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1.
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
2.
We have characterized a Na+/H+ exchanger in the membrane of isolated zymogen granules (ZG) from rat exocrine pancreas and investigated its role in secretagogue-induced
enzyme secretion. ZG Na+/H+ exchanger activity was estimated by measuring Na+ or Li+ influx and consequent osmotic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternatively, intragranule pH was
investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na+- or Li+-dependent ZG lysis was enhanced by increasing inward to outward directed H+ gradients. Na+-dependent ZG lysis was not prevented by an inside-positive K+ diffusion potential generated by valinomycin which argues against parallel operation of separate electrogenic Na+ and H+ permeabilities and for coupled Na+/H+ exchange through an electroneutral carrier. Na+- and Li+-dependent ZG lysis was inhibited by EIPA (EC50∼25 μm) and benzamil (EC50∼100 μm), but only weakly by amiloride. Similarly, absorbance changes due to release of acridine orange from acidic granules into
the medium were obtained with Na+ and Li+ salts only, and were inhibited by EIPA, suggesting the presence of a Na+/H+ exchanger in the membrane. Na+ dependent lysis of ZG was inhibited by 0.5 mm MgATP and MgATP-γ-S by about 60% and 35%, respectively. Inhibition by MgATP was prevented by incubation of ZG with alkaline
phosphatase (100 U/ml), or by the calmodulin antagonists calmidazolium (0.75 μm), trifluoperazine (100 μm) and W-7 (500 μm), suggesting that the ZG Na+/H+ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na+ dependence of secretagogue (CCK-OP)-stimulated amylase secretion was investigated in digitonin permeabilized rat pancreatic
acini and was higher in acini incubated in Na+ containing buffer (30 mm NaCl/105 mm KCl buffer; 6.4 ± 0.4% of total amylase above basal) compared to buffer without Na+ (0 mm NaCl/135 mm KCl buffer; 4.7 ± 0.4% of total amylase above basal, P < 0.03). EIPA (50 μm) reduced CCK-OP-induced amylase secretion in Na+ containing buffer from 7.5 ± 0.6% to 4.1 ± 0.8% (P < 0.02). In the absence of Na+ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 μm EIPA. The data suggest that an amiloride insensitive, EIPA inhibitable Na+/H+ exchanger is present in ZG membranes, which is stimulated by calmodulin antagonists and could be involved in secretagogue-induced
enzyme secretion from rat pancreatic acini.
Received: 7 December 1995/Revised: 2 April 1996 相似文献
3.
K. van Norren R. Gorissen F. Borgese J.M.P.M. Borggreven J.J.H.H.M. De Pont 《The Journal of membrane biology》1997,160(3):193-199
The protein responsible for the Na+/Li+ exchange activity across the erythrocyte membrane has not been cloned or isolated. It has been suggested that a Na+/H+ exchanger could be responsible for the Na+/Li+ exchange activity across the erythrocyte membrane. Previously, we reported that in the trout erythrocyte, the Li+/H+ exchange activity (mediated by the Na+/H+ exchanger βNHE) and the Na+/Li+ exchange activity respond differently to cAMP, DMA (dimethyl-amiloride) and O2. We concluded that the DMA insensitive Na+/Li+ exchange activity originates from a different protein. To further examine these findings, we measured Li+ efflux in fibroblasts expressing the βNHE as the only Na+/H+ exchanger. Moreover, the internal pH of these cells was monitored with a fluorescent probe. Our findings indicate that acidification
of fibroblasts expressing the Na+/H+ exchanger βNHE, induces a Na+ stimulated Li+ efflux activity in trout erythrocytes. This exchange activity, however, is DMA sensitive and therefore differs from the DMA
insensitive Na+/Li+ exchange activity. In these fibroblasts no significant DMA insensitive Na+/Li+ exchange activity was found. These results support the hypothesis that the trout erythrocyte Na+/Li+ exchange activity is not mediated by the Na+/H+ exchanger (βNHE) present in these membranes.
Received: 6 December 1996/Revised: 11 August 1997 相似文献
4.
The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using
continuous short circuit current (I
SC
) measurements in HCO3
−/CO2 buffered solution. Steady state I
SC
averaged 73.8 ± 3.2 μA/cm2 (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na+ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y+ and a basal amino acid transport system y+L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic
amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I
SC
averaging 36.6 ± 5.4 μA/cm2 (n= 19) and 32.0 ± 7.2 μA/cm2 (n= 8), respectively. The response was preserved in the absence of bath Cl− (n= 4), but was abolished either in the absence of apical Na+ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I
SC
response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization
of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na+ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract
passive cationic amino acid leak into the CCD in the presence of electrogenic Na+ transport, consistent with reports of stimulated expression of Na+ and cationic amino acid transporters by aldosterone.
