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1.
MDCK cells display several acid-base transport systems found in intercalated cells, such as Na+-H+ exchange, H+–K+ ATPase and Cl−/HCO−
3 exchange. In this work we studied the functional activity of a vacuolar H+-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips
utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H+ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH+
4 pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na+ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10−8
m (a specific inhibitor of the vacuolar H+-ATPase) was added to the zero Na+ solution. When using a solution with zero Na+, low K+ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H+–K+-ATPase. This result was confirmed by the use of 5 × 10−5
m Schering 28080. The Na+ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10−5
m (a specific blocker of Cl− channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl−/normal Na+ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na+ and Cl− free solution. From these results we conclude that: (i) MDCK cells have two Na+-independent mechanisms of pHi recovery, a concanamycin sensitive H+-ATPase and a K+ dependent, Schering 28080 sensitive H+–K+ ATPase; and, (ii) pHi recovery in Na+-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl−–free medium. This finding supports a role for chloride in the function of the H+ ATPase, which might be electrical shunting or a biochemical interaction.
Received: 24 October 1997/Revised: 19 February 1998 相似文献
2.
In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO3) has been shown to mediate a large fraction of the total K+ transport. In the present study, Cl−-dependent and Cl−-independent K+ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K+ ([K+]
e
and [K+]
i
) concentration. The dependence of ouabain-resistant Cl−-dependent K+ (86Rb) influx on [K+]
e
over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K
m
) of 8.2 ± 1.3 mm and maximal velocity (V
max
) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl−-dependent K+ influx increased both K
m
(12.8 ± 1.7 mm, P < 0.05) and V
max
(20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K+]
e
above 20 mm in isotonic media significantly reduced the Cl−-dependent K+ influx due to a reciprocal decrease of the external Na+ ([Na+]
e
) concentration below 50 mm. Replacing [Na+]
e
by NMDG+ markedly decreased V
max
(3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K
m
(15.7 ± 2.1 mm, P < 0.03) of Cl−-dependent K+ influx. Moreover, NMDG+ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb+ uptake and K+ loss from red cells. Cell swelling did not affect the Na+-dependent changes in Rb+ uptake and K+ loss. In a nominally K+(Rb+)-free medium, net K+ loss was reduced after lowering [Na+]
e
below 50 mm. These results indicate that over 50 mm [Na+]
e
is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K+, Cl−-dependent K+ loss in K+-free media was a linear function of [K+]
i
, with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr−1 (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry
with respect to transported K+ ions. The residual, ouabain-resistant K+ fluxes in NO3 were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were
not different from those for K+ effluxes in isotonic (∼0.014 hr−1) and hypotonic (∼0.022 hr−1) media, but cell swelling resulted in a significant increase in the rate constants.
Received: 19 November 1998/Revised: 23 August 1999 相似文献
3.
F. Gagnon N.O. Dulin J. Tremblay P. Hamet S.N. Orlov 《The Journal of membrane biology》1999,167(2):193-204
P2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca2+]
i
, activation of phospholipases C (PLC) and A2 (PLA2), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling
pathways in the inhibition of Na+,K+,Cl− cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive
to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors,
H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led
to a transient increase of [Ca2+]i that was abolished by a chelator of Ca2+
i
, BAPTA. However, neither BAPTA nor the Ca2+ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca2+-pump, thapsigargin, modified ATP-induced inhibition of Na+,K+,Cl− cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate
production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na+,K+,Cl− cotransport activity. Inhibitors of PLA2 (AACOCF3), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced
inhibition of Na+,K+,Cl− cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na+ channels (amiloride), Cl− channels (NPPB) and Na+/H+ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent
ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na+,K+,Cl− cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition
of Na+,K+,Cl− cotransport and suggest the presence of a novel P2-receptor-coupled signaling mechanism.
Received: 29 July 1998/Revised: 19 October 相似文献
4.
