共查询到20条相似文献,搜索用时 31 毫秒
1.
pH
i
recovery in acid-loaded Ehrlich ascites tumor cells and pH
i
maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO
3
–
-free media and at 25 mm HCO
3
–
, the measured pH
i
(7.26 and 7.82, respectively) was significantly more alkaline than the pH
i
. value calculated assuming the transmembrane HCO
3
–
gradient to be equal to the Cl– gradient. Thus, pH
i
in these cells is not determined by the Cl– gradient and by Cl–/HCO
3
–
exchange.pH
i
recovery following acid loading by propionate exposure, NH
4
+
withdrawal, or CO2 exposure is mediated by amiloride-sensitive Na+/H+ exchange in HCO3
– free media, and in the presence of HCO
3
–
(25 mm) by DIDS-sensitive, Na+-dependent Cl–/HCO
3
–
exchange. A significant residual pH
i
recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H+ pump in pH
i
regulation. pH
i
maintenance at steady-state involves both Na+/H+ exchange and Na+-dependent Cl–/HCO
3
–
exchange.Acute removal of external Cl– induces a DIDS-sensitive, Na+-dependent alkalinization, taken to represent HCO
3
–
influx in exchange for cellular Cl–. Measurements of 36Cl– efflux into Cl–-free gluconate media with and without Na+ and/or HCO
3
–
(10 mm) directly demonstrate a DIDS-sensitive, Na+
dependent Cl–/HCO
3
–
exchange operating at slightly acidic pH
i
(pHo 6.8), and a DIDS-sensitive, Na+-independent Cl–/HCO
3
–
exchange operating at alkaline pH
i
(pH
o
8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.). 相似文献
2.
The pH-sensitivity of transepithelial K+ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pH
iwas measured microfluorometrically with the pH-sensitive dye 2,7-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (I
sc), which is a measure for transepithelial K+ secretion, was calculated from measurements of the transepithelial voltage (V
t)and the transepithelial resistance (R
t) in a micro-Ussing chamber. All experiments were conducted in virtually HCO
3
–
-free solutions. Under control conditions, pH
iwas 7.01±0.04 (n=18), V
twas 9.1±0.5 mV, R
t16.7±0.09 cm2, and I
sc was 587±30 A/cm2 (n=49). Addition of 20 mm propionate– caused a biphasic effect involving an initial acidification of pH
i, increase in V
tand I
sc and decrease in R
tand a subsequent alkalinization of pH
i, decrease of V
tand increase of R
t. Removal of propionate– caused a transient effect involving an alkalinization of pH
i, a decrease of V
tand I
sc and an increase in R
t. pH
iin the presence of propionate– exceeded pH
iunder control conditions. Effects of propionate – on V
t, R
tand I
sc were significantly larger when propionate– was applied to the basolateral side rather than to the apical side of the epithelium. The pH
i-sensitivityof I
sc between pH 6.8 and 7.5 was –1089 A/(cm2 · pH-unit) suggesting that K+ secretion ceases at about pH
i7.6. Acidification of the extracellular pH (pH
o)caused an increase of V
tand I
sc and a decrease of R
tmost likely due to acidification of pH
i. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pH
osensitivity of I
sc between pH 7.4 and 6.4 was –155 A/(cm2 · pH unit). These results demonstrate that transepithelial K+ transport is sensitive to pH
iand pH
oand that vestibular dark cells contain propionate– uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K+ channel (I
sK)in the apical membrane. Whether the effect of pH
ion the I
sK channel is a direct or indirect effect remains to be determined.The authors wish to thank Drs. Daniel C. Marcus, Zhjiun Shen and Hiroshi Sunose for helpful discussions. This work was supported by grants NIH-R29-DC01098 and NIH-R01-DC00212. 相似文献
3.
