共查询到20条相似文献,搜索用时 15 毫秒
1.
F. Pluciennik F. Verrecchia B. Bastide J.C. Hervé M. Joffre J. Délèze 《The Journal of membrane biology》1996,149(3):169-177
A direct cell-to-cell exchange of ions and molecules occurs through specialized membrane channels built by the interaction
of two half channels, termed connexons, contributed by each of the two adjacent cells. The electrical and diffusional couplings
have been investigated by monitoring respectively the cell-to-cell conductance and the fluorescence recovery after photobleaching,
in Sertoli and cardiac cells of young rat. In both cell types, a rapid impairment of the intercellular coupling has been observed
in the presence of testosterone propionate. This interruption of the cell-to-cell communication through gap junction channels
was dose-dependent, observed in the concentration range 1 to 25 μm and was progressively reversed after withdrawing the testosterone ester. Pretreatment with cyproterone acetate, an antiandrogen
which blocks the nuclear testosterone receptor by binding, did not prevent the uncoupling action of the androgen ester. This
observation, together with the rapid time course of the uncoupling and recoupling, and the rather high effective concentration
(micromolar) of the steroid compound, suggests a nongenomic mechanism of action. The uncoupling concentrations were very similar
to those of other steroid compounds known to interrupt gap junctional communication. The uncoupling could result from a direct
interaction of the steroid with the proteolipidic structure of the membrane, that might alter the conformation of the gap
junction channels and their functional state.
Received: 10 April 1995/Revised: 27 October 1995 相似文献
2.
A large conductance, Ca2+-activated K+ channel of the BK type was examined in cultured pituitary melanotrophs obtained from adult male rats. In cell-attached recordings
the slope conductance for the BK channel was ≈190 pS and the probability (P
o
) of finding the channel in the open state at the resting membrane potential was low (<<0.1). Channels in inside-out patches
and in symmetrical 150 mm K+ had a conductance of ≈260 pS. The lower conductance in the cell-attached recordings is provisionally attributed to an intracellular
K+ concentration of ≈113 mm. The permeability sequence, relative to K+, was K+ > Rb+ (0.87) > NH+
4 (0.17) > Cs+≥ Na+ (≤0.02). The slope conductance for Rb+ was much less than for K+. Neither Na+ nor Cs+ carried measurable currents and 150 mm internal Cs+ caused a flickery block of the channel. Internal tetraethylammonium ions (TEA+) produced a fast block for which the dissociation constant at 0 mV (K
D
(0 mV)) was 50 mm. The K
D
(0 mV) for external TEA+ was much lower, 0.25 mm, and the blocking reaction was slower as evidenced by flickery open channel currents. With both internal and external TEA+ the blocking reaction was bimolecular and weakly voltage dependent. External charybdotoxin (40 nm) caused a large and reversible decrease of P
o
. The P
o
was increased by depolarization and/or by increasing the concentration of internal Ca2+. In 0.1 μm Ca2+ the half-maximal P
o
occurred at ≈100 mV; increasing Ca2+ to 1 μm shifted the voltage for the half-maximal P
o
to −75 mV. The Ca2+ dependence of the gating was approximated by a fourth power relationship suggesting the presence of four Ca2+ binding sites on the BK channel.
Received: 23 October/Revised: 15 December 1995 相似文献
3.
O. Strauß K. Steinhausen S. Mergler F. Stumpff M. Wiederholt 《The Journal of membrane biology》1999,169(3):141-153
This combined study of patch-clamp and intracellular Ca2+ ([Ca2+]
i
) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl− channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca2+]
i
and activation of Cl− currents. In contrast, intracellular application of Ca2+ (10 μm) only induced transient activation of Cl− currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of I
CRAC, La3+ (10 μm), despite the fact that both maneuvers led to a decline in [Ca2+]
i
. The InsP3-induced rise in Cl− conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca2+ stores or by removal of Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca2+-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca2+/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl−-conductance in 50% of the cells investigated without affecting [Ca2+]
i
. Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca2+]
i
and Cl− conductance. In summary, elevation of [Ca]
i
by InsP3 leads to activation of Cl− channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for the Ca2+-independent maintenance of this conductance.
Received: 15 October 1998/Revised: 3 March 1999 相似文献
4.
