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1.
The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl− absorption linked to Na+ were investigated in isolated perfused tubules using Cl−-selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl− (a
i
Cl
) is 11.3 ± 0.5 mm, the basolateral (V
1
), apical (V
2
), and transepithelial (V
3
) potential differences are −68 ± 1.2 mV, +62 ± 1.2 mV and −6.4 ± 0.3 mV, respectively. When Na+ absorption is decreased by removal of organic substrates from the lumen, a
i
Cl
falls by 1.3 ± 0.3 mm and V
2
hyperpolarizes by +11.4 ± 1.7 mV. Subsequent removal of Na+ from the lumen causes a
i
Cl
to fall further by 2.3 ± 0.4 mm and V
2
to hyperpolarize further by +15.3 ± 2.4 mV. The contribution of transporters and channels to the observed changes of a
i
Cl
was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide
or hydrochlorothiazide have no effect on a
i
Cl
. The effects of luminal HCO−
3 removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO3 or Cl-OH. The effects of basolateral HCO−
3 removal, of basolateral Na+ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven
Cl-HCO3 exchange. Several lines of evidence favor conductive Cl− transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate
to the lumen or bath, increases the a
i
Cl
by 2.4 ± 0.6 mm or 2.9 ± 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO−
3-free Ringer, the equilibrium potential for Cl− does not differ from the membrane potential V
2
. Finally, the logarithmic changes in a
i
Cl
in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane
potential. These findings strongly support the presence of Cl− channels at the apical and basolateral cell membranes of the proximal tubule.
Received: 14 November 1997/Revised: 6 July 1998 相似文献
2.
The proximal tubule Na+-HCO−
3 cotransporter is located in the basolateral plasma membrane and moves Na+, HCO−
3, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO−
3 anions are transported for each Na+ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane
potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the
cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized
rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized
with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through
the cotransporter was achieved by applying Na+ or HCO−
3 concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference
current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl− independent; its conductance ranged between 0.12 and 0.23 mS/cm2 and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence
of Na+ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a
cotransport ratio of 3HCO−
3:1Na+.
Received: 19 January 1996/Revised: 24 April 1996 相似文献
3.
Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate
that luminal and basolateral ATP via P2Y2 receptors stimulate luminal Ca2+-activated Cl− channels and inhibit Na+ transport. Here we address the mechanism of ATP-mediated inhibition of Na+ transport. M-1 cells had a transepithelial voltage (V
te
) of −31.4 ± 1.3 mV and a transepithelial resistance (R
te
) of 1151 ± 28 Ωcm2. The amiloride-sensitive short circuit current (I
sc
) was −28.0 ± 1.1 μA/cm2. The ATP-mediated activation of Cl− channels was inhibited when cytosolic Ca2+ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca2+]i increase was paralleled by a rapid and transient R
te
decrease (297 ± 51 Ωcm2). In the presence of CPA, basolateral ATP led to an R
te
increase by 144 ± 17 Ωcm2 and decreased V
te
from −31 ± 2.6 to −26.6 ± 2.5 mV. I
sc
dropped from −28.6 ± 2.4 to −21.6 ± 1.9 μA/cm2. Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated
inhibition of Na+ absorption. Lowering [Ca2+]i by removal of extracellular Ca2+ did not alter the ATP effect. PKC inhibition or activation were without effect. Na+ absorption was activated by pHi alkalinization and inhibited by pHi acidification. ATP slightly acidified M-1 cells by 0.05 ± 0.005 pH units, quantitatively not explaining the ATP-induced effect.
In summary this indicates that extracellular ATP via luminal and basolateral P2Y2 receptors inhibits Na+ absorption. This effect is not mediated via [Ca2+]i, does not involve PKC and is to a small part mediated via intracellular acidification.
Received: 9 February 2001/Revised: 17 May 2001 相似文献
4.