Frank Stahl Albrecht Lepple-Wienhues Marianne Koch Michael Wiederholt 《The Journal of membrane biology》1992,127(3):215-225
Summary We investigated intracellular pH (pH
i
) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4,5-dimethylfluorescein (CDMF). The steady-state pH
i
was 7.09±0.04 (n = 12) in CO2/ HCO
3
–
-buffered and 6.86±0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11±0.06 pH units (n = 12) in the presence of CO2/ HCO
3
–
and by 0.91±0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pH
i
recovery, which was almost completely amiloride-sensitive in the absence of CO2/ HCO
3
–
but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25±0.02 (n = 4) in 6 min, while amiloride had no effect. The pH
i
recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pH
i
recovery was almost completely mediated by Na+/H+ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amilorideinsensitive pH
i
recovery was observed in CO2/HCO
3
–
-buffered solution which was mediated by chloride-independent and chloride-dependent Na+ HCO
3
–
cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter). was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pH
i
recovery after NH4Cl prepulse revealed V
max = 0.57 pH units/min, K
m= 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and V
max = 0.19 pH units/min, K
m= 14.3 mmol/liter extracellular sodium for the arniloride-insensitive component. We conclude that Na+/H+ exchange and chloride-independent and chloride-dependent Na+HCO
3
–
cotransport are involved in the pH
i
regulation of cultured human ciliary muscle cells.The expert technical assistance of Astrid Krolik is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft grant DFG Wi 328/11. 相似文献
4.
M. R. Williams G. Duncan P. C. Croghan R. Riach S. F. Webb 《The Journal of membrane biology》1992,129(2):179-187
Summary The intracellular pH (pH
i
) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2,7-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35°C with artificial aqueous humour solution (AAH) containing 16 mM HCO
3
-
and 5% CO2, pH 7.25, pH
i
was 7.19±0.02 (sem, n = 95). On removing HCO
3
-
and CO2 there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97±0.03 (sem, n = 54). Addition of 0.25 mM 4,4-diisothiocyanatostilbene2, 2-disulfonic acid (DIDS) to AAH containing HCO
3
-
and CO2 led to a rapid and pronounced fall in pH. Exposure to Na+-free AAH again led to a marked fall in pH
i
, but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl– in the presence of HCO
3
-
led to a rise in pH
i
, while substitution in the absence of HCO
3
-
led to a fall in pH
i
. The above data indicate a significant role for a sodium-dependent Cl–-HCO
3
-
exchange mechanism in the regulation of pH
i
. Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO
3
-
led to a marked fall in pH
i
, indicating that a Na+/H+ exchange mechanism also has a significant role in the regulation of pH
i
. There is evidence for a lactic acid transport mechanism in bovine lens cells, as addition of lactate to the external medium produced a rapid fall in pH
i
. Larger changes in pH
i
were observed in control compared to HCO
3
-
-free AAH and in the latter case a pronounced alkalinizing overshoot was obtained on removing external lactate. Tissue-cultured bovine lens cells thus possess at least three membrane transport mechanisms that are involved in pH regulation. The buffering capacity of the lens cells was measured by perturbing pH
i
with either NH
4
+
or procaine. The values obtained were similar in both cases and the intrinsic buffering capacity measured in the absence of external HCO
3
-
was 5 mm/pH unit (procaine). However, in the presence of HCO
3
-
and CO2 the buffer capacity increases approximately fourfold, indicating that HCO
3
-
is the principal intracellular buffer.We acknowledge financial support from the Wellcome Trust and the Humane Research Trust for this project. M.R. Williams was in receipt of a Science & Engineering Research Council studentship. 相似文献
5.
Summary The intracellular pH (pH
i
) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H+ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pH
i
of cells placed in an acidic medium (pH
o
below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pH
o
is acutely reduced to 5.5, pH
i
falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H+. The unidirectional influx of H+ exhibits saturation kinetics with respect to extracellular [H+]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andK
m
is 0.74 ± 0.09 × 10–6
m.Steady-state cells with pH
i
above 6.8 continuously extrude H+ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pH
i
6.3) when returned to pH
o
7.3 medium respond by transporting H+, resulting in a rapid rise in pH
i
. The halftime for this process is 1.09 ± 0.22 min. The H+ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pH
i
to its steady-state value. 相似文献
6.