Orlov SN Dulin NO Gagnon F Gekle M Douglas JG Schwartz JH Hamet P 《The Journal of membrane biology》1999,172(3):225-234
We demonstrated recently that in renal epithelial cells from collecting ducts of Madin-Darby canine kidneys (MDCK), Na+,K+,Cl− cotransport is inhibited up to 50% by ATP via its interaction with P2Y purinoceptors (Biochim. Biophys. Acta 1998. 1369:233–239). In the present study we examined which type of renal epithelial cells possesses the highest sensitivity of Na+,K+,Cl− cotransport to purinergic regulation. We did not observe any effect of ATP on Na+,K+,Cl− cotransport in renal epithelial cells from proximal and distal tubules, whereas in renal epithelial cells from rabbit and
rat collecting ducts ATP decreased the carrier's activity by ∼30%. ATP did not affect Na+,K+,Cl− cotransport in C7 subtype MDCK cells possessing the properties of principal cells but led to ∼85% inhibition of this carrier
in C11-MDCK cells in which intercalated cells are highly abundant. Both C7- and C11-MDCK exhibited ATP-induced IP3 and cAMP production and transient elevation of [Ca2+]
i
. In contrast to the above-listed signaling systems, ATP-induced phosphorylation of ERK and JNK MAP kinases was observed in
C11-MDCK only. Thus, our results reveal that regulation of renal Na+,K+,Cl− cotransport by P2Y receptors is limited to intercalated cells from collecting ducts and indicate the involvement of the MAP kinase cascade in
purinergic control of this ion carrier's activity.
Received: 10 June 1999/Revised: 23 August 1999 相似文献
5.
Kedzierska K Bober J Ciechanowski K Gołembiewska E Kwiatkowska E Noceń I Dołegowska B Dutkiewicz G Chlubek D 《Biological trace element research》2005,107(1):21-32
The aim of the study was to verify the hypothesis if copper could influence the activity of sodium-transporting systems in
erythrocyte membrane that could be related to essential hypertension. The examined group of patients consisted of 15 men with
hypertension. The control group was 11 healthy male volunteers. The Na+/H+ exchanger (NHE) activity in erythrocytes was determined according to Orlov et al. The activity of transporting systems (ATP-Na+/K+; co-Na+/K+/Cl−; ex-Na+/Li+; free Na+ and K+ outflow [Na+, K+-outflow]) was determined according to Garay's method. The concentration of copper in plasma was assessed using atomic absorption
spectrometry. The activity of ATP-Na+/K+ (μmol/L red blood cells [RBCs]/h) in hypertensive patients was 2231.5±657.6 vs 1750.5±291 in the control (p<0.05), the activity of co-Na+/K+/Cl− (μmol/L RBCs/h) in hypertensives was 171.3±77.9 vs 150.7±53.9 in the control (NS). Na+-outflow (μmol/L RBCs/h) in hypertensives was 118.3±51.6 vs 113.3±24.4 in the control (NS). The K+-outflow (μmol/L RBCs/h) in hypertensives was 1361.7±545.4 vs 1035.6±188.3 in the control (NS). The activity of ex-Na+/Li+ (μmol/L RBCs/h) in hypertensive patients was 266.1±76.1 vs 204.1±71.6 in the control (p<0.05). NHE activity (mmol/L RBCs/h) in hypertensives was 9.7±2.96 vs 7.7±1.33 in the control (p<0.05). In hypertensive patients, negative correlation was found between the activity of Na+/K+/Cl− co-transport and plasma copper concentration (R
s=−0.579, p <0.05) and between the activity of ex-Na+/Li+ and plasma copper concentration (R
s=−0.508, p<0.05). Plasma copper concentration significantly influences the activity of sodium transporting systems in erythrocyte membrane.
Copper supplementation could be expected to provide therapeutic benefits for hypertensive patients. 相似文献
6.
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
7.
Aporn Bualuang Kanteera Soontharapirakkul Aran Incharoensakdi 《Journal of applied phycology》2010,22(2):123-129
The activity of Na+/H+ exchanger to remove toxic Na+ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na+/H+ exchange activity since exogenously added Na+ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K
m (Na+) and V
max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H+ min−1 mg−1, respectively. For cells grown under salt-stress condition, the apparent K
m (Na+) and V
max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H+ min−1 mg−1, respectively. Three cations with decreasing efficiency namely Li+, Ca2+, and K+ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg2+. The exchange activity was strongly inhibited by Na+-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na+/H+ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na+/H+ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na+/H+ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na+/H+ exchange activity during 15 days of growth. 相似文献
8.
To examine the involvement of Na+,K+,2Cl− cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on 86Rb, 36Cl and 22Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward
and outward 86Rb fluxes were not different. Bumetanide decreased basal 86Rb uptake by 70–75% with a K
i
of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect 36Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to 86Rb and 36Cl influx, bumetanide did not inhibit 22Na uptake by VSMC. BS 86Rb uptake was completely abolished in Na+- or Cl−-free media. In contrast to 86Rb, basal BS 36Cl influx was not affected by Na+
o
and K+
o
. Hyperosmotic and isosmotic shrinkage of VSMC increased 86Rb and 36Cl influx to the same extent. Shrinkage-induced increments of 86Rb and 36Cl uptake were completely abolished by bumetanide with a K
i
or ∼0.3 μm. Shrinkage did not induce BS 86Rb and 36Cl influx in (Na+ or Cl−)- and (Na+ or K+)-depleted media, respectively. In the presence of an inhibitor of Na+/H+ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated 22Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory
volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na+,K+,2Cl− cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K+ influx is mediated by (Na+
o
+ Cl−
o
)-dependent K+/K+ exchange and Na+
o
-dependent K+,Cl− cotransport, respectively.
