共查询到20条相似文献,搜索用时 593 毫秒
1.
2.
John M. Bayliss W. Brian Reeves Thomas E. Andreoli 《The Journal of membrane biology》1990,113(1):49-56
Summary This paper provides the results of studies which characterized conductive36Cl– flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive36Cl– uptake was studied under two different experimental conditions. In the first,36Cl– flux was driven by an inside positive voltage created with oppositely directed Cl– and gluconate gradients. In the second, an inwardly direct K+ gradient was used to drive36Cl– uptake. By these two methods, voltage-sensitive36Cl– uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total36Cl– flux. Separate paired studies demonstrated that the conductive36Cl– uptake was inhibited by the Cl– channel blocker diphenylamine-2-carboxylate (DPC) with an IC50 for DPC of 154 m. The voltagedependent36Cl– uptake had an activation energy of 6.4 kcal/mole. This36Cl– conductance had an anion selectivity sequence of I–>Cl–NO
3
–
gluconate. 相似文献
3.
Summary The present studies examined some of the properties of Cl– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP
Cl/P
K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl– conductance of isolated medullary thick ascending limb segments. The Cl– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca2+ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca2+ concentrations, but was inactivated by reducing KCl concentrations to 50mm. 相似文献
4.
Short-circuit current (I
sc
), transepithelial conductance (G
t
), electrical capacitance (C
T
) and the fluctuation in I
sc
were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na+- and Cl−-free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases
in G
t
, C
T
, I
sc
and generated a Lorentzian I
sc
-noise. The responses could be related to active, electrogenic secretion of Cl−. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable
cAMP analogue or forskolin showed stable increases in G
t
with only moderate peaking of I
sc
. Phosphodiesterase inhibitors also stimulated Cl− secretion with peaking responses in G
t
and I
sc
. All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl− channel, with almost identical corner frequency (40–50 Hz). Repetitive challenge with the hormones led to a refractory behavior
of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused C
T
, a measure of apical membrane area, to increase in a manner roughly synchronous with G
t
. These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular
Cl− channel pool. Apical flufenamate depressed Cl− current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments
could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel
inhibitor.
Received: 20 May 1998/Revised: 8 September 1998 相似文献
5.
《Cell calcium》2019
There is tight interplay between Ca2+ and Cl− flux that can influence brain tumour proliferation, migration and invasion. Glioma is the predominant malignant primary brain tumour, accounting for ˜80% of all cases. Voltage-gated Cl− channel family (ClC) proteins and Cl− intracellular channel (CLIC) proteins are drastically overexpressed in glioma, and are associated with enhanced cell proliferation, migration and invasion. Ca2+ also plays fundamental roles in the phenomenon. Ca2+-activated Cl− channels (CaCC) such as TMEM16A and bestrophin-1 are involved in glioma formation and assist Ca2+ movement from intracellular stores to the plasma membrane. Additionally, the transient receptor protein (TRP) channel TRPC1 can induce activation of ClC-3 by increasing intracellular Ca2+concentrations and activating Ca2+/calmodulin-dependent protein kinase II (CaMKII). Therefore, Ca2+ and Cl−currents can concurrently mediate brain tumour cellular functions. Glioma also expresses volume regulated anion channels (VRACs), which are responsible for the swelling-induced Cl− current, ICl,swell. This current enables glioma cells to perform regulatory volume decrease (RVD) as a survivability mechanism in response to hypoxic conditions within the tumour microenvironment. RVD can also be exploited by glioma for invasion and migration. Effective treatment for glioma is challenging, which can be in part due to prolonged chemotherapy leading to mutations in genes associated with multi-drug resistances (MRP1, Bcl-2, and ABC family). Thus, a potential therapeutic strategy for treatment of glioma can be through the inhibition of selected Cl− channels. 相似文献
6.
7.