J.I. Kourie 《The Journal of membrane biology》1999,167(1):73-83
The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca2+ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of
the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pH
cis
) and luminal pH (pH
trans
) was investigated using the lipid bilayer-vesicle fusion technique. Low pH
cis
6.75–4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pH
cis
7.26–7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65–75 pS) whereas
at low pH
cis
6.75–4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5–40 pS). Similarly, low
pH
trans
4.07, but not pH
trans
6.28, modified the activity of SCl channels. The effects of low pH
cis
are pronounced at 10−3 and 10−4
m [Ca2+]
cis
but are not apparent at 10−5
m [Ca2+]
cis
, where the subconductances of the channel are already prominent. Low pH
cis
-induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of
the subconductance states. The results in this study suggest that low pH
cis
can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction
in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels.
Received: 20 May 1998/Revised: 24 September 1998 相似文献
5.
We used 31P NMR to investigate the temperature-dependence of intracellular pH (pH
i
) in isolated frog skeletal muscles. We found that ln[H+
i
] is a linear function of 1/T
abs paralleling those of neutral water (i.e., H+= OH−) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was unaffected
by inhibition of glycolysis and was not dependent upon the pH or [Na+] in the bathing solution. Insulin stimulation of Na+-H+ exchange shifted the intercept in the alkaline direction but had no effect on the slope. Acid loading followed by washout
resulted in an amiloride-sensitive return to the (temperature dependent) basal pH
i
.
These results show that the temperature dependence of activation of Na+-H+ exchange is similar to that of the intracellular buffers, and suggest that constancy of [H+]/[OH−] with changing temperature is achieved in the short term by intracellular buffering and in the long term by the set-point
of the Na+-H+ exchanger. Proton activation of the exchanger has an apparent standard enthalpy change (ΔH°) under both control and insulin-stimulated
conditions that is similar to the ΔH° of the intracellular buffers and approximately half of the ΔH° for the dissociation
of water. Thus, the temperature-dependent component of the standard free-energy change (ΔF°) is unaffected by insulin stimulation,
suggesting that changes in Arrhenius activation energy (E
a
) may not be a part of the mechanism of hormone stimulation.
Received: 12 February 1997/Revised: 1 October 1997 相似文献
6.
P. Macri S. Breton M. Marsolais J.-Y. Lapointe R. Laprade 《The Journal of membrane biology》1997,155(3):229-237
Collapsed proximal convoluted tubules (PCT) shrink to reach a volume 20% lower than control and do not exhibit regulatory
volume increase when submitted to abrupt 150 mOsm/kg hypertonic shock. The shrinking is accompanied by a rapid depolarization
of the basolateral membrane potential (V
BL) of 8.4 ± 0.5 mV, with respect to a control value of −54.5 ± 1.9 mV (n= 15). After a small and transient hyperpolarization, V
BL further depolarizes to reach a steady depolarization of 19.5 ± 1.5 mV (n= 15) with respect to control. In the post-control period, V
BL returns to −55.8 ± 1.5 mV. The basolateral partial conductance to K+ (t
K
) which is 0.17 ± 0.01 (n= 5) in control condition, decreases rapidly to nonmeasurable values during the hypertonic shock and returns to 0.23 ± 0.03
in the post-control period. The basolateral partial conductance to Cl− (t
Cl), which is 0.05 ± 0.02 (n= 5) in control, also decreases in hypertonicity to a nonmeasurable value and returns to 0.03 ± 0.01 in post control. The
partial conductance mediated by the Na-HCO3 cotransporter (t
NaHCO3), which is 0.48 ± 0.06 (n= 5) in control condition, remains the same at 0.44 ± 0.05 (n= 5) during the hypertonic period. Similarly, the membrane absolute conductance mediated by the Na-HCO3 cotransporter (G
Na-HCO3) does not vary appreciably. Concomitant with cell shrinkage, intracellular pH (pH
i
) decreases from a control value of 7.26 ± 0.01 to 7.13 ± 0.02 (n= 12) and then remains constant. Return to control solution brings back pH
i
to 7.28 ± 0.03. From these results, we conclude that in collapsed PCT, a sustained decrease in cellular volume leads to cell
acidification and to inhibition of K+ and Cl− conductances.
