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1.
Cobra venom cytotoxins (CTX) have been shown to disrupt cells as different as immunocytes, skeletal myocytes, erythrocytes
and tumor cells. Nevertheless, even subpopulations of tumor cells are differentially susceptible to CTX by an order of magnitude.
In the present study, our objective was to compare CTX-specific binding with cytolytic potency for two disparate cell types
in vitro. We investigated the lytic activity of cytotoxin-III from Naja naja atra (NNA, fraction D) using heart cells and human leukemic T-cells (CEM cells). For both cell types, 50% cytolysis, assessed
by tetrazolium dye conversion, occurred with μm concentrations of toxin (EC50= 2.2 μm). We examined the binding of radiolabeled CTX III to both heart cells and CEM cells and found the apparent dissociation constant
(K
Dapp) to be 0.69 μm and 0.75 μm, for CEM and heart cells respectively. The B
max for the CEM cells was 1.0 fmoles/cell and that for heart cells was 5.2 fmoles/cell, both exhibiting positive cooperativity
between the sites (Hill coefficients 1.4, T-cells; 1.6, heart). Relatively modest dissociation constants plus high numbers
of binding sites per cell are consistent with a model of CTX binding to plasma membranes by interaction with phospholipids
in the bilayer. Our results suggest that the lytic activity of this cytotoxin follows its binding to a population of sites
on the cells in a cooperative fashion.
Received: 8 May 1995/Revised: 17 November 1995 相似文献
2.
Protection from Cell Death by mcl-1 Is Mediated by Membrane Hyperpolarization Induced By K+ Channel Activation 总被引:3,自引:0,他引:3
Mcl-1, a member of the Bcl-2 family, has been identified as an inhibitor of apoptosis induced by anticancer agents and radiation in myeloblastic leukemia
cells. The molecular mechanism underlying this phenomenon, however, is not yet understood. In the present study, we report
that hyperpolarization of the membrane potential is required for prevention of mcl-1 mediated cell death in murine myeloblastic FDC-P1 cells. In cells transfected with mcl-1, the membrane potential, measured by the whole-cell patch clamp, was hyperpolarized more than −30 mV compared with control
cells. The membrane potential was repolarized by increased extracellular K+ concentration (56 mV per 10-fold change in K+ concentration). Using the cell-attached patch-clamp technique, K+ channel activity was 1.7 times higher in mcl-1 transfected cells (NP
o
= 22.7 ± 3.3%) than control cells (NP
o
= 13.2 ± 1.9%). Viabilities of control and mcl-1 transfected cells after treatment with the cytotoxin etoposide (20 μg/ml), were 37.9 ± 3.9% and 78.2 ± 2.0%, respectively.
Suppression of K+ channel activity by 4-aminopyridine (4-AP) before etoposide treatment significantly reduced the viability of mcl-1 transfected cells to 49.0 ± 4.6%. These results indicate that as part of the prevention of cell death, mcl-1 causes a hyperpolarization of membrane potential through activation of K+ channel activity.
Received: 30 March 1999/Revised: 20 July 1999 相似文献
3.
The gram-positive bacterium Mycobacterium phlei was treated with detergents. Reconstitution experiments using lipid bilayers suggested that the detergent extracts contain
a channel forming protein. The protein was purified to homogeneity by preparative SDS-PAGE and identified as a protein with
an apparent molecular mass of about 135 kDa. The channel-forming unit dissociated into subunits with a molecular mass of about
22 kDa when it was boiled in 80% dimethylsulfoxid (DMSO). The channel has on average a single channel conductance of 4.5 nS
in 1 m KCl and is highly voltage-dependent in an asymmetric fashion when the protein is added to only one side of the membrane.
Zero-current membrane potential measurements with different salts implied that the channel is highly cation-selective because
of negative point charges in or near the channel mouth. Analysis of the single-channel conductance as a function of the hydrated
cation radii using the Renkin correction factor and the effect of the negative point charges on the single-channel conductance
suggest that the diameter of the cell wall channel is about 1.8 to 2.0 nm. The channel properties were compared with those
of other members of the mycolata and suggest that these channels share common features. Southern blots demonstrated that the
chromosome of M. phlei and other mycolata tested contain homologous sequences to mspA (gene of the cell wall porin of Mycobacterium smegmatis).
