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1.
Summary The intracellular distribution of Na+, K+, Cl– and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La3+ and mechanical stress indicated that La3+ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na+ is sequestered within the nucleus, while K+ and Cl– are more highly concentrated in the cell cytoplasm. Reduction of the [Na+] of the incubation medium by replacement with K+ results in reduced cytoplasmic [Na+], increased [Cl–] and no change in [K+]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was –13.7 mV. Reduction of [Na+] in the medium caused significant depolarization. The measured potential is describable by the Cl– equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed. 相似文献
2.
Selective Changes in Cell Bodies and Growth Cones of Nerve Growth Factor-Differentiated PC12 Cells Induced by Chemical Hypoxia 总被引:2,自引:1,他引:1
Abstract: Cytosolic free Ca2+ concentration ([Ca2+]i) was measured in differentiated PC12 cells to test whether chemical hypoxia selectively alters intracellular Ca2+ in growth cones and cell bodies. Hypoxia increased [Ca2+]i and exaggerated its response to K+ depolarization in both parts of the cells. [Ca2+]i in the cell bodies was greater than that in the growth cones under resting conditions and in response to K+ or hypoxia. Ca2+-channel blockers selectively altered these responses. The L-channel blocker nifedipine reduced [Ca2+]i following K+ depolarization by 67% in the cell bodies but only 25% in the growth cones. In contrast, the N-channel blocker ω-conotoxin GVIA (ω-CgTX) diminished K+-induced changes in [Ca2+]i only in the growth cones. During hypoxia, nifedipine was more effective in the cell bodies than in the growth cones. During hypoxia, ω-CgTX diminished K+-induced changes by 50–75% in both parts of the cell, but only immediately after depolarization. The combination of nifedipine and ω-CgTX diminished the [Ca2+]i response to K+ with or without hypoxia by >90% in the cell body and 70% in the growth cones. Thus, the increased Ca2+ entry with K+ during hypoxia is primarily through L channels in the cell bodies, whereas in growth cones influx through L and N channels is about equal. The results show that chemical hypoxia selectively alters Ca2+ regulation in the growth cone and cell body of the same cell. 相似文献
3.
Using Ca2+-selective microelectrodes, the concentration of free calcium ([Ca2+]) in the cytosol has been measured in root hair cells of Medicago sativa L. in the presence of nodulation (Nod) factors. Growing root hairs of M. sativa displayed a steep apical [Ca2+] gradient, i.e. 604–967 nM in the tip compared with 95–235 nM in the basal region. When tested within the first 5 to 10 μm
of the tip, addition of NodRm-IV(C16:2,S) decreased the cytosolic [Ca2+], whereas an increase was observed when tested behind the tip. Overall, this led to a partial dissipation of the [Ca2+] gradient. The Ca2+ response was specific: it was equally well observed in the presence of NodRm-IV(Ac,C16:2,S), reduced with NodRm-IV(C16:0,S),
but not with chitotetraose, the nonactive glucosamine backbone. In contrast to growing root hairs, non-growing root hairs
without a tip-to-base cytosolic [Ca2+] gradient responded to NodRm-IV(C16:2,S) with an increase in cytosolic [Ca2+] at the tip as well as at the root hair base. We suggest that the response to Nod factors depends on the stage of development
of the root hairs, and that changes in cytosolic [Ca2+] may play different roles in Nod-factor signaling: changes of cytosolic [Ca2+] in the apical part of the root hair may be related to root hair deformation, while the increase in [Ca2+] behind the tip may be essential for the amplification of the Nod signal, for its propagation and transduction to trigger
downstream events.
Received: 5 January 1999 / Accepted: 14 April 1999 相似文献
4.
