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1.
We have characterized a Ca2+-dependent Cl− current (ClCa) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed
with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca2+, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic
acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca2+ strongly reduced the currents indicating that they were Ca2+ dependent. With cells dialyzed with Cs+ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca2+-activated Cl− currents.
With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca2+. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These
results may suggest that ClCa is inhibited by cAMP-dependent protein kinase.
Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a
hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl− and bicarbonate-rich primary fluid by these cells.
Received: 30 November 1998/Revised: 2 March 1999 相似文献
2.
Cell-volume changes induced by terbutaline (a specific β2-agonist) were studied morphometrically in rat fetal distal lung epithelium (FDLE) cells. Cell-volume changes qualitatively
differed with the concentration of terbutaline. Terbutaline of 10−10–10−8
m induced transient cell swelling. Terbutaline of 10−7
m induced transient cell swelling followed by slow cell shrinkage. Terbutaline of 10−6–10−5
m induced rapid cell shrinkage followed by slow cell shrinkage. Terbutaline of 10−3
m induced transient cell shrinkage; then cell volume oscillated during stimulation. Benzamil of 10−6
m suppressed the cell swelling induced by 10−10–10−8
m terbutaline and quinine of 10−3
m inhibited the cell shrinkage induced by 10−6–10−5
m terbutaline. These results suggest that cell swelling would be induced by NaCl influx and the cell shrinkage is by KCl efflux.
Dibutyryl cyclic AMP (DBcAMP) also induced similar cell-volume changes over a wide range of concentrations (10−9–10−3
m): a low concentration induced transient cell swelling; a high concentration, rapid and slow cell shrinkage. Forskolin (10−4
m), like terbutaline (10−5
m), induced rapid cell shrinkage followed by slow cell shrinkage, and this decrease in the cell volume was enhanced by the
presence of benzamil. On the other hand, cell shrinkage was induced by ionomycin (even low concentration; 3 × 10−10
m ionomycin), and after that cell volume remained at a plateau level. Removal of extracellular Ca2+ abolished the cell swelling caused by terbutaline of 10−10–10−8
m. With removal of extracellular Ca2+, the initial, rapid cell shrinkage induced by 10−5
m terbutaline became transient, but we still detected slow cell shrinkage similar to that in the presence of extracellular
Ca2+. Overall, at low concentrations (10−10–10−8
m), terbutaline induced benzamil-sensitive cell swelling in FDLE cells, which was cAMP- and Ca2+-dependent; high concentrations (≥−6) induced quinine-sensitive rapid cell shrinkage, which was Ca2+-dependent; high concentrations (≥−7) induced slow cell shrinkage, which was cAMP-dependent. These findings suggest that terbutaline regulates cell volume in
FDLE cells by cytosolic cAMP and Ca2+ through activation of Na+ and K+ channels.
Received: 13 March 1995/Revised: 17 January 1996 相似文献
3.
Chloride (Cl−) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole
cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl2, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl− current activated by 10−5
m forskolin, 10−3
m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10−6
m phorbol 12-myristate 13-acetate (PMA), 10−3
m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10−7
m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br− > Cl−≫ I− > glutamate. This current was inhibited by 10−3
m diphenylamine-2-carboxylate (DPC) and 10−4
m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10−3
m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic
fibrosis transmembrane conductance regulator (CFTR) Cl− conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl− current blocked by DIDS. To determine the exact location of the Cl− conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded
on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl− removal from the apical solution induced a Cl− efflux which was stimulated by 10−5
m forskolin, 10−7 calcitonin and inhibited by 10−5
m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br− influx through the Cl− pathway. Forskolin and calcitonin had no effect on the basolateral Cl− permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl− conductance in the apical membrane through a cAMP-dependent mechanism.
Received: 5 July 1995/Revised: 21 December 1995 相似文献
4.
