共查询到20条相似文献,搜索用时 25 毫秒
1.
García JJ Reiter RJ Pié J Ortiz GG Cabrera J Sáinz RM Acuña-Castroviejo D 《Journal of bioenergetics and biomembranes》1999,31(6):609-616
We investigated the influence of pinoline (0.01–1.5 mM) on microsomal membrane fluiditybefore and after rigidity was induced by oxidative stress. In addition, we tested the effect ofpinoline in the presence of 1 mM melatonin. The fluidity in rat hepatic microsomes wasmonitored using fluorescence spectroscopy and it was compared to the inhibition ofmalonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) production as a reflection of lipid peroxidation.Below 0.6 mM, pinoline inhibited membrane rigidity in a manner parallel to its inhibitoryeffect on MDA + 4–HDA formation. At concentrations between 1–1.5 mM, pinoline wasless effective in stabilizing microsomal membranes than was predicted from its inhibition oflipid peroxidation. The addition of 1 mM melatonin enhanced the membrane-stabilizing activityof pinoline (0.01–0.6 mM). This cooperative effect was not observed for concentrations ofpinoline between 1–1.5 mM. When pinoline was tested without induced oxidative damage,1–1.5 mM pinoline maintained membrane fluidity at the same level as that recorded afterinduced lipid peroxidation. The results suggest that pinoline may be another pineal moleculethat prevents membrane rigidity mediated by lipid peroxidation and this ability is enhancedby melatonin. 相似文献
2.
García JJ Reiter RJ Cabrera JJ Pié J Mayo JC Sáinz RM Tan DX Qi W Acuña-Castroviejo D 《Journal of cellular biochemistry》2000,76(4):651-657
Lipid peroxidation is a degenerative chain reaction in biological membranes that may be initiated by exposure to free radicals. This process is associated with changes in the membrane fluidity and loss of several cell membrane-dependent functions. 5-methoxytryptophol (ML) is an indole isolated from the mammalian pineal gland. The purpose of this study was to investigate the effects of ML (0. 01mM-10mM) on membrane fluidity modulated by lipid peroxidation. Hepatic microsomes obtained from rats were incubated with or without ML (0.01-10 mM). Then lipid peroxidation was induced by FeCl(3), ADP, and NADPH. Membrane fluidity was determined using fluorescence spectroscopy. Malonaldehyde (MDA) +4-hydroxyalkenals (4-HDA) concentrations were estimated as an indicator of the degree of lipid peroxidation. With oxidative stress, membrane fluidity decreased and MDA+4-HDA levels increased. ML (0.01-3 mM) reduced membrane rigidity and the rise in MDA+4-HDA formation in a concentration-dependent manner. 10 mM ML protected against lipid peroxidation but failed to prevent the membrane rigidity. In the absence of oxidative reagents, ML (0.3-10 mM) decreased membrane fluidity whereas MDA+4-HDA levels remained unchanged. This indicates that ML may interact with membrane lipids. The results presented here suggest that ML may be another pineal indoleamine (in addition to melatonin) that resists membrane rigidity due to lipid peroxidation. 相似文献
3.
M. C. Reyes-Gonzales L. Fuentes-Broto E. Martínez-Ballarín F. J. Miana-Mena C. Berzosa F. A. García-Gil M. Aranda J. J. García 《The Journal of membrane biology》2009,231(2-3):93-99
The ability of several indoleamines to scavenge free radicals is well documented. Our aim was to evaluate the ability of 0.01–3 mm tryptophan (Trp) and 0.1–5 mm 5-hydroxytryptophan (5-OH-Trp) to protect hepatic cell membranes against 0.1 mm FeCl3 plus 0.1 mm ascorbic acid–induced lipid peroxidation and increases in membrane rigidity. Membrane fluidity was evaluated using fluorescence spectroscopy. Lipid and protein oxidation were estimated by quantifying malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HDA) concentrations and carbonyl group content, respectively. Exposure to FeCl3 plus ascorbic acid increased hepatic cell membrane rigidity, MDA + 4-HDA and carbonyl content. The presence of 5-OH-Trp, but not Trp, attenuated these changes. In the absence of oxidative stress, neither indoleamine modified fluidity, MDA + 4-HDA or carbonylation. These results suggest that C5 hydroxylation determines the ability of Trp to preserve membrane fluidity in the presence of oxidative stress. 相似文献
4.
Mouse liver microsomes treated with octylthioglucoside (OTG-microsomes) were examined for copper-stimulated ATPase activity.