Rhizobium Nod factors induce increases in intracellular free calcium and extracellular calcium influxes in bean root hairs 总被引:6,自引:1,他引:5
Luis Cárdenas José A. Feijó Joseph G. Kunkel Federico Sánchez Terena Holdaway-Clarke Peter K. Hepler Carmen Quinto 《The Plant journal : for cell and molecular biology》1999,19(3):347-352
Application of Nod factors to growing, responsive root hairs of the bean Phaseolus vulgaris induces marked changes in both the intracellular cytosolic free calcium (Ca2+) and in the influx of extracellular [Ca2+]. The intracellular [Ca2+], which has been measured by ratiometric imaging in cells microinjected with fura-2-dextran (70 kDa), elevates within 5 min from approximately 400 n m to 1500 n m in localised zones in the root hair apex. Of particular note is the observation that the elevated regions of [Ca2+] appear to shift position during short time intervals. Increases in and fluctuations of the intracellular [Ca2+] are also observed in the perinuclear region after 10–15 min treatment with Nod factors. The extracellular Ca2+ flux, detected with the non-invasive, calcium specific vibrating electrode, is inwardly directed and also increases quickly in response to Nod factors from 13 pmol cm–2 s–1 to 28 pmol cm–2 s–1. Chitin-oligomers, which are structurally similar but biologically inactive when compared to the active Nod factors, fail to elicit changes in either intracellular or extracellular Ca2+. The similar timing and location of the intracellular elevations and the increased extracellular influx provide support for the idea that Ca2+ participates in secretion and cell wall remodelling, which occur in anticipation of root hair deformation and curling. 相似文献
5.
Summary Flocculation in two strains of Saccharomyces uvarum appears to be governed by the ratio of K+ and Ca2+ concentrations ([K+]/[Ca2+]). When the ratio is diminished either by an increased [Ca2+] or by a decreased [K+], the intensity and rate of flocculation are increased.K+ seems to be an antagonist of Ca2+ in the flocculation mechanism since enhancement of [K+] in the medium decreases uptake of Ca2+. Conversely an increase in [Ca2+] decreases the uptake of K+. 相似文献
6.
G. Bkaily D. Econornos L. Potvin J. -L. Ardilouze C. Marriott J. Corcos D. Bonneau Chin Nok Fong 《Molecular and cellular biochemistry》1992,117(1):93-106
The effect of high K– concentration, insulin and the L-type Ca2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca2+]i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca2+-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K+]o induced a rapid transient increase of [Ca2+]i that was followed by a sustained component. The early transient increase of [Ca2+]i by high [+]o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca2+ blocker was found to decrease both the early transient and the sustained increase of [Ca2+]i induced by depolarization of the cell membrane with high [K+]o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca2+]i that was blocked by PN 200-110 or by lowering the extracellular Ca2+ concentration with EGTA. The sustained increase of [Ca2+], induced by high [K+]o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na+ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca2+ current and the delayed outward K+ current. These results suggest that the early increase of (Ca2+]i during depolarization of the cell membrane of heart and VSM cells with high [K+]o is due to the opening and decay of an L-type Ca 2+ channel. However, the sustained increase of [Ca2+]i during a sustained depolarization is due to the activation of a resting (R) Ca 2+ channel that is insensitive to lowering [ATP]i and sensitive to insulin. 相似文献
7.
A. Ziegler F. H. E. Scholz 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1997,167(8):536-542
We analyzed the ionic composition of the hemolymph of Porcellio scaber in four different stages of the molt cycle using capillary electrophoresis and calcium selective mini- and microelectrodes.
The main ions in the hemolymph were K+, Ca2+, Na+, Mg+, and Cl−. The values for total calcium obtained by means of capillary electrophoresis and calcium selective minielectrodes did not
differ significantly from each other. In situ measurements of the free Ca2+ concentration ([Ca2+]) by means of calcium-selective microelectrodes indicated that Ca2+ is not bound in the hemolymph. During molt the [Ca2+] is significantly larger than during intermolt. The [Ca2+] increased by 13%, 19% and 18% during premolt, intramolt, and postmolt, respectively. The concentration of the other cations
and of Cl− decreased significantly between premolt and intramolt. Thus, the rise of the [Ca2+] in the hemolymph is not due to a general increase in all ions, but rather to the resorption of cuticular calcium. Furthermore,
the results suggest that K+, Na+, Mg+, and Cl−are extruded from the hemolymph during and/or after posterior ecdysis.
Accepted: 5 August 1997 相似文献
8.