Summary We have investigated the kinetic properties of the human red blood cell Na+/H+ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H+ (H
i
) and external Na+ (Na
o
) in red blood cells of normal subjects.Red blood cells with different cell Na+ (Na
i
) and pH (pH
i
) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na+ influx were measured by varying pH
i
(from 5.7 to 7.4), external pH (pH
o
), Na
i
and Na
o
and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na+ influx (Na
i
<2.0 mmol/liter cell, Na
o
= 150mm) increased sigmoidally (Hill coefficient 2.5) when pH
i
fell below 7.0 and the external pH
o
was 8.0, but increased linearly at pH
o
6.0. The net Na+ influx driven by an outward H+ gradient was estimated from the difference of Na+ influx at the two pH
o
levels (pH
o
8 and pH
o
6). The H+-driven Na+ influx reached saturation between pH
i
5.9 and 6.1. TheV
max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK
m
for H
i
to activate H+-driven Na+ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na+/H+ exchange. The stoichiometric ratio between H+-driven Na+ influx and Na+-driven H+ efflux was 11. The dependence of Na+ influx from Na
o
was studied at pH
i
6.0, and Na
i
lower than 2 mmol/liter cell at pH
o
6.0 and 8.0. The meanK
m
for Na
o
of the H+-gradient-driven Na+ influx was 55±7mm.An increase in Na
i
from 2 to 20 mmol/liter cell did not change significantly H+-driven net Na+ influx as estimated from the difference between unidirectional22Na influx and efflux. Na+/Na+ exchange was negligible in acid-loaded, DIDS-treated cells. Na+ and H+ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na+ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na+/H+ exchanger.It is concluded that human red cell Na+/H+ exchange performs 11 exchange of external Na+ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H+ and external Na+ is similar to other cells, but it displays a larger variability in theV
max between individuals. 相似文献
7.
Summary Intracellular pH (pH
i
) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pH
i
was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pH
i
only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH
4
+
and bicarbonate buffer solution gassed with 5% CO2/95% O2 was 26 and 46mm/pH, respectively Stimulation with 1 m acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pH
i
. In the presence of amiloride (0.1mm) or the absence of Na+, application of ACh caused a significant decrease in pH
i
and removal of amiloride or replacement with Na+-containing saline, respectively, rapidly increased the pH
i
. Pretreatment with DIDS (0.2mm) did not change the pH
i
of the nonstimulated conditions; however, it significantly enhanced the increase in pH
i
induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na+ removal from the bath solution. We suggest that both Na+/H+ and HCO
3
–
/Cl– exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells. 相似文献
8.
The influence of cytosolic pH (pHi) in controlling K+-channel activity and its interaction with cytosolic-free Ca2+ concentration ([Ca2+]i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K+-channel currents were recorded under voltage clamp while pHi or [Ca2+]i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K+ concentration, current through inward-rectifying K+ channels (IK,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na+-acetate outside imposed acid loads, lowering pHi from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pHi was independent of the weak acid used, and indicated a H+-buffering capacity which rose from 90 mM H+/pH unit near 7.5 to 160 mM H+/pH unit near pHi 7.0. With acid-going pHi, (IK,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pHi lasting 3–5 min and was accompanied by a reduction in (IK,in) before recovery of the initial resting pHi and current amplitude. The pHi-sensitivity of the current was consistent with a single, titratable site for H+ binding with a pKa near 6.3. Acid pHi loads also affected current through the outward-rectifying K+ channels (IK,out) in a manner antiparallel to (IK,in) The effect on IK, out was also scalar, but showed an apparent pKa of 7.4 and was best accommodated by a cooperative binding of two H+. Parallel measurements showed that Na+-butyrate loads were generally without significant effect on [Ca2+]i, except when pHi was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca2+]i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pHi changes and K+-channel responses. The action on [Ca2+]i coincided with a greater variability in (IK,in) stimulation evident at pHi values around 7.0 and below, and with negative displacements in the voltage-dependence of (IK,in) gating. These results distinguish the actions of pHi and [Ca2+]i in modulating (IK,in) they delimit the effect of pHi to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pHi as a second messenger capable of acting in parallel with, but independent of [Ca2+]i in controlling the K+ channels.Abbreviations BCECF
2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein
- [Ca2+]i
cytosolic free Ca2+ concentration
- gK
ensemble (steady-state) K+-channel conductance
- IK,out, IK,in
outward-, inward-rectifying K+ channel (current)
- IN
current-voltage (relation)
- Mes
2-(N-morpholinolethanesulfonic acid
- pHi
cytosolic pH
- V
membrane potential 相似文献
9.