Received: 30 January 1996/Revised: 20 May 1996 相似文献
9.
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
10.
Growth characteristic and ion contents of rice callus under the influence of NaCl and hydroxyproline
Calli of salt tolerant (Bhoora rata) and salt susceptible (GR11) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD50 concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth rate of callus and Na+, K+, Cl−, Mg+2, and Ca+2 contents of the cultured rice tissues were determined at the end of 0, 2, 4 and 6 weeks of incubation. Hydroxyproline resistant
calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing both NaCl and hydroxyproline showed
increased dry weight and enhanced intracellular levels of K+, Mg+2 and Ca+2. The accumulation of Na+ and Cl− ions was less in the hydroxyproline resistant calli. 相似文献
11.
Effects of copper on branchial ionoregulation in the rainbow trout,Salmo gairdneri Richardson 总被引:1,自引:1,他引:0
Darrel Jon Laurén D. G. McDonald 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1985,155(5):635-644
Summary Hard- and softwater acclimated adult rainbow trout were statically exposed to copper (12.5, 25, 50, 100, and 200 ppb) for
two, 12 h periods at neutral and pH 5.0. Unidirectional Na+, and Cl−, and net Na+, Cl−, K+, and ammonia fluxes were monitored as a measure of branchial ionoregulatory disturbance. Copper concentrations as low as
12.5 ppb led to measurable ion losses. Net Na+, Cl−, and K+ losses were concentration dependent and unaffected by prior acclimation to either hard- or softwater at both neutral pH and
pH 5. From 12.5 to 50 ppb net NaCl losses arose primarily as a result of the inhibition ofJ
in, and at higher concentrations,J
out was also stimulated. In softwater,J
in was more resistant to inhibition than in hardwater. However, in hardwater,J
out recovered to normal levels during the second 12 h period, but no such recovery was found in softwater. Plasma NaCl was inversely
correlated with [copper], while plasma glucose and ammonia increased with [copper]. At pH 5.0 and [copper] from 12.5 to 50
ppb, H+ contributed significantly to the total ion loss, while at 100 and 200 ppb, ion losses were no greater at pH 5.0 than at neutral
pH. In no case were the effects of copper and H+ strictly additive. 相似文献
12.
Sulian Lv Lingling Nie Pengxiang Fan Xuchu Wang Dan Jiang Xianyang Chen Yinxin Li 《Acta Physiologiae Plantarum》2012,34(2):503-513
Salicornia europaea is a succulent euhalophyte that belongs to the Chenopodiaceae family. It is found that moderate concentration of NaCl can
dramatically stimulate the growth of S. europaea plants. To elucidate the mechanism underlying the phenomenon, morphological and physiological changes of S. europaea in response to different ions, including cations (Na+, K+, Li+, Cs+) and anions (Cl−, NO3
−, CH3COO−) were investigated, and the effects of Na+, Cl− and K+ on the growth of S. europaea were also studied. Na+ was more effective than K+ and Cl− in stimulating shoot succulence, cell expansion, and stomatal opening. Plants treated with Na+ (including NaCl, Na+, NaNO3) showed better plant growth, increased photosynthesis and less cell membrane damage than those untreated and treated with
200 mM of Cl− and K+ (including KCl and KNO3). Both SEM-X-Ray microanalysis and flame emission results revealed that well developed S. europaea plants had a higher content of sodium but lower potassium and chlorine. It is concluded that sodium plays a more important
role in the growth and development of S. europaea than potassium and chloride. 相似文献
13.
Effect of endothelin-1 and chemically induced hypoxia on Na+−K+−Cl− cotransport activity in cultured rat brain capillary endothelial cells was examined by using86Rb+ as a tracer for K+; bumetanide-sensitive K+ uptake was defined as Na+−K+−Cl− cotransport activity. Endothelin-1, phorbol 12-myristate 13-acetate (PMA), or thapsigargin increased Na+−K+−Cl− cotransport activity. A protein kinase C inhibitor, bisindolylmaleimide, inhibited PMA- and endothelin-1- (but not thapsigargin-)
induced Na+−K+−Cl− cotransport activity, indicating the presence of both protein kinase C-dependent regulatory mechanisms and protein kinase
C-independent mechanisms which involve intracellular Ca2+. Oligomycin, sodium azide, or antimycin A increased Na+−K+−Cl− cotransport activity by 80–200%. Oligomycin-induced Na+−K+−Cl− cotransport activity was reduced by an intracellular Ca2+ chelator (BAPTA/AM) but not affected by bisindolylmaleimide, suggesting the involvement of intracellular Ca2+, and not protein kinase C, in hypoxia-induced Na+−K+−Cl− cotransport activity.