E. M. Novikova V. A. Vodeneev V. S. Sukhov 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2017,11(2):151-167
Electrical signals, including action potential (AP), play an important role in plant adaptation to the changing environmental conditions. Experimental and theoretical investigations of the mechanisms of AP generation are required to understand the relationships between environmental factors and electrical activity of plants. In this work we have elaborated a mathematical model of AP generation, which takes into account the participation of vacuole in the generation of electrical response. The model describes the transporters of the plasma membrane (Ca2+, Cl–, and K+ channels, H+- and Ca2+-ATPases, H+/K+ antiporter, and 2H+/Cl– symporter) and the tonoplast (Ca2+, Cl–, and K+ channels; H+- and Ca2+-ATPases; H+/K+, 2H+/Cl–, and 3H+/Ca2+ antiporters), with due consideration of their regulation by second messengers (Ca2+ and IP3). The apoplastic, cytoplasmic and vacuolar buffers are also described. The properties of the simulated AP are in good agreement with experimental data. The AP model describes the attenuation of electrical signal with an increase in the vacuole area and volume; this effect is related to a decrease in the Ca2+ spike magnitude. The electrical signal was weakly influenced by the K+ and Cl– content in the vacuole. It was also shown that the contribution of vacuolar IP3-dependent Ca2+ channels into the generation of calcium spike during AP was insignificant with the given parameters of the model. The results provide theoretical evidence for the significance of the vacuolar area and volume in plant cell excitability. 相似文献
9.
O. V. Akopova L. I. Kolchinskaya V. I. Nosar V. A. Bouryi I. N. Mankovskaya V. F. Sagach 《Biochemistry. Biokhimii?a》2013,78(1):80-90
The effect of potential-dependent potassium uptake at 0–120 mM K+ on matrix Ca2+ accumulation in rat brain mitochondria was studied. An increase in oxygen consumption and proton extrusion rates as well as increase in matrix pH with increase in K+ content in the medium was observed due to K+ uptake into the mitochondria. The accumulation of Ca2+ was shown to depend on K+ concentration in the medium. At K+ concentration ?30 mM, Ca2+ uptake is decreased due to K+-induced membrane depolarization, whereas at higher K+ concentrations, up to 120 mM K+, Ca2+ uptake is increased in spite of membrane depolarization caused by matrix alkalization due to K+ uptake. Mitochondrial K ATP + -channel blockers (glibenclamide and 5-hydroxydecanoic acid) diminish K+ uptake as well as K+-induced depolarization and matrix alkalization, which results in attenuation of the potassium-induced effects on matrix Ca2+ uptake, i.e. increase in Ca2+ uptake at low K+ content in the medium due to the smaller membrane depolarization and decrease in Ca2+ uptake at high potassium concentrations because of restricted rise in matrix pH. The results show the importance of potential-dependent potassium uptake, and especially the K ATP + channel, in the regulation of calcium accumulation in rat brain mitochondria. 相似文献
10.
A mathematical model of calcium dynamics in vascular smooth muscle cell (SMC) was developed based on data mostly from rat mesenteric arterioles. The model focuses on (a) the plasma membrane electrophysiology; (b) Ca2+ uptake and release from the sarcoplasmic reticulum (SR); (c) cytosolic balance of Ca2+, Na+, K+, and Cl− ions; and (d) IP3 and cGMP formation in response to norepinephrine (NE) and nitric oxide (NO) stimulation. Stimulation with NE induced membrane depolarization and an intracellular Ca2+ ([Ca2+]i) transient followed by a plateau. The plateau concentrations were mostly determined by the activation of voltage-operated Ca2+ channels. NE causes a greater increase in [Ca2+]i than stimulation with KCl to equivalent depolarization. Model simulations suggest that the effect of [Na+]i accumulation on the Na+/Ca2+ exchanger (NCX) can potentially account for this difference. Elevation of [Ca2+]i within a concentration window (150-300 nM) by NE or KCl initiated [Ca2+]i oscillations with a concentration-dependent period. The oscillations were generated by the nonlinear dynamics of Ca2+ release and refilling in the SR. NO repolarized the NE-stimulated SMC and restored low [Ca2+]i mainly through its effect on Ca2+-activated K+ channels. Under certain conditions, Na+-K+-ATPase inhibition can result in the elevation of [Na+]i and the reversal of NCX, increasing resting cytosolic and SR Ca2+ content, as well as reactivity to NE. Blockade of the NCX's reverse mode could eliminate these effects. We conclude that the integration of the selected cellular components yields a mathematical model that reproduces, satisfactorily, some of the established features of SMC physiology. Simulations suggest a potential role of intracellular Na+ in modulating Ca2+ dynamics and provide insights into the mechanisms of SMC constriction, relaxation, and the phenomenon of vasomotion. The model will provide the basis for the development of multi-cellular mathematical models that will investigate microcirculatory function in health and disease. 相似文献
11.