S. Gaillard J. L. Rodeau 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1987,157(4):435-444
Summary Intracellular pH (pHi) regulation was studied in crayfish neurons with pH-, and Na+-sensitive microelectrodes. It was confirmed to involve both a HCO
3
–
-dependent and a HCO
3
–
-independent mechanism. The latter was identified as the amiloride-sensitive Na+/H+ exchange described in vertebrate cells. Its dependence on extracellular pH (pHe) and Na+ concentration ([Na+]e) was studied in CO2-free external solutions at 20°C. The steady state pHi and the rate constant (k) of the exponential pHi recovery following an acid load were determined. At pHe=7.5 and [Na+]e=200 mM, the average steady state pHi was 7.09±0.12 (as compared to 7.30±0.10 in the presence of 5 mM bicarbonate). The dependence of the rate constant of recovery on [Na+]e could be described by Michaelis-Menten kinetics; at pHe=7.5 the apparentK
m andK
max were 39 mM and 1.4 mmol·l–1·min–1, respectively. Decreasing pHe reduced the rate of recovery, the variations ofk with pHe conforming to a simple titration curve with an apparent pK of 7.05±0.21. These kinetic properties of the Na+/H+ exchange in crayfish neurons are similar to those described in vertebrate cells.Preliminary results were presented at the First International Congress of Comparative Physiology and Biochemistry (Liège, Belgium, 1984) 相似文献
10.
Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k
H
+
was determined by addition of vesicles (pH
i
= 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+–3 sec–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k
H
+
were proportionately greater in iBBMV as compared with dBBMV. The presence of Na+/H+ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH
i
< pH
o
) 22Na+ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K+-diffusion potential ([K+]
i
[K+
o
). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K+-ionophore valinomycin, indicating that the native K+ permeability is rate limiting. In the presence of valinomycin, the K+-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K+ permeability. Valinomycin was without effect, but the K+/H+ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K+-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na+/H+ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA). 相似文献
11.
Summary Membrane-permeant weak acids and bases, when applied to the bath, modulate the resting membrane potential and the glucose-induced electrical activity of pancreatic B cells, as well as their insulin secretion. These substances alter the activity of a metabolite-regulated. ATP-sensitive K+ channel which underlies the B-cell resting potential. We now present several lines of evidence indicating that the channel may be directly gated by pH
i
. (1) The time course of K+(ATP) channel activity during exposure to and washout of NH4Cl under a variety of experimental conditions, including alteration of the electrochemical gradient for NH4Cl entry and inhibition of the Na
o
+
H
i
+
exchanger, resembles the time course of pH
i
measured in other cell types that have been similarly treated. (2) Increasing pH
o
over the range 6.25–7.9 increases K+(ATP) channel activity in cell-attached patches where the cell surface exposed to the bath has been permeabilized to H+ by the application of the K+/H+ exchanger nigericin. (3) Increasing pH
i
over a similar range produces similar effects on K+(ATP) channels in inside-out excised patches exposed to small concentrations of ATP
i
. The physiological role of pH
i
in the metabolic gating of this channel remains to be explored. 相似文献
12.