The activity was about 1 μmol Pi/mg protein/hr under optimal conditions [300 mm KCl, 3 mm MgSO4, 10 mm GSH, 0.5 μm CuSO4, 3 mm ATP and 50 mm acetate buffer at pH5.0]. A reducing agent such as GSH or dithiothreitol was required for the activity, and removal of Cu+ from the reaction mixture by bathocuporinedisulfonate resulted in a complete loss of copper-stimulated ATPase activity. Vanadate
inhibited the copper-stimulated ATPase activity. The OTG-microsomes were phosphorylated in a hydroxylamine-sensitive and copper-stimulated
way. Iron used instead of copper also stimulated both ATPase and phosphorylation. These results suggest that microsomes from
mouse liver contain copper/iron-stimulated P-type ATPase.
Received: 2 September 1998/Revised: 16 March 1999 相似文献
5.
Karbownik M Reiter RJ Garcia JJ Cabrera J Burkhardt S Osuna C Lewiński A 《Journal of cellular biochemistry》2001,81(3):507-513
Excessive free iron and the associated oxidative damage are commonly related to carcinogenesis. Among the antioxidants known to protect against iron-induced oxidative abuse and carcinogenesis, melatonin and other indole compounds recently have received considerable attention. Indole-3-propionic acid (IPA), a deamination product of tryptophan, with a structure similar to that of melatonin, is present in biological fluids and is an effective free radical scavenger. The aim of the study was to examine the effect of IPA on experimentally induced oxidative changes in rat hepatic microsomal membranes. Microsomes were preincubated in presence of IPA (10, 3, 2, 1, 0.3, 0.1, 0.01 or 0.001 mM) and, then, incubated with FeCl(3) (0.2 mM), ADP (1.7 mM) and NADPH (0.2 mM) to induce oxidative damage. Alterations in membrane fluidity (the inverse of membrane rigidity) were estimated by fluorescence spectroscopy and lipid peroxidation by measuring concentrations of malondialdehyde+4-hydroxyalkenals (MDA+4-HDA). IPA, when used in concentrations of 10, 3 or 2 mM, increased membrane fluidity, although at these concentrations it did not influence lipid peroxidation significantly. The decrease in membrane fluidity due to Fe(3+) was completely prevented by preincubation in the presence of IPA at concentrations of 10, 3, 2 or 1 mM. The enhanced lipid peroxidation due to Fe(3+) was prevented by IPA only at the highest concentration (10 mM). It is concluded that Fe(3+)-induced rigidity and, to a lesser extent, lipid peroxidation in microsomal membranes may be reduced by IPA. However, IPA in high concentrations increase membrane fluidity. Besides melatonin, IPA may be used as a pharmacological agent to protect against iron-induced oxidative damage to membranes and, potentially, against carcinogenesis. 相似文献
6.
A voltage-activated Ca++ channel has been identified in the apical membranes of cultured rabbit proximal tubule cells using the patch-clamp technique.
With 105 mm CaCl2 solution in the pipette and 180 NaAsp in the bath, the channel had a conductance of 10.4 ± 1.0 pS (n= 8) in on-cell patches, and 9.8 ± 1.1 pS (n= 8) in inside-out patches. In both on-cell and inside-out patches, the channel is active by membrane depolarization. For
this channel, the permeation to Ba++ and Ca++ is highly selective over Na+ and K+ (PCa(Ba):PNa(K) >200:1). The sensitivity to dihydropyridines is similar to that for L-type channels where the channel was blocked by nifedipine
(10 μm), and activated by Bay K 8644 (5 μm). When activated by Bay K 8644, the channel showed subconductance levels. Treatment with forskolin (12.5 μm), phorbol ester (1 μm), or stretching (40 cm water) did not activate this channel. These results indicate that this Ca++ channel is mostly regulated by membrane voltage, and appears to be an epithelial class of L-type Ca++ channel. As such, it may participate in calcium reabsorption during periods of enhanced sodium reabsorption, or calcium signaling
in volume regulation, where membrane depolarization occurs for prolonged periods.
Received: 1 April 1996/Revised: 5 August 1996 相似文献
7.
