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1.
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
2.
It has previously been shown that osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical
collecting duct cells [54] and in a variety of other cell types [20]. In the present study we further characterized the shrinkage-activated
NSC channel in M-1 cells and its mechanism of activation using whole-cell current recordings. Osmotic cell shrinkage induced
by addition of 100 mm sucrose to the bath solution caused a 20-fold increase in whole-cell inward currents from −10.8 ± 1.5 pA to −211 ± 10.2 pA
(n= 103). A similar response was observed when cell shrinkage was elicited using a hypo-osmotic pipette solution. This indicates
that cell shrinkage and not extracellular osmolarity per se is the signal for current activation. Cation substitution experiments revealed that the activated channels discriminate poorly
between monovalent cations with a selectivity sequence NH4 (1.2) ≥ Na+ (1) ≈ K+ (0.9) ≈ Li+ (0.9). In contrast there was no measurable permeability for Ca2+ or Ba2+ and the cation-to-anion permeability ratio was about 14. The DPC-derivatives flufenamic acid, 4-methyl-DPC and DCDPC were
the most effective blockers followed by LOE 908, while amiloride and bumetanide were ineffective. The putative channel activator
maitotoxin had no effect. Current activation was dependent upon the presence of intracellular ATP and Mg2+ and was inhibited by staurosporine (1 μm) and calphostin C (1 μm). Moreover, cytochalasin D (10 μm) and taxol (2 μm) reduced the current response to cell shrinkage. These findings suggest that the activation mechanism of the shrinkage-activated
NSC channel involves protein kinase mediated phosphorylation steps and cytoskeletal elements.
Received: 3 May 2000/Revised: 6 July 2000 相似文献
3.
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
4.
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
5.
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
6.
The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using
continuous short circuit current (I
SC
) measurements in HCO3
−/CO2 buffered solution. Steady state I
SC
averaged 73.8 ± 3.2 μA/cm2 (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na+ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y+ and a basal amino acid transport system y+L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic
amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I
SC
averaging 36.6 ± 5.4 μA/cm2 (n= 19) and 32.0 ± 7.2 μA/cm2 (n= 8), respectively. The response was preserved in the absence of bath Cl− (n= 4), but was abolished either in the absence of apical Na+ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I
SC
response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization
of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na+ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract
passive cationic amino acid leak into the CCD in the presence of electrogenic Na+ transport, consistent with reports of stimulated expression of Na+ and cationic amino acid transporters by aldosterone.
Received: 11 September 2000/Revised: 6 December 2000 相似文献
7.
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
8.
The Ca2+-activated maxi K+ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean
number of open channels (NP
o
) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the
stretch-activation of Ca2+-permeable cation channels, because the maxi K+ channel was not stretch-activated in both the cell-attached configuration using Ca2+-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K+ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K+ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K+ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV
t
) when luminal K+ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV
t
was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced
ΔV
t
to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV
t
to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV
t
was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV
t
=−7.2 ± 0.5 mV in control vs.ΔV
t
=−6.7 ± 0.6 mV in AA). These results suggest that the Ca2+-activated maxi K+ channel is responsible for flow-dependent K+ secretion by coupling with the stretch-activated Ca2+-permeable cation channel in the rabbit CNT.
Received: 21 August 1997/Revised: 20 March 1998 相似文献
9.
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
10.
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
11.
We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH2-terminal domain of the Shaker K+ channel with purified, ryanodine-modified, cardiac Ca2+-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a
holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a
single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec−1μm
−1 for ShB and 2000 ± 200 sec−1μm
−1 for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied
potential with the bulk of the voltage-dependence residing in Koff. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As
is the case with inactivation and block of K+ channels, this is mediated by a large increase in Kon. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites
which will contribute to the ion handling properties of this channel.
Received: 15 December 1997/Revised: 13 March 1998 相似文献
12.
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
13.
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
14.