Properties of large conductance Ca2+-activated K+ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels
had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K+
o
//155 mm K+
i
) and 231 ± 4 pS in external high-K
o
solution (155 mm K+
o
//155 mm K+
i
). The channels were activated by depolarization and by an increase in internal Ca2+ concentration. Potentials of half-maximum channel activation (E50) were −13, −34, −64 and −85 mV in the presence of 10−6, 10−5, 10−4 and 10−3
m internal Ca2+, respectively. Using an internal solution containing 10−4
m Ca2+, averaged KCa currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged KCa currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with
a 50% blocking concentration (IC50) of 0.17 ± 0.02 mm. KCa channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that KCa channels activated by Ca2+ entry during the action potential play an important role in the excitability of motoneurones.
Received: 7 November 1996/Revised: 29 October 1997 相似文献
5.
Andrew Sivak 《In vitro cellular & developmental biology. Plant》1977,13(6):337-343
Summary Cell division is induced in stationary cultures of BALB/c-3T3 mouse embryo cells without renewal of medium by addition of
the tumor promoter, phorbol myristate acetate (PMA), or bovine serum. The addition of dbcAMP (10−3
m) or other inhibitors of cAMP phosphodiesterase, papaverine (6.7×10−6
m), Persantin (5×10−5
m) or RO-20-1724 (10−4
m), prevents cell replication induced by PMA or serum. In contrast, ouabain (10−4
m) and N,N′-dicyclohexylcarbodiimide (10−5
m), inhibitors of Na+−K+-ATPase activity, block the PMA-stimulated effect but do not inhibit serum-stimulated cell division. Several stages in the
cell cycle are sensitive to dbcAMP addition. One is early in the G1 phase at the time of reinitiation of the cell cycle from a stationary (G0) phase, a second is associated with the G1-S transition, and a third with passage of cells from a post-S phase to mitosis. Based on observations of early morphological
changes, responses of plasma membrane ezymes and effects of enzyme inhibitors, the stimulation of cell division in BALB/c-3T3
cells by PMA or serum appears to involve several membrane functions which may act in a cooperative manner.
This work was supported by a USPHS Research Grant CA12503, and a Center Grant ES-00260 awarded to the Institute of Environmental
Medicine. Mrs. Susan Kulina provided the consistent and excellent technical aid necessary to perform this work.
Note added in proof: During the preparation and review of this paper, Boynton reported that PMA appears to sensitize BALB/c-3T3
cells to calcium ion which may play a critical role in the regulation of the DNA synthesis (36). 相似文献
6.
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
7.
Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating
CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to
test whether G-proteins control CFTR Cl− conductance (CFTR G
Cl
) in the native sweat duct (SD). We permeabilized the basolateral membrane with α-toxin so as to manipulate cytosolic nucleotides.
We activated G-proteins and monitored CFTR G
Cl
activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-γ-S (100 μm) also activates CFTR G
Cl
in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-γ-S increased CFTR G
Cl
by 44 ± 20 mS/cm2 (mean ±se; n= 7). GDP (10 mm) inhibited G-protein activation of CFTR G
Cl
even in the presence of GTP-γ-S. The heterotrimeric G-protein activator (AlF4
−) in the cytoplasmic bath activated CFTR G
Cl
(increased by 51.5 ± 9.4 mS/cm2 in the presence of 5 mm ATP without cAMP, n= 6), the magnitude of which was similar to that induced by GTP-γ-S. Employing immunocytochemical-labeling techniques, we
localized Gαs, Gαi, Gαq, and Gβ at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G
Cl
in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G
Cl
activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude
that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt
absorption by controlling CFTR G
Cl
activity.
Received: 9 June 2000/Revised: 5 October 2000 相似文献
8.
R. Fernández J.R. Bosqueiro A.C. Cassola G. Malnic 《The Journal of membrane biology》1997,157(2):193-201
The presence of an electrogenic H+-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies
the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting
duct. Transepithelial PD (V
t
) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion.