We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical
stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded
to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration
([Ca2+]
i
) with peak of 422.7 ± 43.8 nm above an average resting [Ca2+]
i
of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca2+]
i
increase was reduced by 50% in the absence of extracellular Ca2+, while the prolonged [Ca2+]
i
recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride
(100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca2+]
i
recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca2+]
i
response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca2+]
i
transient peak. [Ca2+]
i
transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca2+-free solution, or when Ca2+ stores were depleted by thapsigargin in Ca2+-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect
of cytochalasin D was reversible and did not prevent the [Ca2+]
i
increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical
link via actin filaments, between the plasma membrane and an internal Ca2+ store.
Received: 24 March 1997/Revised: 21 July 1997 相似文献
5.
D.B. Light M.R. Adler J.K. Ter Beest S.A. Botsford R.T. Gronau 《The Journal of membrane biology》1998,166(2):119-132
This study examined whether protein kinase C (PKC) stimulates K+ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine
(H-7, 10 μm), but not with the cyclic nucleotide-dependent kinase antagonists N-(2′-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004,
10 μm) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 μm). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X
or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 μm). The effect of these three antagonists and H-7 was reversed with gramicidin (5 μm in a choline Ringer), indicating PKC was linked to K+ efflux (gramicidin is a cationophore that was used to ensure a high K+ permeability). We also measured cell volume recovery from hypotonic shock (0.5× Ringer) with a Coulter counter and estimated
cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 μm), sphingosine (100 nm), chelerythrine (10 μm), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 μm) nor H-8 (5 μm). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 μm). Furthermore, using the patch clamp technique, we found H-7 (10 μm) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K+ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K+ loss during RVD is regulated, at least in part, by protein kinase C.
Received: 20 January 1998/Revised: 2 September 1998 相似文献
6.
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
7.
The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl
oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by
single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle
sarcoplasmic reticulum (HSR) and [3H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P
o
) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P
o (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS
or 4,4′-DTDP to higher concentrations in single channel recordings and [3H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P
o
> 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P
o
= 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither
4-CMPS nor 4,4′-DTDP affected Ca-[125I]calmodulin binding to HSR. 1 mm MgCl2 reduced P
o
from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P
o
> 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished
or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues
of the calcium release channel.
Received: 22 July 1999/Revised: 15 November 1999 相似文献
8.
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
9.
R. Moreau A.M. Hurst J-Y. Lapointe D. Lajeunesse 《The Journal of membrane biology》1996,150(2):175-184
Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike
phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached
patch configuration (CAP) no channel activity was observed in 2 mm Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon
addition of 50 nm parathyroid hormone (PTH), 5 nm prostaglandin E2 (PGE2) or 0.1 mm dibutyryl cAMP + 1 μm forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated
cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K
channel showed that in symmetrical solutions (140 mm K) the channel has a conductance of 246 ± 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (PK/PNa) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability
(P
o
) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the P
o
vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very
unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mm ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE2 or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (45Ca++) by MG-63 cells was stimulated in the presence of PTH and PGE2, an effect inhibited by Nitrendipine (10 μm). Second, whereas PGE2 stimulated the calcium-activated maxi-K channel in 2 mm Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE2 did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mm Ca Ringer and 10 μm Nitrendipine in CAP configuration, PGE2 (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic
subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of
this channel, it is concluded that activation of this channel by PTH, PGE2 or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx.
Received: 17 September 1995/Revised: 7 December 1995 相似文献
10.
Isoform-Specific Function and Distribution of Na/K Pumps in the Frog Lens Epithelium 总被引:3,自引:1,他引:2
Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies
of the Na/K ATPase. RNase protection assays showed that all cells express α1- and α2-isoforms of the Na/K pump but not the α3-isoform, however the α2-isoform dominates in anterior cells whereas the α1-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the
Na/K pump current (I
P
), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I
P
blockade data indicate the α1-isoform has a dissociation constant of 100 μm DHO whereas for the α2-isoform it is 0.75 μm DHO. Both α1- and α2-isoforms are half maximally activated at an intracellular Na+-concentration of 9 mm. The α1-isoform is half maximally activated at an extracellular K+-concentration of 3.9 mm whereas for the α2-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the α1-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the α2-isoform. Under normal physiological conditions, I
P
in equatorial cells is approximately 0.23 μA/μF, and in anterior cells it is about 0.14 μA/μF. These current densities refer
to the area of cell membrane assuming a capacitance of around 1 μF/cm2. Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 μA/cm2 at the equator vs. 0.5 μA/cm2 at the anterior pole.