Christopher J. Winters W. Brian Reeves Thomas E. Andreoli 《The Journal of membrane biology》1992,128(1):27-39
Summary Cl– channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl– activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl– in the absence of PKA. The PKA requirement for activation of Cl– channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl– concentrations on the properties of Cl– channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl– channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl– concentration from 2 to 50 mm increased both channel activity (P
o) and channel conductance (g
Cl, pS). Likewise, trans PKA increased mTALH Cl– channel activity by increasing the activity of individual channels when the trans solutions were 2 mm Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect g
Cl. Moreover, when mTALH Cl– channels were inactivated by reducing cis Cl– concentrations to 50 mm, cis PKA addition did not affect P
o. These results are consistent with the view that these Cl– channels originated from basolateral membranes of the mTALH.Cl– channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl– concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl– channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl– channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl– channel activity by both intracellular Cl– and by PKA differ in basolateral mTALH Cl– channels compared to apical Cl– channels from either the tracheal or small intestine.We acknowledge the able technical assistance of Steven D. Chasteen. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veterans Administration Merit Review Grants to T.E. Andreoli and to W.B. Reeves. C.J. Winters is a Veterans Administration Associate Investigator. 相似文献
8.
9.
Summary The current-voltage relationship of carrier-mediated, passive and active ion transport systems with one charge-carrying pathway can exactly be described by a simple reaction kinetic model. This model consists of two carrier states (one inside, one outside) and two pairs (forwards and backwards) of rate constants: a voltage-dependent one, describing the transport of charge and a voltage-insensitive one, summarizing all the other (voltage-independent) reactions. For the electrogenic Cl– pump inAcetabularia these four rate constants have been determined from electrical measurements of the current-voltage relationship of the pump (Gradmann, Hansen & Slayman, 1981;in: Electrogenic Ion Pumps, Academic Press, New York). The unidirectional Cl– efflux through the pump can also be calculated by the availiable reaction kinetic parameters.36Cl– efflux experiments on singleAcetabularia cells with simultaneous electrical stimulation (action potentials) and recording, demonstrate the unidirectional Cl– efflux to depend on the membrane potential. After subtraction of an efflux portion which bypasses the pump, agreement is found between the measured flux-voltage relationship and the theoretical one as obtained from the reaction kinetic model and its parameters from the electrical data. 相似文献
10.
《生物化学与生物物理学报:生物膜》1997,1330(2):110-112
Basolateral membranes of Aplysia foregut epithelia contain an ATP-dependent Cl− transporter (Cl− pump). Increased activity of the Cl− pump, coupled to apical and basolateral membrane depolarization, changed the Cl− transport energetics across the apical membrane but did not change the vectorially-opposite Cl− transport energetics across the basolateral membrane. 相似文献
11.
Summary Active HCO
3
t-
secretion in the anterior rectal salt gland of the mosquito larva,Aedes dorsalis, is mediated by a 11 Cl–/HCO
3
–
exchanger. The cellular mechanisms of HCO
3
–
and Cl– transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO2 and HCO
3
–
from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (V
a) and had little effect on basolateral potential (V
bl). Changes in luminal Cl– concentration alteredV
a in a repid, linear manner with a slope of 42.2 mV/decaloga
Cl
l
–. Intracellular Cl– activity was 23.5mm and was approximately 10mm lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl– concentration had no effect onV
bl, indicating an electrically silent basolateral Cl– exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated
was 14.4mm. These results show that under control conditions HCO3 enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl–-selective channel, and HCO
3
–
exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl– exit and HCO3 entry, and the effects of serosal addition of the Cl–/HCO3 exchange inhibitor, DIDS, on
and transepithelial potential (V
ic) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl– and HCO
3
–
movements. 相似文献
12.