Received: 6 February 1996/Revised: 10 October 1996 相似文献
7.
An apical membrane ouabain-sensitive H-K exchange and a barium-sensitive basolateral membrane potassium channel are present
in colonic crypt cells and may play a role in both K absorption and intracellular pH (pHi) regulation. To examine the possible interrelationship between apical membrane H-K exchange and basolateral membrane K movement
in rat distal colon in the regulation of pHi, experiments were designed to assess whether changes in extracellular potassium can alter pHi. pHi in isolated rat crypts was determined using microspectrofluorimetric measurements of the pH-sensitive dye BCECF-AM (2′,7′-bis(carboxyethyl-5(6)-carboxy-fluorescein
acetoxy methylester). After loading with the dye, crypts were superfused with a Na-free solution which resulted in a rapid
and reversible fall in pHi (7.36 ± 0.02 to 6.98 ± 0.03). Following an increase in extracellular [K] to 20 mm, in the continued absence of Na, there was a further decrease in pHi (0.20 ± 0.02, P < 0.01). K-induced acidification was blocked both by 2 mm bath barium, a K channel blocker, and by 0.5 mm lumen ouabain. K-induced acidification was also observed when intracellular acidification was induced by a NH4Cl prepulse. These observations suggest that increased basolateral K movement increases intracellular [K] resulting in a decrease
in pHi that is mediated by a ouabain-sensitive apical membrane H,K-ATPase. Our results demonstrate an interrelationship between
basolateral K movement and apical H-K exchange in the regulation of pHi and apical K entry in rat distal colon.
Received: 31 March 1998/Revised: 8 September 1998 相似文献
8.
Davis BA Hogan EM Cooper GJ Bashi E Zhao J Boron WF 《The Journal of membrane biology》2001,183(1):25-32
Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular
alkali loads by moving K+ and HCO−
3 out of the cell, tending to lower intracellular pH (pHi). With an inwardly directed K/HCO3 gradient, the cotransporter mediates a net uptake of alkali (i.e., K+ and HCO−
3 influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA+) inhibit the inwardly directed cotransporter by interacting at the intracellular K+ site. We computed the equivalent HCO−
3 influx (J
HCO3) mediated by the cotransporter from the rate of pHi increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pHi 8.0, using a dialysis fluid (DF) free of K+, Na+ and Cl−. Our standard artificial seawater (ASW) also lacked Na+, K+ and Cl−. After halting dialysis, we introduced an ASW containing 437 mm K+ and 0.5% CO2/12 mm HCO−
3, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO2 influx, followed by a slower plateau-phase pHi increase, due to inward cotransport of K+ and HCO−
3. With no QA+ in the DF, J
HCO3 was ∼58 pmole cm−2 sec−1. With 400 mm tetraethylammonium (TEA+) in the DF, J
HCO3 was virtually zero. The apparent K
i
for intracellular TEA+ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K+ channels. Introducing 100 mm inhibitor into the DF reduced J
HCO3 to ∼20 pmole cm−2 sec−1 for tetramethylammonium (TMA+), ∼24 for TEA+, ∼10 for tetrapropylammonium (TPA+), and virtually zero for tetrabutylammonium (TBA+). The apparent K
i
value for TBA+ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA+), with an apparent K
i
of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO3 influx in the potency sequence PPTEA+ > TBA+ > TPA+ > TEA+≅ TMA+. The identification of inhibitors of the K/HCO3 cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.
Received: 21 November 2000/Revised: 14 May 2001 相似文献
9.
S.L. Wladkowski W. Lin M. McPheeters S.C. Kinnamon S. Mierson 《The Journal of membrane biology》1998,164(1):91-101
We used Ussing chamber measurements and whole-cell recordings to characterize a chloride conductance in rat lingual epithelium.