Received: 22 December 2000/Revised: 10 April 2001 相似文献
4.
Electric Field Pulses Can Induce Apoptosis 总被引:5,自引:0,他引:5
F. Hofmann H. Ohnimus C. Scheller W. Strupp U. Zimmermann C. Jassoy 《The Journal of membrane biology》1999,169(2):103-109
Injection of electric field pulses of high intensity (kV/cm) and short duration (microsecond range) into a cell suspension
results in a temporary increase of the membrane permeability due to a reversible electric breakdown of the cell membrane.
Here we demonstrate that application of supercritical field pulses between 4.5 and 8.1 kV/cm strength and 40 μsec duration
induce typical features of apoptosis in Jurkat T-lymphoblasts and in HL-60 cells including DNA fragmentation and cleavage
of the poly(ADP ribose) polymerase. Apoptosis induction did not depend on the presence of any particular electrolyte in the
extracellular medium. However, no apoptosis was observed in solutions without a minimum amount of salt. Apoptotic DNA fragmentation
was prevented by the caspase inhibitor zVAD.
Received: 16 December 1998/Revised: 24 February 1999 相似文献
5.
Dye Transfer Between Cells of the Lens 总被引:1,自引:0,他引:1
Dye transfer between lens fiber cells and between lens epithelial cells and underlying fiber cells was studied using a wide
dynamic range-cooled CCD camera, H2O immersion objectives and image analysis techniques. Each lens was decapsulated by a new technique which leaves the epithelial
cells adherent to the lens fiber mass. Lucifer Yellow CH was injected into either single epithelial cells or single fiber
cells using the standard whole cell configuration of the patch voltage clamp technique. The results demonstrate extensive
dye communication between fiber cells at the lens posterior surface, anterior surface, and equatorial surface. Dye transfer
between deep fiber cells was also observed. Dye transfer between ≈10% of epithelial cells and their underlying fiber cells
was apparent when care was taken to yield wide dynamic range images. This was required because the relatively high concentration
of dye in the epithelial cell masks the presence of much lower dye concentrations in the underlying fiber cell. A mathematical
model which includes dye concentration, time, and spatial spread suggests that those epithelial cells that are coupled to
an underlying fiber cell are about as well dye coupled as the epithelial cells themselves. The relatively low dye concentration
in a fiber cell is due to its larger volume and diffusion of the dye along the axis of the fiber away from the fiber/epithelial
junction.
Received: 14 September 1995/Revised: 13 November 1995 相似文献
6.
Pazdernik NJ Matzke EA Jessen-Marshall AE Brooker RJ 《The Journal of membrane biology》2000,176(1):31-40
Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This
motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of
videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical
morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane
ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved
forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with
this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca2+]
i
) associated with periodic Ca2+ oscillations during prolonged ACh applications. The early transient rise in [Ca2+]
i
was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly
dependent on external calcium. The Ca2+ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine.
Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca2+]
i
oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca2+-sensitive K+ currents. These K+ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin,
but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca2+ from intracellular stores which in turn activate Ca2+-dependent (BK-type) K+ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway
with glioma cell migration.
Received: 17 December/Revised: 17 March 2000 相似文献
7.
The Involvement of Sphingolipids in Multidrug Resistance 总被引:13,自引:0,他引:13
Administration of most chemotherapeutic agents eventually results in the onset of apoptosis, despite the agents' variety
in structure and molecular targets. Ceramide, the central molecule in cellular glycosphingolipid metabolism, has recently
been identified as an important mediator of this process. Indeed, one of the events elicited by application of many cytotoxic
drugs is an accumulation of this lipid. Treatment failure in cancer chemotherapy is largely attributable to multidrug resistance,
in which tumor cells are typically cross-resistant to multiple chemotherapeutic agents. Different cellular mechanisms underlying
this phenomenon have been described. Of these the drug efflux pump activity of P-glycoprotein and the multidrug resistance-associated
proteins are the most extensively studied examples. Recently, an increased cellular capacity for ceramide glycosylation has
been recognized as a novel multidrug resistance mechanism. Indeed, virtually all multidrug-resistant cells exhibit a deviating
sphingolipid composition, most typically, increased levels of glucosylceramide. On the other hand, several direct molecular
interactions between sphingolipids and drug efflux proteins have been described. Therefore, in addition to a role in the multidrug
resistance phenotype by which ceramide accumulation and, thus, the onset of apoptosis are prevented, an indirect role for
sphingolipids might be envisaged, by which the activity of these efflux proteins is modulated. In this review, we present
an overview of the current understanding of the interesting relations that exist between sphingolipid metabolism and multidrug
resistance.