M. Missori M. Papi G. Maulucci G. Arcovito G. Boumis A. Bellelli G. Amiconi M. De Spirito 《European biophysics journal : EBJ》2010,39(6):1001-1006
Ischemic heart disease is the leading cause of serious morbidity and mortality in Western society. One of the therapeutic
approaches is based on the use of thrombolitic drugs that promote clot lysis. Even if the mechanisms leading to clot lysis
are not completely understood, it is widely accepted that they depend on the complex biochemical reactions that occur among
fibrin fibers and fibrinolitic agents, and by their ready diffusion into the fibers. Here we investigate the effects of specific
anions on the architecture of protofibrils within fibrin fibers in fibrin gels prepared in a para-physiological solution.
The results obtained through small-angle X-ray scattering (SAXS) demonstrate that the characteristic axial and longitudinal
repeat distances among protofibrils are strongly affected by the action of Cl− and F− anions. 相似文献
13.
Janove Sehlin 《Bioscience reports》1987,7(1):67-72
Microdissected -cell-rich pancreatic islets fromob/ob-mice were used in studies of transmembrane36Cl– efflux. The mean rate coefficient for36Cl– efflux was stable at 0.158 min–1 during the initial 10 min. Depolarization of the -cell plasma membrane by acute increases in extracellular K+ (5–130mM) stimulated the36Cl– efflux in a concentration-dependent manner. Glucose-induced (20mM) and K+-induced increases in36Cl– efflux were largely overlapping, but even at 135.9 mM K+, glucose slightly further enhanced the36Cl– efflux rate. The data suggest (1) that pancreatic -cells are equipped with a voltage-dependent Cl– permeability, (2) that glucose-induced increase in Cl– permeability may, at least partly, be mediated by primary membrane depolarization, and (3) that glucose in addition may activate other mechanisms for -cell Cl– transport. 相似文献
14.
15.
O. Zegarra-Moran O. Sacco L. Romano G.A. Rossi L.J.V. Galietta 《The Journal of membrane biology》1997,156(3):297-305
The perforated-patch technique was used to study the response of human bronchial cells to extracellular nucleotides. ATP
or UTP (100 μm) elicited a complex response consisting of a large transient membrane current increase followed by a relatively small sustained
level. These two phases were characterized by different current kinetics. Throughout the transient phase (2–3 min) the membrane
current (I
p
) displayed slow activation and deactivation kinetics at depolarizing and hyperpolarizing potentials respectively. At steady-state
(I
s
) the relaxation at hyperpolarizing potential disappeared whereas at positive membrane potentials the current became slightly
deactivating. The I
s
amplitude was dependent on the extracellular Ca2+ concentration, being completely inhibited in Ca2+-free medium. Cell pre-incubation with the membrane-permeable chelating agent BAPTA/AM prevented completely the response to
nucleotides, thus suggesting that both I
p
and I
s
were dependent on intracellular Ca2+. The presence of a hypertonic medium during nucleotide stimulation abolished I
s
leaving I
p
unchanged. On the contrary, niflumic acid, a blocker of Ca2+-activated Cl− channels, prevented completely I
p
without reducing significantly I
s
. 1,9-dideoxyforskolin fully inhibited I
s
but also reduced I
p
. Replacement of extracellular Cl− with aspartate demonstrated that the currents activated by nucleotides were Cl− selective. I
p
resulted five times more Cl− selective than I
s
with respect to aspartate. Taken together, our results indicate that ATP and UTP activate two types of Cl− currents through a Ca2+-dependent mechanism.
Received: 15 August 1996/Revised: 6 December 1996 相似文献
16.