S.E. Gasanov M.A. Alsarraj N.E. Gasanov E.D. Rael 《The Journal of membrane biology》1997,155(2):133-142
Membrane-active toxins from snake venom have been used previously to study protein-lipid interactions and to probe the physical
and biochemical states of biomembranes. To extend these studies, we have isolated from Naja naja kaowthia (cobra) venom a cytotoxin free of detectable phospholipase A2 (PLA2). The amino acid composition, pI (10.2), and net charge of the cytotoxin compares well with membrane-active toxins isolated
from venoms of other cobras. The cytotoxin, shown by a spin label method, associates with PLA2 in buffers at pH values between 7.0 and 5.0, but not at pH 4.0. It is suggested that cytotoxin and PLA2 (pI close to 4.8) associate electrostatically in the native venom. The effect of the cytotoxin on model phospholipid membranes
was studied by EPR of spin probes in oriented lipid multilayers and 1H-NMR of sonicated liposomes. The cytotoxin did not significantly affect the packing of lipids in pure phosphatidylcholine
(PC) membranes and in PC membranes containing 10 mol% phosphatidic acid (PA) or cardiolipin (CL). However, the cytotoxin induced
an increase in membrane permeability and formation of nonbilayer structures in PC membranes containing 40 mol% of PA or CL.
The purified cytotoxin was cytocidal to Jurkat cells, but had little effect on normal human lymphocytes. However, both Jurkat
cells and normal lymphocytes were killed equivalently when treated with 10−9
m PLA2 and 10−5
m cytotoxin in combination. From its effect on model membranes and Jurkat cells, it is suggested that purified cytotoxin preferentially
targets and disrupts membranes that are rich in acidic phospholipids on the extracellular side of the plasma membrane.
Received: 20 March 1996/Revised: 25 September 1996 相似文献
8.
The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm
cis/150 mm
trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much
lower concentrations (1.1–2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to
420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P
Cl/P
NMDG permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current
flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of
the pores formed by 100–500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P
Cl/P
NMDG permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect
midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties.
Received: 23 February 2001/Revised: 15 June 2001 相似文献
9.
The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and
membrane recycling, induces a cortical [Ca2+]
i
transient and exocytosis of dense core vesicles (``trichocysts') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca2+ ([Ca2+]
o
= 50 μm). When [Ca2+]
o
is kept at 30 nm (<[Ca2+]rest
i
), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion,
visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur
in absence of any sufficient Ca2+
o
. Upon readdition of Ca2+
o
or some other appropriate Me2+
o
at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca2+, Sr2+ or Mn2+ are vesicular, but when formed in presence of Mg2+, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast,
in presence of [Mg2+]
o
= 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with
still condensed contents are detached from the cell surface (``frustrated exocytosis') within ∼15 min. They undergo cytoplasmic
streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable
to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic
membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A
similar number of trichocysts can be detached by merely increasing [Mg2+]
o
to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin
and appropriate [Me2+] to maintain docking sites in a functional state, (ii) requirement of Ca2+
o
or of some other Me2+
o
to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty' signal to go to the regular endocytotic
pathway (with fading fluorescence), and (iv) occurrence of a ``filled' signal for trichocysts to undergo detachment and redocking
(with fluorescence) after ``frustrated exocytosis'.
Received: 20 January 2000/Revised: 5 May 2000 相似文献
10.
Inhibition of large-conductance calcium-activated potassium channel by 2-methoxyestradiol in cultured vascular endothelial (HUV-EC-C) cells 总被引:4,自引:0,他引:4
2-Methoxyestradiol, an endogenous metabolite of 17β-estradiol, is known to have antitumor and antiangiogenic actions. The
effects of 2-methoxyestradiol on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived
from human umbilical vein. In the whole-cell patch-clamp configuration, 2-methoxyestradiol (0.3–30 μm) reversibly suppressed the amplitude of K+ outward currents. The IC
50 value of the 2-methoxyestradiol-induced decrease in outward current was 3 μm. Evans blue (30 μm) or niflumic acid (30 μm), but not diazoxide (30 μm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol
(3 μm) to the bath did not modify the single-channel conductance of large-conductance Ca2+-activated K+ (BKCa) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 μm) produced a shift in the activation curve of BKCa channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BKCa channels is primarily mediated by a decrease in the number of long-lived openings. 2-Methoxyestradiol-induced inhibition
of the channel activity was potentiated by membrane stretch. In contrast, neither 17β-estradiol (10 μm) nor estriol (10 μm) affected BKCa channel activity, whereas 2-hydroxyestradiol (10 μm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 μm) caused membrane depolarization and Evans blue (30 μm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress
the activity of BKCa channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional
activity of endothelial cells.
Received: 27 November 2000/Revised: 13 April 2001 相似文献
11.