Received: 16 June 2000/Revised: 16 August 2000 相似文献
8.
Imre Gombos Zsolt Bacsó Cynthia Detre Henrietta Nagy Katalin Goda Márton Andrásfalvy Gábor Szabó János Matkó 《Cytometry. Part A》2004,61(2):117-126
BACKGROUND: Lipid rafts are cholesterol- and glycosphingolipid-rich microdomains in the cellular plasma membranes that play critical roles in compartmentalization (concentration, coupling, and isolation) of receptors and signal molecules. Therefore, detecting constitutive or induced raft associations of such proteins is of central interest in cell biology. This has mostly been done with time- and cell-consuming immunobiochemical techniques affected by several sources of artifacts. A flow cytometric analysis of immunocytochemical staining under differential circumstances of detergent treatment offers a new alternative to this method. METHODS: Membrane microdomains are resistant to nonionic detergents due to extensive, strong interactions between their molecular constituents. We used this feature to develop a rapid flow cytometric assay of differential detergent resistance based on immunocytochemical labeling of extracellular domain epitopes in membrane proteins. Data evaluation is based on comparative detection of their detergent solubility without and with cholesterol depletion of cell membranes, resolved by moderate concentrations of nonionic detergents. RESULTS: Nonionic detergents Triton X-100 and Nonidet-40 (0.05-0.1%) in cold or Brij-98 (0.1-0.5%) at 37 degrees C efficiently resolved detergent solubility or resistance of many lymphocyte cell surface proteins. Kinetic data revealed that a short (5-10 min) detergent treatment is sufficient for this assay. Comparison of detergent solubility in untreated and cholesterol-depleted cells differentiated membrane proteins associated with or excluded from raft microdomains, respectively. Confocal microscopy showed that this mild detergent treatment leaves the cytoskeleton of the cells intact, with a detectable expression of raft marker detergent-resistant proteins attached to it. An induced association with rafts of immunoglobulin E receptors upon antigen cross-linking was also easily detectable in rat mast cells by this approach. CONCLUSIONS: A protocol is proposed for a rapid (5-10 min) test of detergent resistance of membrane proteins in cells. The approach requires only a small amount of cells (10(4)/sample) and offers a good resolution of detergent solubility or resistance of membrane proteins, also in terms of the underlying mechanisms, with an advantage of applicability for all conventional bench-top flow cytometers. 相似文献
9.
The functional consequence of the casein kinase I-catalyzed phosphorylation of the lens gap junctional protein connexin49
was investigated using a sheep primary lens cell culture system. To determine whether the phosphorylation of connexin49 catalyzed
by endogenous casein kinase I results in an altered junctional communication between lens cells, the effect of the casein
kinase I-specific inhibitor CKI-7 on Lucifer Yellow dye transfer between cells in the lens culture was examined. Dye transfer
was analyzed in cultures of different ages because we have demonstrated previously that the expression of connexin49 increases
as the cultures age while that of connexin43, which is likely not a substrate for casein kinase I, has been shown to decrease
[Yang & Louis (1999) Invest. Ophthalmol. Vis. Sci. 41: 2568–2564]. In 9-day old lens cultures, in which gap junctions are
composed primarily of connexin43, CKI-7 had little effect on the rate of dye transfer between lens cells. In contrast, treatment
of 15-day and 28-day old cultures with CKI-7 resulted in a significant increase in the rate of dye transfer. Thus, the extent
of this CKI-7-dependent increase in cell-to-cell communication was positively correlated with the level of expression of connexin49,
the major casein kinase I substrate in lens plasma membranes. These results suggest that the casein kinase I-catalyzed phosphorylation
of connexin49 decreases cell communication between connexin49-containing gap junctions in the lens.
