia, a condition which very often accompanies
obesity and which is a direct result of leptin resistance. In the present study, moderate and high concentrations of leptin
(16 and 160 ng/ml) were mostly utilized in order to investigate the role of this cytokine in oxidative stress levels in human
monocytes. Leptin was found to increase oxidative species production as measured with 2′,7′-dichlorodihydrofluorescein diacetate
(general marker of oxidative species, but not O2−.) and dihydroethidium (marker of O2−.). Surprisingly, it also augmented superoxide dismutase activity. Inhibition of the Na+–H+ exchanger isoform 1 (NHE1) also inhibited leptin-induced superoxide anion production but at the same time amplified leptin-induced
production of other oxidative species. Signaling proteins such as phosphoinositide 3 kinase and conventional isoforms of protein
kinase C (α-, βi-, βii-), as well as NADPH oxidase, also participated in leptin signaling. Finally, leptin was found to increase glutathionylation
levels of NHE1-bound heat shock protein 70 kDa (Hsp70) but not Hsp70 binding to NHE1. 相似文献
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 GABAA-receptors. It has been established that the sensitive to GABAA-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 GABAA-receptor ligands is suggested. 相似文献
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. 相似文献
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. 相似文献
To understand salt stress, the full impact of salinity on plant cell physiology has to be resolved. Electrical measurements suggest that salinity inhibits the proton pump and opens putative H+/OH? channels all over the cell surface of salt sensitive Chara australis (Beilby and Al Khazaaly 2009; Al Khazaaly and Beilby 2012). The channels open transiently at first, causing a characteristic noise in membrane potential difference (PD), and after longer exposure remain open with a typical current-voltage (I/V) profile, both abolished by the addition of 1 mM ZnCl2, the main known blocker of animal H+ channels. The cells were imaged with confocal microscopy, using fluorescein isothiocyanate (FITC) coupled to dextran 70 to illuminate the pH changes outside the cell wall in artificial fresh water (AFW) and in saline medium. In the early saline exposure, we observed alkaline patches (bright fluorescent spots) appearing transiently in random spatial distribution. After longer exposure, some of the spots became fixed in space. Saline also abolished or diminished the pH banding pattern observed in the untreated control cells. ZnCl2 suppressed the alkaline spot formation in saline and the pH banding pattern in AFW. The osmotic component of the saline stress did not produce transient bright spots or affect banding. The displacement of H+ from the cell wall charges, the H+/OH? channel conductance/density, and self-organization are discussed. No homologies to animal H+ channels were found. Salinity activation of the H+/OH? channels might contribute to saline response in roots of land plants and leaves of aquatic angiosperms. 相似文献