ATP-sensitive K+ (KATP) channels have been characterized in pituitary GH3 cells with the aid of the patch-clamp technique. In the cell-attached configuration, the presence of diazoxide (100 μm) revealed the presence of glibenclamide-sensitive KATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mm) or sodium cyanide (300 μm) increased KATP channel activity, while nicorandil (100 μm) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of KATP channels in a concentration-dependent manner with an IC50 value of 30 μm. The activation of phospholipase A2 caused by mellitin (1 μm) was found to enhance KATP channel activity and further application of aristolochic acid (30 μm) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4′-dithiodipyridine (100 μm) also induced KATP channel activity. Diazoxide, mellitin and 4,4′-dithiodipyridine activated the KATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of
diazoxide (100 μm) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly
indicates that KATP channels similar to those seen in pancreatic β cells are functionally expressed in GH3 cells. In addition to the presence of Ca2+-activated K+ channels, KATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane
potential.
Received: 19 June 2000/Revised: 13 September 2000 相似文献
12.
Single-channel properties of a delayed rectifier voltage-gated K+ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E50, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal
activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant
of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105
mm K+, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C.
In Na+-rich solution with 2.5 mm extracellular K+γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K+ over Na+. γ depended on extracellular K+ concentration (K
D
= 19.6 mm) and temperature (Q
10= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested
with a half-maximal inhibiting concentration (IC50) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX,
IC50= 6.8 nm) and mast cell degranulating peptide (MCDP, IC50= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and
DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane
potential.
Received: 2 June 1995/Revised: 13 October 1995 相似文献
13.
Mucosal crude microsomes, prepared from proximal rat small intestine, exhibited significant Mg-dependent, Zn-ATPase activity;
V
max
= 23 μmoles Pi/mg protein/hr, K
m
= 160 nm, and Hill Coefficient, n= 1.5. Partial purification (∼10-fold) was achieved by detergent extraction, and centrifugation through 250 mm sucrose: V
max
= 268 units, K
m
= 1 nm, and n= 6. In partially purified preparations, the assay was linear with time to 60 min, and with protein concentration to 1 μg/300
μl. Activities at pH 8 and 8.5 were higher than at pH 7.2. The ATP K
m
was 0.7 mm, with an optimal ATP/Mg ratio of ∼2. Ca elicited ATPase activity but did not augment the Zn-dependent activity. In partially
purified preparations, the homologous salts of Co, Cd, Cu, and Mn exhibited no detectable activity. Vanadate inhibition studies
yielded two component kinetics with a K
i
of 12 μm for the first component, and 96 μm for the second component, in partially purified preparations. Tissue distribution analyses revealed gradients of activity.
In the proximal half of the small intestine, Mg/Zn activity increased progressively from crypt to villus tip. In long axis
studies, this activity decreased progressively from proximal to distal small bowel.
Received: 12 September 2000/Revised 6 January 2001 相似文献
14.
These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch
clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch
clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (KCa) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current
and the KCa activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V
m
) and 145 ± 13 pS at negative V
m
. Glibenclamide (50 μm), Ba2+ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50= 46 μm. The identity of this K+ channel with an ATP-sensitive K+ channel (KATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the KCa and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked KATP.
Received: 12 June 1997/Revised: 21 October 1997 相似文献
15.
R. Wondergem M. Cregan L. Strickler R. Miller J. Suttles 《The Journal of membrane biology》1998,161(3):257-262
These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma
(HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and 3H-thymidine uptake. Glibenclamide added at 75 and 150 μm for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth
at concentrations as low as 1 μm (IC50= 73 μm) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range
in which glibenclamide decreased open probability of membrane KATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined
by flow cytometry using 10−5
m bromodeoxyuridine. Glibenclamide (100 μm) increased the percentage of cells in G0/G1 from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens
membrane KATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We
conclude that the sulfonylurea receptor and the corresponding membrane KATP channel are involved in mechanisms controlling HTB-9 cell growth. However, KATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle.
Received: 12 June 1997/Revised: 21 October 1997 相似文献
16.
M.-P. Blanchard N. Klauke S. Zitzmann H. Plattner 《The Journal of membrane biology》1999,169(3):155-165
We analyzed [Ca2+]
i
transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben
& Plattner, 1994. J. Cell Biol.
127:935–945; Plattner et al., 1994. J. Membrane Biol.