The tubules were perfused with electrolyte solutions to which 2 × 10−7
m bafilomycin or 4.6 × 10−8
m concanamycin were added. No significant increase in lumen-negative V
t
upon perfusion with these inhibitors as compared to control, was observed as well as when 10−3
m amiloride, 10−5
m benzamil or 3 mm Ba2+ were perfused alone or in combination. The effect of an inhibition of electrogenic H+ secretion, i.e., increase in lumen-negative V
t
by 2–4 mV, was observed only when Cl− channels were blocked by 10−5
m 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this
segment from 1.21 ± 0.14 (n= 8) to 0.62 ± 0.03 (8) nmol.cm−2.sec−1 as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate
that: (i) the participation of the vacuolar H+ ATPase in the establishment of cortical late distal tubule V
t
is minor in physiological conditions, but can be demonstrated after blocking Cl− channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H+ secretion in this segment is reduced by a Cl− channel blocker, supporting coupling of H+-ATPase with Cl− transport.
Received: 6 July 1996/Revised: 27 December 1996 相似文献
9.
The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers.
The activity of native RyRs, with cytoplasmic (cis) [Ca2+] of 10−7
m (in the absence of Mg2+ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm
cis Mg2+ (10−7
m
cis Ca2+) or by 10 mm
cis Mg2+ (10−3
m
cis Ca2+), or activated by 4 mm ATP (10−7
m
cis Ca2+), also responded to 1 mm
cis 4,4′-DTDP with activation and then loss of activity. P
o
and mean open time (T
o
) of the maximally activated channels were lower in the presence of Mg2+ than in its absence, and the number of openings within the long time constant components of the open time distribution was
reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg2+, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca2+ or caffeine (Eager et al., 1997), the activation produced by 1 mm
cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between
the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation.
Received: 8 September 1997/Revised: 20 January 1998 相似文献
10.
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
11.
Glucagon is known to increase intracellular cAMP levels and enhance glucose-induced electrical activity and insulin secretion
in pancreatic β-cell perfused with Krebs-Ringer bicarbonate solution. The present experiments were aimed at evaluation of
the hypothesis that changes in β-cells ATP-sensitive K+ (K(ATP)) channel activity are involved in the glucagon-induced enhancement of electrical activity. Channel activity was recorded
using the cell-attached configuration of the patch-clamp technique. Addition of glucagon (2.9 × 10−7
m) in the presence of 11.1 mm glucose caused closure of K(ATP) channels followed by an increase in the frequency of biphasic current transients (action currents) due to action potential
generation in the cell. Three calmodulin-antagonists (W-7, chlorpromazine, and trifluoperazine) restored with similar efficacy
K(ATP) channel activity in cells being exposed to glucagon. At 2.8 mm glucose, glucagon did not affect K(ATP) channel activity until Ca2+ was released from Nitr-5 by flash photolysis, at which point channel activity was transiently suppressed. Similar effects
were seen when db-cAMP was used instead of glucagon.These results support the view that glucagon and other cAMP-generating
agonists enhance glucose-induced β-cell electrical activity through a Ca2+/calmodulin dependent-closure of K(ATP) channels.
Received: 26 May 1998/Revised: 18 September 1998 相似文献
12.
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
13.
Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures.
In whole cells, depolarization to voltages higher than +80 mV evoked a large K+ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC50= 5.3 mm). Raising the internal Ca2+ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V
1/2) of the K+ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca2+-activated (BK
Ca
) K+ channel with a conductance of 296 pS (130 mm K+ at both sides of the patch). V
1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca2+, respectively, at the internal face of the patch. In cell-attached patches the open probability (P
o
) of BK
Ca
channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K+ currents with 10 nm Ca2+ in the pipette. Application of 20 μm cytochalasin D increased P
o
of BK
Ca
channels in cell-attached patches within minutes. These data suggest that the activation of BK
Ca
channels in meningioma cells does not only depend on voltage and internal Ca2+ but is also controlled by the cytoskeleton.
Received 18 June 1999/Revised: 18 January 2000 相似文献
14.