SUN LI JINGLING YU MINJIE ZHU FUGENG ZHAO SHENG LUAN 《Plant, cell & environment》2012,35(11):1998-2013
Cadmium (Cd2+) interferes with the uptake, transport and utilization of several macro‐ and micronutrients, which accounts, at least in part, for Cd2+ toxicity in plants. However, the mechanisms underlying Cd2+ interference of ionic homeostasis is not understood. Using biophysical techniques including membrane potential measurements, scanning ion‐selective electrode technique for non‐invasive ion flux assays and patch clamp, we monitored the effect of Cd2+ on calcium (Ca2+) and potassium (K+) transport in root hair cells of rice. Our results showed that K+ and Ca2+ contents in both roots and shoots were significantly reduced when treated with exogenous Cd2+. Further studies revealed that three cellular processes may be affected by Cd2+, leading to changes in ionic homeostasis. First, Cd2+‐induced depolarization of the membrane potential was observed in root hair cells, attenuating the driving force for cation uptake. Second, the inward conductance of Ca2+ and K+ was partially blocked by Cd2+, decreasing uptake of K+ and Ca2+. Third, the outward K+ conductance was Cd2+‐inducible, decreasing the net content of K+ in roots. These results provide direct evidence that Cd2+ impairs uptake of Ca2+ and K+, thereby disturbing ion homeostasis in plants. 相似文献
12.
Various cations have been examined for their effects on phagocytosis. Media with high [Ca2+] and low [K+] favor phagocytosis, which is inhibited in media with high [K+], [Rb+], or [Ba2+] and low [Ca2+]. Microscopical observations of inhibited cells demonstrate that swimming behavior is not modified but they cannot perform the initial step of phagocytosis, attachment to food; when Ca2+ is added, cells attach to and ingest food, demonstrating rapid reversal of inhibition. Attachment is shown to be a linear function of the ratio [K+]/[Ca2+]1/2 or [Rb+]/[Ca2+]1/2 in the medium. The Ca2+ influx inhibitor Verapamil blocks attachment, as does La3+; the latter is believed to compete with Ca2+ for access to the Ca2+ channel. Likewise, treatment of cells with media containing no added Ca2+ inhibits attachment, and addition of 10 μM Ca2+ allows 90% of these cells to attach to and ingest food. The ionophore A23187, known to transport Ca2+ into a wide variety of cells, provokes lysosomal streaming movements typical of attachment. Based upon these observations, Ca2+ influx plays an essential role in attachment; K+ efflux also appears to be necessary since tetraethylammonium chloride blocks attachment. Treatment of cells with Tetrodotoxin, an inhibitor of Na+ transport, or suspending them in media containing no added Na+ does not affect attachment or ingestion, indicating that Na+ is not implicated in these processes. An hypothesis is presented which implicates Ca2+ in both direct adhesion and exocytosis phenomena during attachment. 相似文献
13.
Bucking C Fitzpatrick JL Nadella SR McGaw IJ Wood CM 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2011,181(5):615-630
Recent studies focusing on the consequences of feeding for ion and water balance in freshwater fish have revealed the need for similar comparative studies in seawater fish. A detailed time course sampling of gastrointestinal (GI) tract contents following the ingestion of a single meal of a commercial diet revealed the assimilation of both water and dietary ions (Na+, Cl?, K+, Ca2+, Mg2+) along the GI tract of seawater-acclimated rainbow trout (Oncorhynchus mykiss) which had been fasted for 1 week. Consumption of the meal did not change the drinking rate. There was a large secretion of fluid into the anterior intestine and caecae (presumably bile and/or pancreatic secretions). As a result, net assimilation (63%) of the ingested water along the GI tract was lower than generally reported for fasted trout. Mg2+ was neither secreted into nor absorbed from the GI tract on a net basis. Only K+ (93% assimilated) and Ca2+ (43% assimilated) were absorbed in amounts in excess of those provided by ingested seawater, suggesting that dietary sources of K+ and Ca2+ may be important to seawater teleosts. The oesophagus–stomach served as a major site of absorption for Na+, Cl?, K+, Ca2+, and Mg2+, and the anterior intestine and caecae as a major site of net secretion for all of these ions, except Cl?. Despite large absorptive fluxes of these ions, the ionic composition of the plasma was maintained during the digestion of the meal. The results of the present study were compared with previous work on freshwater-acclimated rainbow trout, highlighting some important differences, but also several similarities on the assimilation of water and ions along the gastrointestinal tract during digestion. This study highlights the complicated array of ion and water transport that occurs in the intestine during digestion while revealing the importance of dietary K+ and Ca2+ to seawater-acclimated rainbow trout. Additionally, this study reveals that digestion in seawater-acclimated rainbow trout appears to compromise intestinal water absorption. 相似文献
14.