Terutaka Ozawa Yoshitaka Saito Akinori Nishiyama 《The Journal of membrane biology》1988,105(3):273-280
Summary Anion exchange transport in the mouse lacrimal gland acinar cell membrane was studied by measuring the intracellular H+ (pHi) and Cl– (aCli) activities with double-barreled ion-selective microelectrodes. In a HCO
3
–
-free solution of pH 7.4 (HEPES/Tris buffered), pHi was 7.25 andaCli was 33mm. By an exposure to a HCO
3
–
(25mm HCO
3
–
/5% CO2, pH 7.4) solution for 15 min,aCli was decreased to 25mm and pHi was transiently decreased to about 7.05 within 1 min, then slowly relaxed to 7.18 in 15 min. Intracellular HCO
3
–
concentration [HCO
3
–
]i, calculated by the Henderson-Hasselbalch's equation, was 11mm at 1 min after the exposure and then slowly increased to 15mm. Readmission of the HCO
3
–
-free solution reversed the changes inaCli and pHi. The intracellular buffering power was about 40mm/pH. An addition of DIDS (0.2mm) significantly inhibited the rates of change inaCli, pHi, and [HCO
3
–
]i caused by admission/withdrawal of the HCO
3
–
, solution and decreased the buffer value. Replacement of all Cl– with gluconate in the HCO
3
–
solution increased pHi, and readmission of Cl– decreased pHi. The rates of these changes in pHi were reduced by DIDS by 32–45% but not by amiloride (0.3mm). In the HCO
3
–
solution, a stimulation of intracellular HCO
3
–
production by exposing the tissue to 25mm NH
4
+
increasedaCli significantly. While in the HCO
3
–
-free solution or in the HCO
3
–
, solution containing DIDS, exposure to NH
4
+
had little effect onaCli. All of these findings were consistent with the presence of a reversible, disulfonic stilbene-sensitive Cl–/HCO
3
–
exchanger in the basolateral membrane of the acinar cells. The possibility of anion antiport different from one-for-one Cl–/HCO
3
–
exchange is discussed. 相似文献
13.
Internal pH (pH
i
) is in Vero cells regulated mainly by three antiports. Na+/H+ antiport and Na+-dependent Cl+/HCO
3
+
antiport increase pH
i
in acidified cells, and Na+-independent Cl+/HCO
3
+
antiport lowers pH
i
in cells after alkalinization. The activities of the antiporters were altered in cells after exposure to 41–45°C. Under such conditions the Na+/H+ antiport and the Na+-dependent Cl+/HCO
3
+
antiport were both stimulated, whereas the Na+-independent Cl+/HCO
3
+
antiport was inhibited in such a way that a higher pH value was required to activate it. This alteration was also induced by some other forms of cellular stress, but did most likely not involve stress proteins as protein synthesis was not required. The possibility of regulation by alteration in protein phosphorylation is discussed.We are grateful to Mrs. Jorunn Jacobsen for her skillful handling of the cell cultures. This work was supported by the Norwegian Research Council for Science and Humanities, the Norwegian Cancer Society and the Lærdal Foundation. 相似文献
14.
Wenhui Wang Robert M. Henderson John Geibel Stanley White Gerhard Giebisch 《The Journal of membrane biology》1989,112(3):277-289
Summary Isolated early distal tubule cells (EDC) of frog kidney were incubated for 20–28 hr in the presence of aldosterone and then whole-cell K+ currents were measured at constant intracellular pH by the whole-cell voltage-clamp technique. Aldosterone increased barium-inhibitable whole-cell K+ conductance (gK+) threefold. This effect was reduced by amiloride and totally abolished by ouabain. However, aldosterone could still raisegK+ in ouabain-treated cells in the presence of furosemide.We tested whether changes in intracellular pH (pH
i
) could be a signal for cells to regulategK+. After removal of aldosterone, the increase ingK+ was preserved by subsequent incubation for 8 hr at pH 7.6 but abolished at pH 6.6. In the complete absence of aldosterone, incubation of cells at pH 8.0 for 20–28 hr raised pH
i
and doubledgK+.Using the patch-clamp technique, three types of K+-selective channels were identified, which had conductances of 24, 45 and 59 pS.Aldosterone had no effect on the conductance or open probability (P
o) of any of the three types of channels. However, the incidence of observing type II channels was increased from 4 to 22%. Type II channels were also found to be pH sensitive,P
o was increased by raising pH.These results indicate that prolonged aldosterone treatment raises pH
i
and increasesgK+ by promoting insertion of K+ channels into the cell membrane. Channel insertion is itself triggered by raising both pH
i
and increasing the activity of the Na+/K+ pump in early distal cells of frog kidney.