Niflumic acid (NFA) and flufenamic acid (FFA), nonsteroidal anti-inflammatory aromatic compounds known to inhibit Cl− conductances in other tissues, reduced transepithelial short-circuit current (I
sc
) in the intact dorsal anterior rat tongue epithelium when added from the serosal side, and reduced whole-cell currents in
rat fungiform taste cells. In both Ussing chamber and patch-clamp experiments, the effect of NFA was mimicked by replacement
of bath Cl− with methanesulfonate or gluconate. In low Cl− bath solution, the effect of NFA on whole-cell current was reduced. Replacement of bath Ca2+ with Ba2+ reduced the whole-cell Cl− current. We conclude that a Ca2+-activated Cl− conductance is likely present in the basolateral membrane of the rat lingual epithelium, and is present in the taste receptor
cells from fungiform papillae. Further experiments will be required to identify the role of this conductance in taste transduction.
Received: 8 September 1997/Revised: 27 March 1998 相似文献
10.
Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might
play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH
(pH
i
) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived
from rabbit NPE. pH
i
was measured using the fluorescent dye BCECF and the pH
i
responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared. ACTZ markedly inhibited the rapid
pH
i
changes elicited by bicarbonate/CO2 removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and
inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pH
i
. We considered whether CA-IV might facilitate H+ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mm) reduced pH
i
0.52 ± 0.10 pH units. In the presence of DBI, the magnitude of pH
i
reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 ± 0.09 pH units. ACTZ similarly reduced the magnitude of the pH
i
reduction. DBI also reduced by ∼40% the rate of pH
i
recovery in cells acidified by an ammonium chloride (20 mm) prepulse; a reduction in pH
i
recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pH
i
alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive
to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We
suggest that the CA-IV isozyme might catalyze rapid equilibration of H+ and HCO−
3 with CO2 in the unstirred layer outside the plasma membrane, preventing local accumulation of H+ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pH
i
changes that might alter the function of other ion transporters and channels in the NPE.
Received: 24 April 1997/Revised: 4 November 1997 相似文献
11.
Kummerow D Hamann J Browning JA Wilkins R Ellory JC Bernhardt I 《The Journal of membrane biology》2000,176(3):207-216
The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive
fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K+ efflux and Cl− loss. When human erythrocytes were suspended in a physiological NaCl solution (pH
o
= 7.4), the measured pH
i
was 7.19 ± 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution,
an immediate alkalinization increased the pH
i
to 7.70 ± 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed
to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl− content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison
of the calculated H+ influx with the measured unidirectional K+ efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were
enhanced by decreasing the ionic strength of the solution resulting in a H+ influx and a K+ efflux in LIS solution of 108.2 ± 20.4 mmol (l
cells
hr)−1 and 98.7 ± 19.3 mmol (l
cells
hr)−1, respectively. For bovine and porcine erythrocytes, in LIS media, H+ influx and K+ efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions.
Quinacrine, a known inhibitor of the mitochondrial K+(Na+)/H+ exchanger, inhibited the K+ efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K+(Na+)/H+ exchanger in the human erythrocyte membrane.
Received: 22 December 1999/Revised: 10 April 2000 相似文献
12.