H.S. Cheng P.Y. Leung S.B. Cheng Chew P.S. Leung S.Y. Lam W.S. Wong Z.D. Wang H.C. Chan 《The Journal of membrane biology》1998,164(2):155-167
The present study investigated both HCO−
3 and Cl− secretions in a human pancreatic duct cell line, CAPAN-1, using the short-circuit current (I
sc
) technique. In Cl−/HCO−
3-containing solution, secretin (1 μm) or forskolin (10 μm) stimulated a biphasic rise in the I
sc
which initially reached a peak level at about 3 min and then decayed to a plateau level after 7 min. Removal of external
Cl− abolished the initial transient phase in the forskolin-induced I
sc
while the plateau remained. In HCO−
3/CO2-free solution, on the contrary, only the initial transient increase in I
sc
was prominent. Summation of the current magnitudes observed in Cl−-free and HCO−
3-free solutions over a time course of 10 min gave rise to a curve which was similar, both in magnitude and kinetics, to the
current observed in Cl−/HCO−
3-containing solution. Removal of external Na+ greatly reduced the initial transient rise in the forskolin-induced I
sc
response, and the plateau level observed under this condition was similar to that obtained in Cl−-free solution, suggesting that Cl−-dependent I
sc
was also Na+-dependent. Bumetanide (50 μm), an inhibitor of the Na+-K+-2Cl− cotransporter, and Ba2+ (1 mm), a K+ channel blocker, could reduce the forskolin-induced I
sc
obtained in Cl−/HCO−
3-containing or HCO−
3-free solution. However, they were found to be ineffective when external Cl− was removed, indicating the involvement of these mechanisms in Cl− secretion. On the contrary, the HCO−
3-dependent (in the absence of external Cl−) forskolin-induced I
sc
could be significantly reduced by carbonic anhydrase inhibitor, acetazolamide (45 μm). Basolateral application of amiloride (100 μm) inhibited the I
sc
; however, a specific Na+-H+ exchanger blocker, 5-N-methyl-N-isobutylamiloride (MIA, 5–10 μm) was found to be ineffective, excluding the involvement of
the Na+-H+ exchanger. However, an inhibitor of H+-ATPase, N-ethylmaleimide did suppress the I
sc
(IC50= 22 μm). Immunohistochemical studies also confirmed the presence of a vacuolar type of H+-ATPase in these cells. H2DIDS (100 μm), an inhibitor of Na+-HCO−
3 cotransporter, was without effect. Apical addition of Cl− channel blocker, diphenylamine-2,2′-dicarboxylic acid (DPC, 1 mm), but not disulfonic acids, DIDS (100 μm) or SITS (100 μm), exerted an inhibitory effect on both Cl− and HCO−
3-dependent forskolin-induced I
sc
responses. Histochemical studies showed discrete stainings of carbonic anhydrase in the monolayer of CAPAN-1 cells, suggesting
that HCO−
3 secretion may be specialized to a certain population of cells. The present results suggest that both HCO−
3 and Cl− secretion by the human pancreatic duct cells may occur concurrently and independently.
Received: 17 October 1997/Revised: 3 April 1998 相似文献
15.
To study vacuolar chloride (Cl−) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl− uptake into isolated tonoplast vesicles was measured using the Cl−-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm,
respectively, and showed a high sensitivity towards Cl−, with a Stern-Volmer constant of 173 m
−1 in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched,
if at all, by other anions. However, the fluorescence intensity and Cl−-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as
the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K
m
of 17.2 mm and a V
max
of 4.8 mm min−1. Vacuolar Cl− transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm
p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl− transport was actually significantly increased by 24%. To determine absolute fluxes of Cl− using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be
1.13 × 107 m−1. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl− transport of 31 nmol m−2 sec−1 was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques.
Received: 18 February 2000/Revised: 30 June 2000 相似文献
16.