Received: 31 July 2000/Revised: 12 January 2001 相似文献
10.
Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid
mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal
membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition:
at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made
accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium
addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM)
was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca2+, Mg2+, Cd2+ and La3+ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to
be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited
vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely
restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle
is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely
lipidic and protein-mediated membrane fusion is discussed.
Received: 27 September 1999/Revised: 22 March 2000 相似文献
11.
Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the
treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular
K+ and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after
a delay, the depolarization of the cell membrane and the full depletion of intracellular K+. These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K+ efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature
but only the delay was affected by pH. Cry1C-induced K+ efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative
characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect
cell line.
Received: 4 October 1999/Revised: 21 December 1999 相似文献
12.
Kim HD Turner JT Burnett JE Joo NS Han BH Lim RW Bowen JW Kirk RG Lee P 《The Journal of membrane biology》2000,175(3):235-244
We examined the influence of serum and furosemide on K movement and cell volume in HCD57 cells, a murine erythroleukemia
cell line, which require erythropoietin (EPO) for survival. We found that maintenance of cell volume depends on the concentration
of serum in the culture medium. In isotonic medium containing 20% serum, HCD57 cells maintain their steady-state volume. In
contrast, the cells shrink progressively as medium serum content is reduced. In serum-free medium, raising external K to 75
mm prevents cell shrinkage and a further increase in K to 145 mm results in swelling, revealing a role for K permeability in the regulation of cell volume. Of particular interest has been
a serendipitous finding with furosemide. Below an external K concentration of 2.1 ± 0.3 mm in medium containing 2% serum, furosemide inhibits K uptake, probably stemming from its well known inhibitory action on KCl
cotransport. However, above that K concentration, furosemide stimulates K uptake in a dose-dependent manner. Moreover, furosemide
potentiates cell shrinkage induced by serum withdrawal. These findings suggest that the transport machinery mediating cellular
shrinkage, once primed by serum depletion, becomes receptive to a second stimulus.
Received: 13 December 1999/Revised: 15 March 2000 相似文献
13.
p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in
HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected
cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in
control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward
rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated
by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion
permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small
but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity.
Received: 17 November 1997/Revised: 9 February 1998 相似文献
14.
CACO-2 BBE was used to determine the response of a gastrointestinal epithelium to tumor necrosis factor-α (TNF). Incubation
of CACO-2 BBE with TNF did not produce any effect on transepithelial resistance (TER) within the first 6 hr but resulted in
a 40–50% reduction in TER and a 30% decrease in I
sc
(short circuit current) relative to time-matched control at 24 hr. The decrease in TER was sustained up to 1 week following
treatment with TNF and was not associated with a significant increase in the transepithelial flux of [14C]-d-mannitol or the penetration of ruthenium red into the lateral intercellular space. Dilution potential and transepithelial
22Na+ flux studies demonstrated that TNF-treatment of CACO-2 BBE cell sheets increased the paracellular permeability of the epithelium
to Na+ and Cl−. The increased transepithelial permeability did not associate with an increase in the incidence of apoptosis. However, there
was a TNF-dependent increase in [3H]-thymidine labeling that was not accompanied by a change in DNA content of the cell sheet. The increase in transepithelial
permeability was concluded to be across the tight junction because: (i) 1 mm apical amiloride reduced the basolateral to apical flux of 22Na+, and (ii) dilution potential studies revealed a bidirectionally increased permeability to both Na+ and Cl−. These data suggest that the increase in transepithelial permeability across TNF-treated CACO-2 BBE cell sheets arises from
an alteration in the charge selectivity of the paracellular conductive pathway that is not accompanied by a change in its
size selectivity.
Received: 4 March 1997/Revised: 3 November 1997 相似文献
15.