Present address: Department of Physiology, The University of Leeds, Leeds, LS2 9NQ, England 相似文献
15.
Wenhui Wang Yang Wang Stefan Silbernagl Hans Oberleithner 《The Journal of membrane biology》1988,101(1):259-265
Summary Experiments were performed in intact proximal tubules of the doubly perfused kidney and in fused proximal tubule cells ofRaha esculenta to evaluate the dependence of intracellular pH (pHi) on cell membrane potential applying pH-sensitive and conventional microelectrodes. In proximal tubules an increase of the K– concentration in the peritubular perfusate from 3 to 15 mmol/liter decreased the peritubular cell membrane potential from –55±2 to –38±1 mV paralleled by an increase of pH
i
, from 7.54±0.02 to 7.66±0.02. The stilbene derivative DIDS hyperpolarized the cell membrane potential from –57 ± 2 to –71 ±4 mV and led to a significant increase of the K–-induced cell membrane depolarization, but prevented the K–-induced intracellular alkalinization. Fused proximal tubule cells were impaled by three microelectrodes simultaneously and cell voltage was clamped stepwise while pH
i
changes were monitored. Cell membrane hyperpolarization acidified the cell cytoplasm in a linear relationship. This voltage-induced intracellular acidification was reduced to about one-third when HCO3 ions were omitted from the extracellular medium. We conclude that in proximal tubule cells pH
i
depends on cell voltage due to the rheogenicity of the HCO
3
–
transport system. 相似文献
16.
Ochratoxin A (OTA) is a nephrotoxin which blocks plasma membrane anion conductance in Madin-Darby canine kidney (MDCK) cells. Added to the culture medium, OTA transforms MDCK cells in a manner similar to exposure to alkaline stress. By means of video-imaging and microelectrode techniques, we investigated whether OTA (1 mol/liter) affects intracellular pH (pH.), Cl– (Cl
i
–
) or cell volume of MDCK cells acutely exposed to normal (pHnorm=7.4) and alkaline (pHalk=7.7) conditions. At pHnorm, OTA increased Cl
i
–
by 2.6±0.4 mmol/liter (n=14, P<0.05) but had no effect on pH
i
. At pHalk, application of OTA increased Cl
i
–
by 8.6±2.6 mmol/liter (n=10, P< 0.05) and raised pH
i
by 0.11±0.03 (n= 8, P<0.05). The Cl–HCO
3
–
exchange inhibitor DNDS (4,4-dinitro-stilbene-2, 2-disulfonate; 10 mol/liter) eliminated the OTA-induced changes of pH
i
and Cl
i
–
. OTA did not affect cell volume under both pHnorm and pHalk conditions.We conclude that the OTA-induced blockade of plasma membrane anion conductance increases Cl
i
–
without changing cell volume. The driving force of plasma membrane Cl–/HCO
3
–
exchange dissipates, leading to a rise of pH
i
when cells are exposed to an acute alkaline load. Thus, OTA interferes with pH
i
and Cl
i
–
homeostasis leading to morphological and functional alterations in MDCK cells.The work was supported by the Deutsche Forschungsgemeinschaft (DFG, Si 170/7-1).We thank the Zeiss Company (Oberkochen, Germany) for providing the Attofluor video-imaging system for the intracellular Ca2+ measurements.This study was carried out with the technical assistance of Sigrid Mildenberger and Ruth Freudinger. 相似文献
17.