Received: 17 May 2000/Revised: 11 August 2000 相似文献
11.
T. Ishikawa 《The Journal of membrane biology》1996,153(2):147-159
A Ca2+-activated Cl− conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When
the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca2+ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of
the cells with pipette solutions containing 300 nm or less than 1 nm free Ca2+ strongly reduced the Cl− currents, indicating the currents were Ca2+-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent.
The anion permeability sequence of the Cl− channels was: NO−
3 (2.00) > I− (1.85) ≥ Br− (1.69) > Cl− (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette
solutions was increased from 0 to 10 mm, the Ca2+-dependency of the Cl− current amplitude shifted to lower free Ca2+ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca2+-activated Cl− currents. This suggests that Ca2+/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca2+-activated Cl− currents. The outward Cl− currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable
Cl− conductance controlled by cytosolic Ca2+ and ATP levels in rat submandibular acinar cells.
Received: 9 January 1996/Revised: 20 May 1996 相似文献
12.
The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic
monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated
currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca2+ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC50) was reversibly increased from 3 μm to about 30 μm. This Ca2+-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure
to 3 mm Mg2++ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca2+ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca2+-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the
affinity of the channel for CAM. The affinity shift induced by Ca2+-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR,
ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated
by an as yet unidentified factor distinct from CAM.
Received: 26 December 1995/Revised: 14 March 1996 相似文献
13.
The chloride conductance of inner medullary collecting duct cells (mIMCD-3 cell line) has been investigated using the whole
cell configuration of the patch clamp technique. Seventy-seven percent of cells were chloride selective when measured with
a NaCl-rich bathing solution and a TEACl-rich pipette solution. Seventy-five percent of chloride-selective cells (90/144)
had whole cell currents which exhibited an outwardly-rectifying (OR) current-voltage (I/V) relationship, while the remaining cells exhibited a linear (L) I/V relationship. The properties of the OR and L chloride currents were distinct. OR currents (mean current densities at ±60
mV of 66 ± 5 pA/pF and 44 ± 3 pA/pF), were time- and voltage-independent with an anion selectivity (from calculated permeability
ratios) of SCN− (2.3), NO−
3 (1.8), ClO−
4 (1.7), Br− (1.7), I− (1.6), Cl− (1.0), HCO−
3 (0.5), gluconate− (0.2). Bath additions of NPPB, flufenamate, glibenclamide (all 100 μm) and DIDS (500 μm) produced varying degrees of block of OR currents with NPPB being the most potent (IC50 of approximately 50 μm) while DIDS was the least effective. Linear chloride currents had similar current densities to the OR chloride currents and
were also time- and voltage-independent. The anion selectivity sequence was SCN− (2.5), NO−
3 (1.9), Br− (1.4), I− (1.1), Cl− (1.0), ClO−
4 (0.5), HCO−
3 (0.5), gluconate− (0.3). In contrast to the OR conductance, glibenclamide was the most potent and DIDS the least potent blocker of L currents.
An IC50 of >100 μm was observed for NPPB block. Neither OR of L chloride currents were affected by acutely or chronically increased intracellular
cAMP and were not affected when intracellular Ca2+ levels were increased or decreased. The molecular identity and physiological role of OR and linear currents in mIMCD-3 cells
are discussed.
Received: 13 June 1995/Revised: 15 September 1995 相似文献
14.
X. Wu H. Yang P. Iserovich J. Fischbarg P.S. Reinach 《The Journal of membrane biology》1997,158(2):127-136
The relationship between relative cell volume and time-dependent changes in intracellular Ca2+ concentration ([Ca2+]
i
) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light
scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic
time (τ
vr
) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca2+]
i
initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell
volume recovery and [Ca2+]
i
transients were inhibited by either: (a) Ca2+-free medium; (b) 5 mm Ni2+ (inhibitor of plasmalemma Ca2+ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca2+ content); or (d) 100 μm ryanodine (inhibitor of Ca2+ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca2+ influx and the emptying of intracellular Ca2+ stores during the [Ca2+]
i
transients, Mn2+ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn2+ permeability 6-fold. However, Mn2+ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca2+]
i
transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca2+ release from an intracellular store leads to a subsequent increase in plasmalemma Ca2+ influx, and that both are required for cells to undergo RVD.
Received: 7 November 1996/Revised: 6 January 1997 相似文献
15.
GABAA channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline
and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches).