Yoshiji Okazaki 《Journal of plant research》1996,109(1):107-112
Internodal cells of a brackish water charophyte,Lamprothamnium succinctum (A. Br. in Ash.) R.D.W. regulate the turgor pressure in response to changes in both the cellular and the external osmotic
pressures. During turgor regulation upon hypotonic treatment, net effluxes of K+ and Cl− from the vacuole, membrane depolarization, a transient increase in the electrical membrane conductance and a transient increase
in concentration of cytoplasmic Ca2+ are induced. Activation of the plasmalemma Ca2+ channels and the Ca2+-controlled passive effluxes of K+ and Cl− through the plasmalemma ion channels are postulated. 相似文献
15.
A. Lepple-Wienhues S. Berweck M. Böhmig C.P. Leo B. Meyling C. Garbe M. Wiederholt 《The Journal of membrane biology》1996,151(2):149-157
K+ channels, membrane voltage, and intracellular free Ca2+ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found
an inwardly rectifying K+ channel and a calcium-activated K+ channel. The inwardly rectifying K+ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The
K+ channel blockers quinidine, tetraethylammonium chloride and Ba2+ and elevated extracellular K+ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation.
Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced
a transient peak in intracellular Ca2+ followed by a long-lasting Ca2+ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca2+, illustrating a transmembrane flux of Ca2+ following its electrochemical gradient. We conclude that K+ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the
influx of Ca2+, a messenger in the mitogenic signal cascade of human melanoma cells.
Received: 9 May 1995/Revised: 30 January 1996 相似文献
16.
Christian Lohr Eric Tucker Lynne A. Oland Leslie P. Tolbert 《Developmental neurobiology》2002,52(2):85-98
Changes in the intracellular Ca2+ concentration ([Ca2+]i) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K+ concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K+ (1 min) with a transient increase in [Ca2+]i of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K+, the amplitude of the [Ca2+]i transients averaging 592 nM. At later stages, all cells responded to 25 mM K+ with [Ca2+]i transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca2+]i transients. The amplitudes of these [Ca2+]i transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca2+]i transients were greatly reduced in Ca2+‐free, EGTA‐buffered saline, and in the presence of the Ca2+ channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca2+ influx through voltage‐activated Ca2+ channels into antennal lobe glial cells. The development of the depolarization‐induced Ca2+ transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 85–98, 2002 相似文献
17.
T. Takeo S. Suga J. Wu Y. Dobashi T. Kanno M. Wakui 《Journal of cellular physiology》1998,174(3):387-397
Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl− and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (VH = 60 mV) and outward (VH = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl− concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl− and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl− and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl− currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (IK(delay)). IK(delay) was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the IK(delay). It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl− and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation. J. Cell. Physiol. 174:387–397, 1998. © 1998 Wiley-Liss, Inc. 相似文献
18.