The activation by abscisic acid (ABA) of current through outward-rectifying K+ channels and its dependence on cytoplasmic pH (pHi) was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled and H+-selective microelectrodes to record membrane potentials and pHi during exposures to ABA and the weak acid butyrate. Potassium channel currents were monitored under voltage clamp and, in some experiments, guard cells were loaded with pH buffers by iontophoresis to suppress changes in pHi. Following impalements, stable pHi values ranged between 7.53 and 7.81 (7.67±0.04, n = 17). On adding 20 M ABA, pHi rose over periods of 5–8 min to values 0.27±0.03 pH units above the pHi before ABA addition, and declined slowly thereafter. Concurrent voltage-clamp measurements showed a parallel rise in the outward-rectifying K+ channel current (IK, out) and, once evoked, both pHi and IK, out responses were unaffected by ABA washout. Acid loads, imposed with external butyrate, abolished the ABA-evoked rise in IK, out. Butyrate concentrations of 10 and 30 mM (pH0 6.1) caused pHi to fall to values near 7.0 and below, both before and after adding ABA, consistent with a cytoplasmic buffer capacity of 128±12 mM per pH unit (n = 10) near neutrality. Butyrate washout was characterised by an appreciable alkaline overshoot in pHi and concomitant swell in the steady-state conductance of IK, out. The rise in pHi and iK, out in ABA were also virtually eliminated when guard cells were first loaded with pH buffers to raise the cytoplasmic buffer capacity four- to sixfold; however, buffer loading was without appreciable effect on the ABA-evoked inactivation of a second, inward-rectifying class of K+ channels (IK, in). The pHi dependence of IK, out was consistent with a cooperative binding of at least 2H+ (apparent pKa = 8.3) to achieve a voltage-independent block of the channel. These results establish a causal link previously implicated between cytoplasmic alkalinisation and the activation of IK, out in ABA and, thus, affirm a role for H+ in signalling and transport control in plants distinct from its function as a substrate in H+-coupled transport. Additional evidence implicates a coordinate control of IK, in by cytoplasmic-free [Ca2+] and pHi.Abbreviations ABA
abscisic acid
- [Ca2+]i
cytoplasmic free [Ca2+]i
- EK
K+ equilibrium potential
- IK, out, IK, in
outward-, inward-rectifying K+ channel (current)
- I-V
current-voltage (relation)
- Mes
2-(N-morpholino)ethanesulfonic acid
- pHi
cytoplasmic pH
- Tes
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino}ethanesulfonic acid
- Vm
membrane potential
We are grateful to G. Thiel (Pflanzenphysiologisches Institut, Universität Göttingen, Germany) for helpful discussions. This work was possible with equipment grants-in-aid from the Gatsby Charitable Foundation, the Royal Society and the University of London Central Research Fund. F.A. holds a Sainsbury Studentship. 相似文献
18.
Summary Recently we proposed that cytoplasmic acidification of low K+ (LK) sheep erythrocytes may stimulate ouabain-resistant Cl–-dependent K+ flux (K+Cl– cotransport), also known to be activated by cell swelling, treatment with N-ethylmaleimide (NEM), or removal of cellular bivalent cations. Here we studied the dependence of K+ transport on intracellular and extracellular pH (pH
i
, pH
o
) varied either simultaneously or independently using the Cl–/HCO
3
–
exchange inhibitor 4,4, diisothiocyanatostilbene-3,2-disulfonic acid (DIDS). In both control and NEM-treated LK cells volumes were kept near normal by varying extracellular sucrose. Using DIDS as an effective pH clamp, both K+ efflux and influx of Rb+ used as K+ congener were strongly activated at acid pH
i
and alkaline pH
o
. A small stimulation of K+ (Rb+) flux was also seen at acid pH
i
in the absence of DIDS, i.e., when pH
i
pH
o
. Anti-L
l
serum, known to inhibit K+Cl– cotransport, prevented the pH
i
-stimulated K+ (Rb+) fluxes. Subsequent to NEM treatment at pH 6, K+ (Rb+) fluxes were activated only by raising pH, and thus were similar to the pH activation profile of K+ (Rb+) fluxes in DIDS-treated cells with pH
o
varied at constant physiologic pH
i
. Anti-L
l
, which inhibited NEM-stimulated K+ (Rb+) fluxes, failed to do so in NEM-plus DIDS-treated cells. Thus, NEM treatment interferes with the internal but not with the external pH-sensitive site. 相似文献
19.