The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application
of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay
that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased
with GABA concentration from less than 10 pS (0.5 μm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC
50 value of 33 μm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of
1 μm diazepam, the GABA EC
50 decreased to 0.2 μm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA
antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 μm GABA the bicuculline IC
50 was 19 μm. The effect of GABA on channel conductance shows that the role of the ligand in GABAA receptor channel function is more complex than previously thought.
Received: 23 October 2000/Revised: 27 February 2001 相似文献
16.
The regulation of the voltage-activated chloride current conductance (G
Cl
) in toad skin was investigated by the use of the SH reagents N-ethylmaleimide (NEM) and p-chloro-mercuricbenzenesulfonic
acid PCMBS. This anion pathway is controlled by a voltage-sensitive gating regulator. Mucosal application of NEM decreased
the voltage-activation in a time and concentration dependent manner, half-maximal inhibition being exerted at a concentration
of 30 μm within 20 min. At concentrations higher than 100 μm, the voltage-activated G
Cl
was near-completely and irreversibly inhibited in less than 10 min. Resting, deactivated conductance was essentially unaffected.
NEM had no effect on active sodium transport (measured as I
sc
) under conditions, which fully dissipated the voltage-activated G
Cl
. After complete inhibition of the voltage-activated G
Cl
with NEM, chloride conductance could still be stimulated by CPT-cAMP as in control tissues. Under these conditions, NEM at
concentrations above 1 mm decreased G
Cl
reversibly. Mucosal application of PCMBS at 500 μm inhibited the activated conductance by 35%, which was slightly reversible. Inhibition of voltage-activated G
Cl
, which was observed after mucosal addition of the membrane-impermeable NEM analogue, eosin-5-maleimide, was completely reversible
after washout. This suggests that the binding site for the maleimide is not accessible from the external face of the apical
membrane. Brief application of NEM at lower concentrations (1–3 min, ≤100 μm) led to partial inhibition of G
Cl
, followed by occasionally complete recovery upon washout of NEM. Recovery of voltage-activated G
Cl
was progressively attenuated and eventually disappeared after subsequent brief applications of NEM. This could reflect recruitment
of permeation/control sites from a finite pool. The data are discussed in the frame of a working model for the voltage-activated
Cl−-pathway, that contains two principle components, i.e., an anion-selective permeation path which is controlled by regulatory
protein(s).
Received: 18 December 1996/Revised: 28 April 1997 相似文献
17.
Y.-H. Tai J. Flick S.A. Levine J.L. Madara G.W.G. Sharp M. Donowitz 《The Journal of membrane biology》1996,149(1):71-79
Elevation in intracellular Ca2+ acting via protein kinase C (PKC) is shown to regulate tight junction resistance in T84 cells, a human colon cancer line and a model Cl− secretory epithelial cell. The Ca2+ ionophore A23187, which was used to increase the intracellular Ca2+ concentration, caused a decrease in tight junction resistance in a concentration- and time-dependent manner. Dual Na+/mannitol serosal-to-mucosal flux analysis performed across the T84 monolayers treated with 2 μm A23187 revealed that A23187 increased both fluxes and that in the presence of ionophore there was a linear relationship between
the Na+ and mannitol fluxes with a slope of 56.4, indicating that the decrease in transepithelial resistance was due to a decrease
in tight junction resistance. Whereas there was no effect of 0.1 μm A23187, 1 or 2 μm produced a 55% decrease in baseline resistance in 1 hr and 10 μm decreased resistance more than 80%. The A23187-induced decrease in tight junction resistance was partially reversible by
washing 3 times with a Ringer's-HCO3 solution containing 1% BSA. The A23187 effect on resistance was dependent on intracellular Ca2+; loading the T84 cells with the intracellular Ca2+ chelator BAPTA significantly reduced the decrease in tight junction resistance caused by A23187. This intracellular Ca2+ effect was mediated by protein kinase C and not calmodulin. While the protein kinase C antagonist H-7 totally prevented the
action of A23187 on tight junction resistance, the Ca2+/calmodulin inhibitor W13 did not have any effect. Sphingosine, another inhibitor of PKC, partially reduced the A23187-induced
decline in tight junction resistance. The PKC agonist PMA mimicked the A23187 effect on resistance, although the effect was
delayed up to 1 hr after exposure. In addition, however, PMA also caused an earlier increase in resistance, indicating it
had an additional effect in addition to mimicking the effect of elevating Ca2+. The effects of a phospholipase inhibitor (mepacrine) and of inhibitors of arachidonic acid metabolism (indomethacin for
the cyclooxygenase pathway, NDGA for the lipoxygenase pathway, and SKF 525A for the epoxygenase pathway) on the A23187 action
were also examined. None of these agents altered the A23187-induced decrease in resistance. Monolayers exposed to 2 μm A23187 for 1 hr were stained with fluorescein conjugated phalloidin, revealing that neighboring cells did not part one from
another and that A23187 did not have a detectable effect on distribution of F-actin in the perijunctional actomyosin ring.