Ostuni MA Ducroc R Péranzi G Tonon MC Papadopoulos V Lacapere JJ 《Biology of the cell / under the auspices of the European Cell Biology Organization》2007,99(11):639-647
Background information. TSPO (translocator protein), known previously as PBR (peripheral‐type benzodiazepine receptor), is a 18 kDa protein expressed in the mitochondrial membrane of a variety of tissues. TSPO has been reported to be over‐expressed in human colorectal tumours and cancer cell lines, but its function is not well characterized. Results. We investigated the expression and function of TSPO in the human colon cancer cells HT‐29. Immunohistochemical studies revealed that TSPO is localized in mitochondria, and its endogenous ligand, the polypeptide diazepam‐binding inhibitor, in the cytosol. Radioligand binding studies using the specific high‐affinity drug ligand [3H]PK 11195 and membrane fraction demonstrated saturable binding, with Kd and Bmax values of 13.5±1.5 nM and 10.1±1.0 pmol/mg respectively. PK 11195 induced a rapid and transient dose‐dependent rise in intracellular [Ca2+], which was unaffected by extracellular Ca2+, but was blocked by the PTP (permeability transition pore) inhibitor, cyclosporin A, and by the TSPO partial agonist, flunitrazepam. Using HT‐29 clone 19A cell line, which forms cell monolayers, we demonstrated that TSPO ligand stimulated a Ca2+‐dependent transepithelial Cl? secretion. This secretion was inhibited: (i) after removal of extracellular Cl?; (ii) by apical addition of the Cl? channel blocker NPPB [5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate]; and (iii) by basolateral addition of the Na+–K+–2Cl? co‐transporter inhibitor bumetanide. Furthermore, the intracellular Ca2+ chelator BAPTA/AM [bis‐(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetra‐acetic acid tetrakis(acetoxymethyl ester)] and cyclosporin A abolished the rise in PK 11195‐induced Cl? secretion. Conclusions. These findings indicate that TSPO is located in mitochondrial membranes of HT‐29 and reveal that its activation induces a rise in cytosolic Ca2+, leading to the stimulation of Cl? secretion. 相似文献
19.
45Ca2+ fluxes across the plasma membrane of zygotes of the fucoid alga, Pelvetia fastagiata (J. Ag.) De Toni, were studied in artificial sea waters of various potassium concentrations. Except for two cases, hyperpolarization of the cell membrane (with low [K+]) increases, and depolarization (with high [K+]) decreases the influx of Ca2+ over the range of [K+] studied (1–100 mM). The fractional increases of influx during hyperpolarization are close to the fractional increases in membrane potential but the decreases during depolarization are much smaller than those in membrane potential. In two anomalous cases, the influxes of 45Ca2+ at a potassium concentration of 30 mM were about 20% higher than the control value instead of being 10% lower.The effluxes of 45Ca2+ are increased by both hyperpolarization and by depolarization. On balance (and excepting the two anomalous cases) the net result of hyperpolarization should be to increase and that of depolarization to decrease intracellular [Ca2+]. 相似文献
20.
Maria Ereciska David Nelson Fiorenzo Dagani Judith Deas† Ian A. Silver† 《Journal of neurochemistry》1993,61(4):1356-1368
Abstract: Effects of nigericin were investigated in rat brain synaptosomes, cultured neurons, and C6 glioma cells to characterize the relations among ATP synthesis, [Na+]i., [K+]i, and [Ca2+]i, and pH under conditions when [H+]i is substantially increased and transmembrane electrical potential is decreased. Intracellular acidification and loss of K+ were accompanied by enhanced oxygen consumption and lactate production and a decrease in cellular energy level. Changes in the last three parameters were attenuated by addition of 1 mM ouabain. In synaptosomes treated with nigericin, neither respiration nor glycolysis was affected by 0.3 μM tetrodotoxin, whereas 1 mM amiloride reduced lactate production by 20% but did not influence respiration. In C6 cells, amiloride decreased the nigericin-stimulated rate of lactate generation by about 50%. The enhancement by nigericin of synaptosomal oxygen uptake and glycolytic rate decreased with time. However, there was only a small reduction in respiration and none in glycolysis in C6 cells. Measurements with ion-selective microelectrodes in neurons and C6 cells showed that nigericin also caused a rise in [Ca2+], and [Na+]., The increase in [Na+], in C6 cells was partially reversed by 1 mM amiloride. It is concluded that nigericin-induced loss of K+ and subsequent depolarization lead to an increase in Na+ influx and stimulation of the Na+/K+ pump with a consequent rise in energy utilization; that acidosis inhibits mitochondrial ATP production; that a rise in [H+] does not decrease glycolytic rate when the energy state (a fall in [ATP] and rises in [ADP] and [AMP]) is simultaneously reduced; that a fall in [K+], depresses both oxidative phosphorylation and glycolysis; and that the nigericin-induced alterations in ion levels and activities of energy-producing pathways can explain some of the deleterious effects of ischemia and hypoxia. 相似文献