In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed
if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles. Here we report the use of acridine
orange to quantify passive proton fluxes. Right-side out vesicles were exposed to pH jumps. From the decay of the applied
ΔpH the proton fluxes and proton permeability coefficients (PH+) were calculated. As in the intact Elodea plasma membrane, the proton permeability of the vesicle membrane is pH sensitive, an effect of internal pH as well as external
pH on PH+ was observed. Under near symmetric conditions, i.e., zero electrical potential and zero ΔpH, PH+ increased from 65 × 10−8 at pH 8.5 to 10−1 m/sec at pH 11 and the conductance from 13 × 10−6 to 30 × 10−4 S/m2. At a constant pH
i
of 8 and a pH
o
going from 8.5 to 11, PH+ increased more than tenfold from 2 to 26 × 10−6 m/sec. The calculated values of PH+ were several orders of magnitude lower than those obtained from studies on intact leaves. Apparently, in plasma membrane
purified vesicles the transport system responsible for the observed high proton permeability in vivo is either (partly) inactive
or lost during the procedure of vesicle preparation. The residue proton permeability is in agreement with values found for
liposome or planar lipid bilayer membranes, suggesting that it reflects an intrinsic permeability of the phospholipid bilayer
to protons. Possible implications of these findings for transport studies on similar vesicle systems are discussed.
Received: 5 April 1995/Revised: 28 March 1996 相似文献
13.
High speed video imaging microscopy and the pH-sensitive fluorophore2′,7′,-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
were used to examine acid-base functions of beta-intercalated cells of the rabbit cortical collecting duct. The presence of
intercalated cells was established and the properties of apical and basolateral acid-base transporters assessed by monitoring
cell pH during acid loading and luminal and basolateral ion substitutions. We showed that treatment of beta-intercalated cells
with ammonium chloride (20 mm) induced a profound decrease of their intracellular pH from 6.98 ± 5.93 ± 0.08. pH recovery occurred after different lag
periods ranging between 2 to 15 min (0.22 ± 0.04 dpH/dt). We demonstrated that this pH recovery mechanism was independent
of basolateral Na+ and apical HCO−
3 and K+. It was also not affected by apical and basolateral addition of NEM, by basolateral DIDS and by apical application of the
H-KATPase inhibitor SCH28080. The process of pH recovery was however, critically dependent on basolateral HCO−
3. These results are best explained by acid-induced insertion and/or activation of chloride-bicarbonate exchangers that are
functional properties with their apical analogues.
Received: 11 January 1994/Revised: 13 June 1997 相似文献
14.
A mathematical model of the HCO−
3-secreting pancreatic ductal epithelium was developed using network thermodynamics. With a minimal set of assumptions, the
model accurately reproduced the experimentally measured membrane potentials, voltage divider ratio, transepithelial resistance
and short-circuit current of nonstimulated ducts that were microperfused and bathed with a CO2/HCO−
3-free, HEPES-buffered solution, and also the intracellular pH of duct cells bathed in a CO2/HCO−
3-buffered solution. The model also accurately simulated: (i) the effect of step changes in basolateral K+ concentration, and the effect of K+ channel blockers on basolateral membrane potential; (ii) the intracellular acidification caused by a Na+-free extracellular solution and the effect of amiloride on this acidification; and (iii) the intracellular alkalinization
caused by a Cl−-free extracellular solution and the effect of DIDS on this alkalinization. In addition, the model predicted that the luminal
Cl− conductance plays a key role in controlling both the HCO−
3 secretory rate and intracellular pH during HCO−
3 secretion. We believe that the model will be helpful in the analysis of experimental data and improve our understanding of
HCO−
3-transporting mechanisms in pancreatic duct cells.
Received: 18 October 1995/Revised: 5 July 1996 相似文献
15.
Depolarization-activated H+-selective currents were studied using whole-cell and excised-patch voltage clamp methods in human monocytic leukemia THP-1
cells, before and after being induced by phorbol ester to differentiate into macrophage-like cells. The H+ conductance, g
H, activated slowly during depolarizing pulses, with a sigmoidal time course. Fitted by a single exponential following a delay,
the activation time constant, τact was roughly 10 sec at threshold potentials, decreasing at more positive potentials. Tail currents upon repolarization decayed
mono-exponentially at all potentials. The tail current time constant, τtail, was voltage dependent, decreasing with hyperpolarization from 2–3 sec at 0 mV to ∼200 msec at −100 mV. Surprisingly, although
τact depended strongly on pH
o
, τtail was completely independent of pH
o
. H+ currents were inhibited by Zn2+. Increasing pH
o
or decreasing pH
i
shifted the voltage-activation relationship to more negative potentials, tending to activate the g
H at any given voltage. Studied in excised, inside-out membrane patches, H+ currents were larger and activated much more rapidly at lower bath pH (i.e., pH
i
). In THP-1 cells differentiated into macrophages, the H+ current density was reduced by one-half, and τact was slower by about twofold. The properties of H+ channels in THP-1 cells and in other macrophage-related cells are compared.