158:197–208). Wild-type cells (7S), nondischarge strain nd9–28°C and trichocyst-free strain ``trichless' (tl), respectively,
displayed similar, though somewhat diverging time course and plateau values of [Ca2+]
i
transients with moderate [Ca2+]
o
in the culture/assay fluid (50 μm or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca2+]
o
= 30 nm (c.f. [Ca2+]
rest
i
=∼50 to 100 nm), veratridine produced only a small cortical [Ca2+]
i
transient. This increased in size and spatial distribution at [Ca2+]
o
= 50 μm of 1 mm. Interestingly with unusually high yet nontoxic [Ca2+]
o
= 10 mm, [Ca2+]
i
transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With
[Ca2+]
o
= 30 nm, the restricted residual response observed is due to Ca2+ mobilization from subplasmalemmal stores. (ii) With moderate [Ca2+]
o
= 50 μm to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca2+
o influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl
cells, where free docking sites allow for rapid Ca2+ spread, and least in 7S cells, whose perfectly assembled docking sites may ``consume' a large part of the [Ca2+]
i
increase. (iii) With unusually high [Ca2+]
o
, mobilization of cortical stores and/or Ca2+
o
influx may be impeded by the known membrane stabilizing effect of Ca2+
o
counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and,
hence, not to be toxic side-effects, as confirmed by retention of injected calcein.
(v) Finally, Mn2+ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific
Me2+ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine
is of particular and continous interest.
Received: 8 December 1998/Revised: 2 March 1999 相似文献
17.
18.
L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral
membrane vesicles l-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective.
The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is
saturable with respect to external lactate with a K
m
of 39.2 ± 4.8 mm and a J
max of 8.9 ± 0.7 nmoles mg protein−1 sec−1. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains
a H+-lactate cotransporter, whereas in the apical membrane both H+-lactate and Na+-lactate cotransporters are present, even if they exhibit a low transport rate.
Received: 22 October 1996/Revised: 11 March 1997 相似文献
19.
Swelling-activated Cl− currents (I
Cl,swell
) have been characterized in a mouse renal inner medullary collecting duct cell line (mIMCD-K2). Currents activated by exposing
the cells to hypotonicity exhibited characteristic outward rectification and time- and voltage-dependent inactivation at positive
potentials and showed an anion selectivity of I− > Br− > Cl− > Asp−. NPPB (100 μm) inhibited the current in a voltage independent manner, as did exposure to 10 μm tamoxifen and 500 μm niflumic acid (NFA). In contrast, DIDS (100 μm) blocked the current with a characteristic voltage dependency. These characteristics of I
Cl,swell
in mIMCD-K2 cells are essentially identical to those of heterologously expressed cardiac CLC-3.
A defining feature of CLC-3 is that activation of PKC by PDBu inhibits the conductance. In mIMCD-K2 cells preincubation with
PDBu (100 nm) prevented the activation of I
Cl,swell
by hypotonicity. However, PDBu inhibition of I
Cl,swell
was reversed after PDBu withdrawal, but this was refractory to subsequent PDBu inhibition. Activation of either the cystic
fibrosis transmembrane conductance regulator (CFTR) or Ca2+ activated Cl− conductance (CaCC), which are coexpressed in mIMCD-K2 cells prior to PDBu treatment, abolished the PDBu inhibition of I
Cl,swell
. Control of I
Cl,swell
by PKC therefore depends on the physiological status of the cell.
In intact mIMCD-K2 layers in Ussing chambers, forskolin stimulation of an inward short-circuit current (due to transepithelial
Cl− secretion via apical CFTR) was inhibited by cell swelling upon hypotonic exposure at the basolateral surface. Activation
of I
Cl,swell
is therefore capable of regulating transepithelial Cl− secretion and suggests that I
Cl,swell
is located at the basolateral membrane. PDBu exposure prior to or during hypotonic challenge was ineffective in reversing
the swelling-activated inhibition of Cl− secretion, but tamoxifen (100 μm) abolished the hypotonic inhibition of forskolin-stimulated short-circuit current (I
sc
).
RT-PCR analysis confirmed expression of mRNA for members of the CLC family, including both CLC-2 and 3, in the mIMCD-K2 cell
line.
Received: 24 February 2000/Revised: 26 May 2000 相似文献
20.
Muscarinic m3 receptor-mediated changes in cytosolic Ca2+ concentration ([Ca2+]l) occur by activation of Ca2+ release channels present in the endoplasmic reticulum membrane and Ca2+ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation
of a voltage-insensitive, cation-selective channel of low conductance (3.2 ± 0.6 pS; 25 mm Ca2+ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced
activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side),
suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised
inside-out membrane patches from nonstimulated A9m3 cells GTP (10 μm) and GDP (10 μm) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mm Ca2+ as charge carrier) respectively. In contrast, ATP (10 μm), UTP (10 μm) or CTP (10 μm) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the
activation of a cation channel that conducts calcium.
Received: 14 November 1996/Revised: 4 April 1997 相似文献