Portions were presented at “27th Annual Meeting, The American Society for Neurochemistry” Philadelphia, Pennsylvania, March
2–6, 1996. 相似文献
14.
M. M. Reddy J. R. Riordan P. M. Quinton 《In vitro cellular & developmental biology. Plant》1988,24(9):905-910
Summary The present investigation was undertaken to examine the usefulness of cultured human sweat duct cells for ion transport and
related studies in the genetic disease, cystic fibrosis. Electrical properties of cultured duct (CD) cells were compared with
electrical properties of microperfused duct (MPD) cells. The resting apical membrane potential (V
a
) of the CD cells was −26.4±0.9 mV,n=158 cells as compared to −24.3±0.6 mV,n=105 of MPD cells. The Na+−K+ pump inhibitor ouabain, when applied to the apical surface of the CD cells and basolateral surface of MPD cells, depolarized
both CD cells (from −28.6±3.6 to −16.8±2.4 mV,n=5) and MPD cells (from −23.8±0.5 mV to −19.5±1.8 mV,n=6). The Na+ conductance inhibitor amiloride applied to the apical surface hyperpolarized the apical membrane potentials (Va) of CD cells and MPD cells by −13.2±1.4 mV,n=43 and −34.3±3.1 mV,n=19), respectively, indicating the presence of amiloride sensitive Na+ channels in both groups of cells. However, the amiloride sensitivity of CD cells was dependent on the age of the culture.
Cl− substitution at the apical side by the impermeant anion gluconate depolarized the V
a
of CD cells and MPD cells by 12.2±0.9 mV,n=32 and 37.9±4.3 mV,n=12, respectively. The effect of β-adrenergic agonist isoproterenol (IPR), was inconsistent. In CD cells, IPR either hyperpolarized
(ΔV
a
=−8.3±1.2mV,n=5) or depolarized (ΔV
a
=8.2±2.3 mV,n=4) or had no effect,n=2. In contrast, most of the MPD cells did not respond to IPR, but three cells had a varied response to IPR. Our results suggest
that CD cells, like MPD cells, retain significant Na+ and Cl− conductances. CD cells seem to have developed a higher sensitivity to β-adrenergic stimulation in tissue culture as compared
to MPD cells.
This work was supported by grants from the National Institutes of Health, Bethesda, MD, DK26547, Getty Oil Co., the Gillette
Co., Cystic Fibrosis Research Inc., and the U.S. National Cystic Fibrosis Foundation. 相似文献
15.
Summary The intact human reabsorptive sweat duct (RD) has been a reliable model for investigations of the functional role of “endogenous”
CFTR (cystic fibrosis transmembrane conductance regulator) in normal and abnormal electrolyte absorptive function. But to
overcome the limitations imposed by the use of fresh, intact tissue, we transformed cultured RD cells using the chimeric virus
Ad5/SV40 1613 ori-. The resultant cell line, RD2(NL), has remained differentiated forming a polarized epithelium that expressed
two fundamental components of absorption, a cAMP activated Cl− conductance (Gcl) and an amiloride-sensitive Na+ conductance (GNa). In the unstimulated state, there was a low level of transport activity; however, addition of forskolin (10−5
M) significantly increased the Cl− diffusion potential (Vt) generated by a luminally directed Cl− gradient from − 15.3 ± 0.7 mV to −23.9 ± 1.1 mV,n=39; and decreased the transepithelial resistance (Rt) from 814.8 ± 56.3 Ω.cm2 to 750.5 ± 47.5 Ω.cm2,n=39, (n=number of cultures). cAMP activation, anion selectivity (Cl−>I−>gluconate), and a dependence upon metabolic energy (metabolic poisoning inhibited GCl), all indicate that the GCl expressed in RD2(NL) is in fact CFTR-GCl. The presence of an apical amiloride-sensitive GNa was shown by the amiloride (10−5
M) inhibition of GNa as indicated by a reduction of Vt and equivalent short circuit current by 78.0 ± 3.1% and 77.9 ± 2.6%, respectively, and an increase in Rt by 7.2 ± 0.8%,n=36. In conclusion, the RD2(NL) cell line presents the first model system in which CFTR-GCl is expressed in a purely absorptive tissue. It provides an opportunity to study the properties and role of CFTR in the context
of absorptive function in immortalized epithelial cells. 相似文献
16.