Received: 19 September 1995/Revised: 14 March 1996 相似文献
16.
Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric
fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011
pH units/min in the presence of Na+, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na+ independent H+ transport mechanisms in HEPES Ringer. In CO2/HCO−
3 Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO2, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl− or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl−/HCO−
3 exchange. This exchange was not Na+ dependent. When Na+ was added to cells incubated in 0 Na+ Ringer while blocking Na+/H+ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na+/HCO−
3 cotransport carrying HCO−
3 into these cells, which was abolished by DIDS. These experiments, thus, show that Na+/H+ and Cl−/HCO−
3 exchange and Na+/HCO−
3 cotransport participate in cell pH regulation in T84 cells.
Received: 3 April 2000/Revised: 22 June 2000 相似文献
17.
L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral
membrane vesicles l-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective.
The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is
saturable with respect to external lactate with a K
m
of 39.2 ± 4.8 mm and a J
max of 8.9 ± 0.7 nmoles mg protein−1 sec−1. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains
a H+-lactate cotransporter, whereas in the apical membrane both H+-lactate and Na+-lactate cotransporters are present, even if they exhibit a low transport rate.
Received: 22 October 1996/Revised: 11 March 1997 相似文献
18.
The rat primary cultured-airway monolayer had been an excellent model for deciphering the ion channel after nystatin permeabilization
of its basolateral or apical membrane (Hwang et al., 1996). After apical membrane permeabilization of rat primary cultured-airway
monolayer, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive outwardly rectifying depolarization-induced
Cl− (BORDIC) currents were observed across the basolateral membrane in symmetrical NMG-Cl solution in this study. No significant
Cl− current induced by the application of voltage clamping was observed across the apical membrane in symmetrical NMG-Cl solution
after basolateral membrane permeabilization. The halide permeability sequence for BORDIC current was Br−≒ I− > Cl−. BORDIC current was not affected by basolaterally applied bumetanide (0.5 mm). Basolateral DIDS (0.2 mm) but not apical DIDS inhibited CFTR mediated short-circuit current (I
sc
) in an intact monolayer of rat airway epithelia, a T84 human colonal epithelial cell line, and a Calu-3 human airway epithelial
cell line. This is the first report showing that depolarization induced Cl− current is present on the basolateral membrane of airway epithelia.
Received: 7 October 1999/Revised: 24 April 2000 相似文献
19.
20.
It is well known, that in mammalian small intestine, cAMP increases Cl− permeability of the apical membrane of enterocytes as part of its secretory action. Paradoxically, this is usually accompanied
by an increase of the transepithelial resistance. In the present study we report that in the presence of bumetanide (to block
basolateral Cl− uptake) cAMP always decreased the transepithelial resistance. We examined whether this decrease in resistance was due to
a cAMP-dependent increase of the paracellular electrolyte permeability in addition to the increase of the Cl− permeability of the apical cell membrane. We used diffusion potentials induced by serosal replacement of NaCl, and transepithelial
current passage to evoke transport number effects. The results revealed that cAMP (but not carbachol) could increase the Cl− permeability of the tight junctions in rat ileum. Moreover, we observed a variation in transepithelial resistance of individual
tissue preparations, inversely related to the cation selectivity of the tissue, suggesting that Na+ permeability of the tight junctions can vary between preparations.
Received: 7 September 1996/Revised: 5 November 1996 相似文献