Our previously published whole-cell patch-clamp studies on the cells of the intralobular (granular) ducts of the mandibular glands of male mice revealed the presence of an amiloride-sensitive Na+ conductance in the plasma membrane. In this study we demonstrate the presence also of a Cl– conductance and we show that the sizes of both conductances vary with the Cl– concentration of the fluid bathing the cytosolic surface of the plasma membrane. As the cytosolic Cl– concentration rises from 5 to 150 mmol/liter, the size of the inward Na+ current declines, the decline being half-maximal when the Cl– concentration is approximately 50 mmol/liter. In contrast, as cytosolic Cl– concentration increases, the inward Cl– current remains at a constant low level until the Cl– concentration exceeds 80 mmol/liter, when it begins to increase. Studies in which Cl– in the pipette solution was replaced by other anions indicate that the Na+ current is suppressed by intracellular Br-, Cl– and NO
3
-
but not by intracellular I-, glutamate or gluconate. Our studies also show that the Cl– conductance allows passage of Cl– and Br- equally well, I-less well, and NO
3
-
, glutamate and gluconate poorly, if at all. The findings with NO
3
-
are of particular interest because they show that suppression of the Na+ current by a high intracellular concentration of a particular anion does not depend on actual passage of that anion through the Cl– conductance. In mouse granular duct cells there is, thus, a reciprocal regulation of Na+ and Cl– conductances by the cytosolic Cl– concentration. Since the cytosolic Cl– concentration is closely correlated with cell volume in many epithelia, this reciprocal regulation of Na+ and Cl– conductances may provide a mechanism by which ductal Na+ and Cl transport rates are adjusted so as to maintain a stable cell volume.This project was supported by the National Health and Medical Research Council of Australia. We thank Professor P. Barry (University of New South Wales) for assistance with the junction potential measurements. 相似文献
17.
Adam Sun Eric B. Grossman Michael Lombardi Steven C. Hebert 《The Journal of membrane biology》1991,120(1):83-94
Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K+ dependence of the apical, furosemide-sensitive Na+:Cl– cotransporter and on transport-related oxygen consumption (QO2). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K+ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K+)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na+ and Cl–, but not K+, while in the presence of AVP the apical cotransporter required all three ions.86Rb+ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,22Na+ uptake was unaffected by extracellular K+ in the absence of AVP while after AVP exposure22Na+ uptake was strictly K+-dependent. The AVP-induced coupling of K+ to the Na+:Cl– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular22Na+ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na+:Cl– cotransport to Na+:K+:2Cl– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K+ to the apical Na+:Cl– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed. 相似文献
18.
Christopher J. Winters W. Brian Reeves Thomas E. Andreoli 《The Journal of membrane biology》1991,122(1):89-95
Summary We examined the interactions of cAMP-dependent protein kinase and varying aqueous Cl– concentrations in modulating the activity of Cl– channels obtained by fusing basolaterally enriched renal outer medullary vesicles into planar lipid bilayers. Under the present experimental conditions, thecis andtrans solutions face the extracellular and intracellular aspects of these Cl– channels, respectively. Raising thetrans Cl– concentration from 2 to 50mm increased the channel open-time probability, raised the unit channel conductance, and affected the voltage-independent determinant (G) of channel activity but not the gating charge (Winters, C.J., Reeves, W.B., Andreoli, T.E. 1990.J. Membrane Biol.
118:269–278). With 2mm
trans KCl,trans addition of the catalytic subunit of PKA (C-PKA) plus ATP increased channel open-time probability and altered the voltage-independent determinant of channel activity without affecting either unit channel conductance or gating charge. The effect was ATP specific, did not occur with (C-PKA plus ATP) addition tocis solutions, and was abolished by denaturing C-PKA. Finally, (C-PKA plus ATP) activation of channel activity was not detected with relatively high (50mm)trans Cl– concentrations. These data indicate that (C-PKA plus ATP) might modulate Cl– channel activity by phosphorylation at or near the Cl–-sensitive site on the intracellular face of these channels. 相似文献
19.