The results indicate that elevation in intracellular Ca2+ decreases tight junction resistance in the T84 monolayer, acting through protein kinase C by a mechanism which does not involve visible changes in the perijunctional actomyosin
ring.
Received: 14 July 1995/Revised: 25 September 1995 相似文献
18.
We show that rabbit skeletal RyR channels in lipid bilayers can be activated or inhibited by NO, in a manner that depends
on donor concentration, membrane potential and the presence of channel agonists. 10 μm
S-nitroso-N-acetyl-penicillamine (SNAP) increased RyR activity at −40 mV within 15 sec of addition to the cis chamber, with a 2-fold increase in frequency of channel opening (F
o
). 10 μm SNAP did not alter activity at +40 mV and did not further activate RyRs previously activated by 2 mm
cis ATP at +40 or −40 mV. In contrast to the increase in F
o
with 10 μm SNAP, 1 mm SNAP caused a 2-fold reduction in F
o
but a 1.5-fold increase in mean open time (T
o
) at −40 mV in the absence of ATP. 1 mm SNAP or 0.5 mm sodium nitroprusside (SNP) induced ∼3-fold reductions in F
o
and T
o
at +40 or −40 mV when channels were activated by 2 mm
cis ATP or in channels activated by 6.5 μm peptide A at −40 mV (peptide A corresponds to part of the II–III loop of the skeletal dihydropyridine receptor). Both SNAP-induced
activation and SNAP/SNP-induced inhibition were reversed by 2 mm dithiothreitol. The results suggest that S-Nitrosylation or oxidation of at least three classes of protein thiols by NO each produced characteristic changes in RyR
activity. We propose that, in vivo, initial release of NO activates RyRs, but stronger release increases [NO] and inhibits
RyR activity and contraction.
Received: 27 August 1999/Revised: 25 October 1999 相似文献
19.
A K+ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid
bilayers. This channel was identified as an ATP-sensitive K+ channel (KATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na2. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0
mV and with a 5/250 KCl gradient, the main conductance of the KATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil
stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels.
In general, KATP channels were characterized by brief openings followed by long closings (open probability, P
o
≈ 0.02); only occasionally (3 out of 12 experiments) did the KATP channels have a high open probability (P
o
≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl2 to the internal side of the channel.
Received: 7 November 1995/Revised: 10 June 1996 相似文献
20.
P.M. Dunn 《The Journal of membrane biology》1998,165(2):133-143
The actions of clotrimazole and cetiedil, two drugs known to inhibit the Gardos channel, have been studied on single intermediate
conductance calcium-activated potassium (IKCa) channels in inside out patches from human red blood cells, and compared with those of TEA and Ba2+ applied to the cytoplasmic face of the membrane. TEA produced a fast block which was observed as a reduction in the amplitude
of the single channel current. This effect was weakly voltage dependent with the fraction of the membrane potential sensed
by TEA at its binding site (δ) of 0.18 and a K
d
at 0 mV of 20.5 mm. Ba2+ was a very potent blocker of the channel, breaking the single channel activity up into bursts, interspersed with silent periods
lasting several seconds. The effect of Ba2+ was very voltage sensitive, δ= 0.44, and a K
d
at 0 mV of 0.15 μm. Clotrimazole applied to the inner face of the membrane at a concentration ≤1 μm produced a slow block resulting in bursts of channel activity separated by quiescent periods lasting many seconds. The effect
of clotrimazole was mimicked by a quaternary derivative UCL 1559, in keeping with an action at the cytoplasmic face of the
channel. A high concentration of cetiedil (100 μm) produced only a weak block of the channel. The kinetics of this action were very slow, with burst and inter-burst intervals
lasting several minutes. While inhibition of the Gardos channel by cetiedil is unlikely to involve an intracellular site of
action, if clotrimazole is able to penetrate the membrane, part of its effect may result from binding to an intracellular
site on the channel.
Received; 18 February 1998/Received: 5 June 1998 相似文献