We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control
in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to
measure the intracellular [Na+]i in these cells loaded with the Na+ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na+]i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations
up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 μm RG-13022) and cPLA2 activity (10 μm AACOCF3) obviated EGF-induced increases in [Na+]i. In contrast, PGE2 (10 μg/ml) and cAMP (2 mm) increased [Na+]i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 μm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na+]i. Similarly, EGF failed to increase [Na+]i following inhibition of: 1) PKA activity (10 μm H-89); 2) Erk1/2 (15 μm PD98059) or 3) p38 (15 μm SB203580) activity. Stimulation protein kinase C activity (0.1 μm PMA) transiently increased [Na+]i followed by a decline towards its baseline value. EGF-induced increases in [Na+]i were unaltered by inhibition of K+ conductance (100 μm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA2 activity. Their stimulation increases PGE2 and cAMP levels resulting in PKA and NKCC activation.
Received: 18 December 2000/Revised: 24 May 2001 相似文献
17.
A. Chorvatova A. Guyot C. Ojeda O. Rougier A. Bilbaut 《The Journal of membrane biology》1998,162(1):39-50
The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the
whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which
began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These
effects were abolished by Losartan (10−5
m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized
in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method,
a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal
stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau
current. In Cl−-free solution, the outward current reversed at −78.5 mV, a value close to E
K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K+-free solution, the transient outward current, sensitive to Cl− channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E
Cl. Its magnitude changed in the same direction as the driving force for Cl−. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca2+-free solution (47% of cells) and was reversibly blocked by Cd2+ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca2+ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin
II is due to Ca2+-dependent K+ and Cl− currents. These two membrane currents are co-activated in response to an internal increase of [Ca2+]
i
originating from intra- and extracellular stores.
Received: 29 May 1997/Revised: 4 November 1997 相似文献
18.
Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of
Cl−-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats.
In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl− currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl− currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10−5
m Ca2+ via the patch pipette also led to a transient activation of Cl− currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3
led to release of Ca2+ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a
mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which
show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl−-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the
second messenger IP3.
Received: 17 July 1995/Revised: 31 January 1996 相似文献
19.
In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl− secretion to secretagogues acting via cAMP. Using a Ca2+ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca2+]
i
via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase
in [Ca2+]
i
in normal (16HBE14o− cell line and primary lung culture) and in cystic fibrosis (CFTE29o− cell line) human airway epithelia. The potency order of nucleotides on [Ca2+]
i
variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca2+]
i
response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o− cells, the forskolin-induced [Ca2+]
i
response increased with increasing temperature. In CFTE29o− cells, forskolin had no effect on [Ca2+]
i
at body temperature-forskolin-induced [Ca2+]
i
response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead
of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca2+]
i
response. Together, these results demonstrate that once activated, CFTR regulates [Ca2+]
i
by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia.
Received: 3 April 2000/Revised: 30 June 2000 相似文献
20.
P. A. Sansberro H. Y. Rey L. A. Mroginski M. M. Collavino 《Journal of Plant Growth Regulation》1998,17(2):101-105
The effects of benzyladenine (BAP), kinetin (KIN), zeatin (ZEA), isopentenyladenine (2iP), and thidiazuron (TDZ) were studied
on in vitro growth of rudimentary embryos of Ilex paraguariensis St. Hil. Heart stage zygotic embryos were removed from seeds of immature, light green fruits and cultured aseptically on
quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar, and supplemented with or without three concentrations
of BAP, KIN, ZEA, 2iP, or TDZ. Cultures were incubated in darkness at 27 ± 2°C. Media containing 4.4 × 10−6
m BAP, 4.6 × 10−6
m KIN, or 4.9 × 10−6
m 2iP were totally ineffective in inducing embryo growth after culture for 28 days. However, lower concentrations of these
compounds (4.4 × 10−8
m BAP, 4.6 × 10−8
m KIN, 4.5 × 10−8
m ZEA, or 4.9 × 10−8
m 2iP) promoted embryo growth. TDZ at 9.9 × 10−9
m, 9.9 × 10−8
m, or 9.9 × 10−7
m induced embryo growth at similar rates. The maximum percentage of embryos converted to seedlings was achieved when the medium
was supplemented with 4.5 × 10−7
m ZEA.
Received August 1, 1997; accepted February 19, 1998 相似文献