Summary The potential dependence of unidirectional36Cl fluxes through toad skin revealed activation of a conductive pathway in the physiological region of transepithelial potentials. Activation of the conductance was dependent on the presence of Cl– or Br– in the external bathing solution, but was independent of whether the external bath was NaCl-Ringer's, NaCl-Ringer's with amiloride, KCl-Ringer's or choline Cl-Ringer's To partition the routes of the conductive Cl– ion flow, we measured in the isolated epithelium with double-barrelled microelectrodes apical membrane potentialV
a
, and intracellular Cl– activity,a
Cl
c
, of the principal cells indentified by differential interference contrast microscopy. Under short-circuit conditionsI
sc=27.0±2.0 A/cm2, with NaCl-Ringer's bathing both surfaces,V
a was –67.9±3.8mV (mean ±se,n=24, six preparations) anda
Cl
c
was 18.0±0.9mM in skins from animals adapted to distilled water. BothV
a anda
Cl
a
were found to be positively correlated withI
sc (r=0.66 andr=0.70, respectively). In eight epithelia from animals adapted to dry milieu/tap waterV
a anda
Cl
c
were measured with KCl Ringer's on the outside during activation and deactivation of the transepithelial Cl– conductance (G
Cl) by voltage clamping the transepithelial potential (V) at 40 mV (mucosa positive) and –100 mV. AtV=40 mV; i.e. whenG
Cl was deactivated,V
a
was –70.1±5.0 mV (n=15, eight preparations) anda
Cl
c
was 40.0±3.8mm. The fractional apical membrane resistance (fR
a) was 0.69±0.03. Clamping toV=–100 mV led to an instantaneous change ofV
a
to 31.3±5.6 mV (cell interior positive with respect to the mucosal bath), whereas neithera
Cl
c
norfR
a changed significantly within a 2 to 5-min period during whichG
Cl increased by 1.19±0.10 mS/cm2. WhenV was stepped back to 40 mV,V
a
instantaneously shifted to –67.8±3.9 mV whilea
Cl
c
andfR
a remained constant during deactivation ofG
Cl. Similar results were obtained in epithelia impaled from the serosal side. In 12 skins from animals adapted to either tap water or distilled water the density of mitochondria-rich (D
MRC) cells was estimated and correlated with the Cl current (I
Cl though the fully activated (V=–100mV) Cl– conductance). A highly significant correlation was revealed (r=–0.96) with a slope of –2.6 nA/m.r. (mitochondria-rich cell and an I-axis intercept not significantly different from zero. In summary, the voltage-dependent Cl currents were not reflected infR
a anda
Cl
a
of the principal cells but showed a correlation with the m.r. cell density. We conclude that the pricipal cells do not contribute significantly to the voltage-dependent Cl conductance. 相似文献
20.
Using the inside-out patch clamp technique, we identified a Cl? channel in patches from the membrane of cultured human hematopoietic myeloblastic leukemia ML-1 cells. The Cl? channel was not seen at negative membrane potentials in excised patches until the membrane potential was depolarized to greater than +40 mV. The channel was also activated by addition of cAMP-dependent protein kinase (PKA) catalytic subunit at physiological membrane potential (?40 mV). Biophysical studies of the Cl? channel revealed that the current-voltage (I-V) relationship of the Cl? channel was outwardly rectifying in symmetrical 142 mm Cl? solutions. Single channel conductances were 48 pS for the outward current measured at +60 mV and 27 pS for the inward current at ?60 mV. The open time constant of the channel was dependent on the membrane potential and was significantly prolonged at positive membrane potentials. Channels activated by cAMP-dependent protein kinase spent a significantly longer time in the open state compared to those channels activated by depolarization pulses. Pharmacological properties of the Cl? channel were also studied. Two anion transport inhibitors, anthracene-9-carboxylic acid (9-AC) and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) caused a flickering block of the channel. Half-inhibitory concentrations (IC50) for 9-AC and DIDS were 174 ± 20 and 70±16 μm, respectively. Blockade of the Cl? channel by 9-AC or DIDS was completely reversible. Our findings suggest that outwardly rectifying Cl? channels (ORCC) are present in human hematopoietic myeloblasts. The function of ORCC may be involved in hormone-regulated cell growth, cell volume regulation and immune responses. 相似文献