The balance of K+, Na+, and Cl− fluxes across the cell membrane with the Na+/K+ pump, ion channels, and Na+K+2Cl− (NKCC) and Na+-Cl− (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional
K+, Na+, and Cl− fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if
the following experimental data are known: K+, Na+, and Cl− concentrations in cell water; total Cl− flux; total K+ influx; and the ouabain-inhibited pump component of the Rb+(K+) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported
in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells
induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral
permeability of Na+ channels, whereas K+ and Cl− channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K+, Na+, and Cl− account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining
the nonequilibrium steady-state distribution of Cl−, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors. 相似文献
17.
18.
Hyperthermia induces transient changes in [Na+]
i
and [K+]
i
in mammalian cells. Since Cl− flux is coupled with Na+ and K+ in several processes, including cell volume control, we have measured the effects of heat on [Cl−]
i
using the chloride indicator, MQAE, with flow cytometry. The mean basal level of [Cl−]
i
in Chinese hamster ovary cells was 12 mm. Cells heated at 42.0° or 45.0°C for 30 min had about a 2.5-fold increase in [Cl−]
i
above unheated control values when measured immediately after heating. There was about a 3-fold decrease in [Na+]
i
under the same conditions, as measured by Sodium Green. The magnitude of the increase in [Cl−]
i
depended upon time and temperature. The [Cl−]
i
recovered in a time-dependent fashion to control values by 30 min after heating. When cells were heated at 45.0°C for 30
min in the presence of 1.5 mm furosemide, the heat-induced [Cl−]
i
increase was completely blocked. Since furosemide inhibits the Na+/K+/2Cl− cotransporter, Cl− channels, and even Cl−HCO3 exchange, these ion transporters may be involved in the heat-induced increase in [Cl−]
i
.
Received: 15 June 1995/Revised: 9 April 1996 相似文献
19.
The accumulation of inorganic and organic osmolytes and their role in osmotic adjustment were investigated in roots and leaves
of vetiver grass (Vetiveria zizanioides) seedlings stressed with 100, 200, and 300 mM NaCl for 9 days. The results showed that, although the contents of inorganic
(K+, Na+, Ca2+, Mg2+, Cl−, NO3−, SO42− and H2PO3−)) and organic (soluble sugar, organic acids, and free amino acids) osmolytes all increased with NaCl concentration, the contribution
of inorganic ions (mainly Na+, K+, and Cl−) to osmotic adjustment was higher (71.50–80.56% of total) than that of organic solutes (19.43–28.50%). The contribution of
inorganic ions increased and that of organic solutes decreased in roots with the enhanced NaCl concentration, whereas the
case in leaves was opposite. On the other hand, the osmotic adjustment was only effective for vetiver grass seedlings under
moderate saline stress (less than 200 mM NaCl). 相似文献
20.
Koop JH Grieshaber MK 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2000,170(1):75-83
Fluctuating salinities at different sites on the German salt-polluted rivers Werra and Weser were compared with extracellular
ion levels of specimens of Gammarus tigrinus (Sexton; Amphipoda, Crustacea), collected at the same sites. G. tigrinus regulated haemolymph concentrations of inorganic anions (Cl−, SO2−
4, PO3−
4) and cations (Na+, K+, Mg2+, Ca2+) during fluctuations of salt pollution in the upper Weser. This capacity to regulate varying levels of salt pollution in
the upper Weser, correlated well with the distribution of the brackish amphipods in this river ecosystem. G. tigrinus tolerated periods of Na+ and Cl− stress (>380 mmol l−1) without compensating these maxima by regulating extracellular Na+ and Cl−. However, during such bursts of Na+ and Cl− stress in Werra and Weser, the ability to regulate extracellular [K+] at river water K+ stress of ≥6.0 mmol l−1 may explain why this brackish species has been more successful in these rivers than its competitors like Gammarus pulex. The present investigation demonstrates that the water salinity affects the [NO−
3] in the haemolymph of G. tigrinus. With increasing hypo-osmotic stress the animals accumulate increasing amounts of NO−
3. A simultaneous increase in stream water [NO−
3] causes an additional accumulation of NO−
3 in the haemolymph. The high extent of accumulation indicates that active ion transport systems may be involved. The accumulation
of NO−
3 in the haemolymph has low physiological consequences to G. tigrinus, but when hypo-osmotically stressed under anoxic conditions, nitrite formed by the reduction of nitrate may have an adverse
affect on the metabolism of G. tigrinus.
Accepted: 4 October 1999 相似文献