The effect of cyanide (CN−) on voltage-activated or cAMP-induced passive chloride conductance (G
Cl
) was analyzed in isolated toad skin. Comparatively low concentrations of CN− inhibited G
Cl
almost completely and fully reversibly, regardless of whether it was applied from the mucosal or serosal side. The IC50 was 180 ± 12 μm for voltage-activated G
Cl
and 305 ± 30 μm for the cAMP-inducted conductance. At [CN] <100 μm, the initial inhibition frequently declined partly in the continuous presence of CN−. Inhibition was independent of the presence of Ca2+. Inhibition was stronger at more alkaline pH, which suggests that dissociated CN− is the effective inhibitor. The onset of the inhibition of voltage-activated or cAMP-induced G
Cl
by CN− occurred with half-times of 34 ± 10 sec, whereas reversibility upon washout was twice as fast (18 ± 7 sec). If [CN−] <200 μm was applied under inactivating conditions (serosa −30 mV), the reduction of G
Cl
was stronger upon subsequent voltage-activation than under steady-state activated conditions. This effect was essentially
complete less than 30 sec after apical addition of CN−, but G
t
recovered thereafter partially in the continuous presence of CN−. Dinitrophenol inhibited G
Cl
similarly, while omission of oxygen did not affect it. These observations, as well as the time course of inhibition and the
full reversibility, suggest that interference of CN− with oxidative phosphorylation and subsequent metabolic depletion is not the reason for the inhibition of G
Cl
. We propose that the inhibition is directly on G
Cl
, presumably by competition with Cl− at a rate-limiting site in the pathway. Location and molecular nature of this site remain to be identified.
Received: 8 February 1999/Revised: 22 September 1999 相似文献
15.
Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial
glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (p
Gly
) was 2.3 ± 0.23 × 10−6 cm sec−1 with MIP vs. 0.92 ± 0.086 × 10−6 cm sec−1 in control oocytes. The p
Gly
of MIP was independent of concentration from 5 × 10−5 to 5 × 10−2
m, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mm Hg++, 0.2 mm DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mm Cu++, respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This
could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based
on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase
(0.12 ± 0.03 vs. 0.06 ± 0.01 pmol min−1 cell−1) without changing the binding of glycerol to the kinase (K
M
∼ 10 μm).
Received: 23 May 1997/Revised: 4 August 1997 相似文献
16.
Basolateral K+ Conductance Establishes Driving Force for Cation Absorption by Outer Sulcus Epithelial Cells 总被引:1,自引:0,他引:1
Outer sulcus epithelial cells were recently found to actively reabsorb cations from the cochlear luminal fluid, endolymph,
via nonselective cation channels in the apical membrane. Here we determined the transport properties of the basolateral membrane
with the whole-cell patch clamp technique; the apical membrane contributed insignificantly to the recordings. Outer sulcus
epithelial cells exhibited both outward and inward currents and had a resting membrane potential of −90.4 ± 0.7 mV (n= 78), close to the Nernst potential for K+ (−95 mV). The reversal potential depolarized by 54 mV for a tenfold increase in extracellular K+ concentration with a K+/Na+ permeability ratio of 36. The most frequently observed K+ current was voltage independent over a broad range of membrane potentials. The current was reduced by extracellular barium
(10−5 to 10−3
m), amiloride (0.5 mm), quinine (1 mm), lidocaine (5 mm) and ouabain (1 mm). On the other hand, TEA (20 mm), charybdotoxin (100 nm), apamin (100 nm), glibenclamide (10 μm), 4-aminopyridine (1 mm) and gadolinium (1 mm) had no significant effect. These data suggest that the large K+ conductance, in concert with the Na+,K+-ATPase, of the basolateral membrane of outer sulcus cells provides the driving force for cation entry across the apical membrane,
thereby energizing vectorial cation absorption by this epithelium and contributing to the homeostasis of endolymph. 相似文献
17.
T. Miyashita H. Tatsumi H. Furuta N. Mori M. Sokabe 《The Journal of membrane biology》2001,182(2):113-122
We identified a Ca2+-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the
patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K+= Na+ > Ca2+≫ Cl−. This channel was weakly voltage-dependent but was strongly activated by Ca2+ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca2+ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca2+ concentration ([Ca2+]i), activated the channel at an estimated [Ca2+]i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches.
Based on this Ca2+ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing
Na+ under certain pathological conditions that will increase [Ca2+]i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells.
Received: 24 October 2000/Revised: 10 April 2001 相似文献
18.
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
19.
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
20.
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