G. Bertram A. Wessing 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1994,164(3):238-246
The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pHi) and luminal pH (pHlu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pHo 6.9). In proximal segments both pHi (7.43±0.20) and pHlu (7.10±0.24) were significantly lower than in distal segments (pHi 7.70±0.29, pHlu 8.09±0.15). Steady-state pHi of proximal segments was much less sensitive to changes in pHo than pH of the luminal fluid (pHlu/pHo was 0.49 while pHi/pHo was 0.18; pHo 6.50–7.20). Re-alkaliniziation from an NH4Cl-induced intracellular acid load (initial pHi recovery rate 0.55±0.34 pH·min-1) was nearly totally inhibited by 1 mmol·l-1 KCN (96% inhibition) and to a large degree (79%) by 1 mol·l-1 bafilomycin A1. In contrast, both vanadate (1 mmol·l-1) and amiloride (1 mmol·l-1) inhibited pHi recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na+ from the bathing saline had no effect on pHi recovery, indicating that a Na+/H+ exchange is not significantly involved in pHi regulation. Instead pHi regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.Abbreviations DMSO
dimethylsulphoxide
- DNP
2,4-dinitrophenol
- NMDG
N-methyl-D-glucamine
- pHi
intracellular pH
- pHlu
pH of the luminal fluid
- pHo
pH of the superfusion medium
- I
intrinsic intracellular buffer capacity 相似文献
20.
J. Gregory Fitz Steven D. Lidofsky Richard A. Weisiger Ming-Hong Xie Mary Cochran Thomas Grotmol Bruce F. Scharschmidt 《The Journal of membrane biology》1991,122(1):1-10
Summary Recent studies in hepatocytes indicate that Na+-coupled HCO
3
–
transport contributes importantly, to regulation of intracellular pH and membrane HCO
3
–
transport. However, the direction of net coupled Na+ and HCO
3
–
movement and the effect of HCO
3
–
on Na+ turnover and Na+/K+ pump activity are not known. In these studies, the effect of HCO
3
–
on Na+ influx and turnover were measured in primary rat hepatocyte cultures with22Na+, and [Na+]
i
was measured in single hepatocytes using the Na+-sensitive fluorochrome SBFI. Na+/K+ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na+-dependent or ouabain-suppressible86Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K+ on membrane potential difference (PD) and [Na+]
i
. In hepatocyte monolayers, HCO
3
–
increased22Na+ entry and turnover rates by 50–65%, without measurably altering22Na+ pool size or cell volume, and HCO
3
–
also increased Na+/K+ pump activity by 70%. In single cells, exposure to HCO
3
–
produced an abrupt and sustained rise in [Na+]
i
, from 8 to 12mm. Na+/K+ pump activity assessed in single cells by PD excursions during transient K+ removal increased 2.5-fold in the presence of HCO
3
–
, and the rise in [Na+]
i
produced by inhibition of the Na+/K+ pump was similarly increased 2.5-fold in the presence of HCO
3
–
. In intact perfused rat liver, HCO
3
–
increased both Na+/K+ pump activity and O2 consumption. These findings indicate that, in hepatocytes, net coupled Na+ and HCO
3
–
movement is inward and represents a major determinant of Na+ influx and Na+/K+ pump activity. About half of hepatic Na+/K+ pump activity appears dedicated to recycling Na+ entering in conjunction with HCO
3
–
to maintain [Na+]
i
within the physiologic range. 相似文献