The Ach-evoked Ca2+-activated K+ Current in Mouse Mandibular Secretory Cells. Single Channel Studies
Although acetylcholine (ACh) is able to activate voltage- and Ca2+-sensitive K+ (BK) channels in mouse mandibular secretory cells, our recent whole cell studies have suggested that these channels, like
those in sheep parotid secretory cells, do not contribute appreciably to the conductance that carries the ACh-evoked whole
cell K+ current. In the present study, we have used cell-attached patch clamp methods to identify and characterize the K+ channel type responsible for carrying the bulk of this current. When the cells were bathed in a NaCl-rich solution the predominant
channel type activated by ACh (1 μmol/l or 50 nmol/l) had a conductance only of 40 pS; it was not blocked by TEA but it was
sensitive to quinine and it conducted Rb+ to an appreciable extent. BK channels, which could be seen in some but not all patches from resting cells, also showed increased
activity when ACh was added to the bath, but they were much less conspicuous during ACh stimulation than the 40-pS channels.
When the cells were bathed in a KCl-rich rather than a NaCl-rich solution, a small-conductance K+ channel, sensitive to quinine but not to TEA, was still the most conspicuous channel to be activated by ACh although its
conductance was reduced to 25 pS. Our studies confirm that the ACh-evoked whole-cell K+ current is not carried substantially by BK channels and show that it is carried by a small-conductance K+ channel with quite different properties.
Received: 28 September 1995/Revised: 26 December 1995 相似文献
16.
T.G. Hammond F.O. Goda G.L. Navar W.C. Campbell R.R. Majewski D.L. Galvan F. Pontillon J.H. Kaysen T.J. Goodwin S.W. Paddock P.J. Verroust 《The Journal of membrane biology》1998,162(2):157-167
In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized
function known as ``degradative endocytosis.' The endosomal pathways of the renal proximal tubule and the visceral yolk sac
share highly specialized structures for ``degradative endocytosis.' These endosomal pathways also have a unique distribution
of their H+-ATPase, predominantly in the subapical endosomal pathway. Previous studies provide only indirect evidence that H+-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is
H+-ATPase dependent in baby hamster kidney cells, and H+-ATPase subunits bind fusion complex proteins in detergent extracts of fresh rat brain. To determine directly whether homotypic
endosomal fusion is H+-ATPase dependent, we inhibited v-type H+-ATPase during flow cytometry and cuvette-based fusion assays reconstituting endosomal fusion in vitro. We report that homotypic
fusion in subapical endosomes derived from rat renal cortex, and immortalized visceral yolk sac cells in culture, is inhibited
by the v-type H+-ATPase specific inhibitor bafilomycin A1. Inhibition of fusion by H+-ATPase is mediated by the membrane potential as collapsing the pH gradient with nigericin had no effect on homotypic endosomal
fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution
assay this data provides the first direct evidence for a role of v-type H+-ATPase in mammalian homotypic endosomal fusion.
Received: 29 October 1996/Revised: 8 December 1997 相似文献
17.
Effects of Hyperglycemia and Protein Kinase C on Connexin43 Expression in Cultured Rat Retinal Pigment Epithelial Cells 总被引:4,自引:0,他引:4
Malfait M Gomez P van Veen TA Parys JB De Smedt H Vereecke J Himpens B 《The Journal of membrane biology》2001,181(1):31-40
Previous results demonstrated that the intercellular communication mediated by gap junctions in retinal pigment epithelial
(RPE) cells from the healthy Long Evans (LE) rat strain is higher than that from the dystrophic Royal College of Surgeons
(RCS) rat strain. We examined connexin (Cx) expression in both cell types. At the mRNA level, a qualitatively similar expression
pattern was found whereby Cx26, Cx32, Cx36, Cx43, Cx45 and Cx46 were all expressed. At the protein level, only Cx43 and Cx46
were detected. Expression of both isoforms was higher in LE-RPE as compared to RCS-RPE by a factor of 1.25 and 2 respectively.
Phosphorylation of Cx43 was increased upon activation of protein kinase C (PKC) by 1 μM phorbol 12-myristate 13-acetate (PMA).
The phosphorylation status was not changed in hyperglycemic conditions, but this treatment strongly decreased total Cx43 levels
to about 75 and 40% (in LE-RPE and RCS-RPE cells respectively) of the control level in LE-RPE cells. This decrease could be
overcome by PKC downregulation. These results demonstrate that PKC activation and hyperglycemic conditions have different
effects on Cx43 and that PKC is involved in the metabolic pathway induced by hyperglycemic conditions.
Received: 21 July 2000/Revised: 19 January 2001 相似文献
18.
A.R. Rittenhouse D.H. Vandorpe C. Brugnara S.L. Alper 《The Journal of membrane biology》1997,157(2):177-191
Clotrimazole (CLT), a member of the antifungal imidazole family of compounds, has been found to inhibit both calcium (Ca2+)-activated 86Rb and potassium (K) fluxes of human red cells and to inhibit red cell binding of 125I-charybdotoxin (ChTX) [11]. We have now used patch-clamp techniques to demonstrate reversible inhibition of whole cell KCa2+ currents in murine erythroleukemia (MEL) cells by submicromolar concentrations of CLT. Inhibition was equivalent whether
currents were elicited by bath application of the Ca2+ ionophore A23187 or by dialyzing cells with a pipette solution containing micromolar concentrations of free Ca2+. The extent of inhibition of whole cell MEL KCa2+ currents was voltage-dependent, decreasing with increasing test potential. We also determined the single channel basis of
the CLT inhibition in MEL cells by demonstrating the inhibition of a calcium-activated, ChTX-sensitive K channel by CLT in
outside-out patches. The channel was also blocked by the des-imidazolyl metabolite of CLT, 2-chlorophenyl-bisphenyl-methanol
(MET II) [15], thus demonstrating that the imidazole ring is not required for the inhibitory action of CLT. Single KCa2+ channels were also evident in inside-out patches of MEL cells. Block of K current by CLT was not unique to MEL cells. CLT
also inhibited a component of the whole cell K current in PC12 cells. Channel specificity of block by CLT was determined by
examining its effects on other types of voltage-sensitive currents. CLT block showed the following rank order of potency:
K currents in PC12 cells > Ca2+ currents in PC12 cells ≫ Na currents in sympathetic neurons. These results demonstrate that direct inhibition of single KCa2+ by CLT can be dissociated from inhibition of cytochrome P-450 in MEL cells.
Received: 10 September 1996/Revised: 12 December 1996 相似文献
19.
High speed video imaging microscopy and the pH-sensitive fluorophore2′,7′,-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
were used to examine acid-base functions of beta-intercalated cells of the rabbit cortical collecting duct. The presence of
intercalated cells was established and the properties of apical and basolateral acid-base transporters assessed by monitoring
cell pH during acid loading and luminal and basolateral ion substitutions. We showed that treatment of beta-intercalated cells
with ammonium chloride (20 mm) induced a profound decrease of their intracellular pH from 6.98 ± 5.93 ± 0.08. pH recovery occurred after different lag
periods ranging between 2 to 15 min (0.22 ± 0.04 dpH/dt). We demonstrated that this pH recovery mechanism was independent
of basolateral Na+ and apical HCO−
3 and K+. It was also not affected by apical and basolateral addition of NEM, by basolateral DIDS and by apical application of the
H-KATPase inhibitor SCH28080. The process of pH recovery was however, critically dependent on basolateral HCO−
3. These results are best explained by acid-induced insertion and/or activation of chloride-bicarbonate exchangers that are
functional properties with their apical analogues.
Received: 11 January 1994/Revised: 13 June 1997 相似文献
20.
M.E. Forero M. Marín A. Corrales I. Llano H. Moreno M. Camacho 《The Journal of membrane biology》1999,170(2):173-180
Whole cell patch-clamp recordings were used to study the electrical properties of the macrophage-like cell line J774.1, after
infection with Leishmania amazonensis. Infection induced a significant increase in cell size and membrane capacitance, suggesting that parasite invasion leads to
the addition of plasma membrane to the host cell. By 24 hr after infection, the host cell membrane potential was significantly
more hyperpolarized than control cells, and this difference remained for the subsequent 72 hr post-infection. The hyperpolarization
was paralleled by an increase in the density of inward rectifying K+ currents. The shape of the conductance vs. voltage curve, the kinetic properties and the pharmacological profile of these currents were not significantly altered by
infection. These results suggest that infection by L. amazonensis causes an increase in the number of functional inward rectifying K+ channels, leading to hyperpolarization of the host cell membrane.
Received: 19 January 1999/Revised: 20 April 1999 相似文献