Received: 11 September 2000/Revised: 6 December 2000 相似文献
5.
M.Z. Abedin D.I.N. Giurgiu Z.R. Abedin E.A. Peck X. Su P.R. Smith 《The Journal of membrane biology》2001,182(2):123-134
Gallbladder Na+ absorption is linked to gallstone formation in prairie dogs. Na+/H+ exchange (NHE) is one of the major Na+ absorptive pathways in gallbladder. In this study, we measured gallbladder Na+/H+ exchange and characterized the NHE isoforms expressed in prairie dogs. Na+/H+ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na+ flux and apical 22Na+ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J
Na
ms was higher than J
Na
sm with J
Na
net absorption. Mucosal DMA inhibited transepithelial Na+ flux in a dose-dependent fashion, causing J
Na
ms equal to J
Na
sm and blocking J
Na
net absorption at 100 μm. Basal 22Na+ uptake rate was 10.9 ± 1.0 μmol · cm−2· hr−1 which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression
of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na+/H+ exchange accounts for a substantial fraction of gallbladder apical Na+ entry and most of net Na+ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na+ uptake and transepithelial Na+ absorption.
Received: 9 February 2001/Revised: 11 April 2001 相似文献
6.
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
7.
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
8.
We have used computer modeling to investigate how pancreatic duct cells can secrete a fluid containing near isotonic (∼140
mm) NaHCO3. Experimental data suggest that NaHCO3 secretion occurs in three steps: (i) accumulation of HCO−
3 across the basolateral membrane of the duct cell by Na(HCO3)
n
cotransporters, Na+/H+ exchangers and proton pumps; (ii) secretion of HCO−
3 across the luminal membrane on Cl−/HCO−
3 antiporters operating in parallel with Cl− channels; and (iii) diffusion of Na+ through the paracellular pathway. Programming the currently available experimental data into our computer model shows that
this mechanism for HCO−
3 secretion is deficient in one important respect. While it can produce a relatively large volume of a HCO−
3-rich fluid, it can only raise the luminal HCO−
3 concentration up to about 70 mm. To achieve secretion of 140 mm NaHCO3 by the model it is necessary to: (i) reduce the conductive Cl− permeability and increase the conductive HCO−
3 permeability of the luminal membrane of the duct cell, and (ii) reduce the activity of the luminal Cl−/HCO−
3 antiporters. Under these conditions most of the HCO−
3 is secreted via a conductive pathway. Based on our data, we propose that HCO−
3 secretion occurs mainly by the antiporter in duct segments near the acini (luminal HCO−
3 concentration up to ∼70 mm), but mainly via channels further down the ductal tree (raising luminal HCO−
3 to ∼140 mm).
Received: 15 November 1999/Revised: 29 March 2000 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.
We here report on studies on the frog skin epithelium to identify the nature of its excretory H+ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry
using V-ATPase-directed antibodies. Bafilomycin A1 (10 μm) blocked H+ excretion (69 ± 8% inhibition) and therefore Na+ absorption (61 ± 17% inhibition after 60 min application, n= 6) in open-circuited skins bathed on their apical side with a 1 mm Na2SO4 solution, ``low-Na+ conditions' under which H+ and Na+ fluxes are coupled 1:1. The electrogenic outward H+ current measured in absence of Na+ transport (in the presence of 50 μm amiloride) was also blocked by 10 μm bafilomycin A1 or 5 μm concanamycin A. In contrast, no effects were found on the large and dominant Na+ transport (short-circuit current), which develops with apical solutions containing 115 mm Na+ (``high-Na+ conditions'), demonstrating a specific action on H+ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit
E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds
to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane
extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody
labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations
now deliver conclusive evidence that H+ excretion is mediated by a V-ATPase being the electrogenic H+ pump in frog skin.
Received: 21 May 1996/Revised: 24 December 1996 相似文献
11.
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
12.
The NHE-1 isoform of the Na+/H+ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed
since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensor™
microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, EMD 94309, EMD 96785 and HOE
642 which are inhibitors of the isoform 1 of the Na+/H+ exchanger. The experiments were performed with Chinese hamster ovary cells (CHO K1) which are enriched in the NHE-1 isoform
of the Na+/H+ antiporter. The Na+/H+ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton
exchange rate was measured as a function of the NaCl concentration in the range of 10–138 mm NaCl stimulation. The proton exchange rate followed Michaelis-Menten kinetics with a K
M
= 30 ± 4 mm for Na+. Addition of either one of the four inhibitors decreased the acidification rate. The IC50 values of the four compounds could be determined as 23 ± 7 nm for EMD 84021, 5 ± 1 nm for EMD 94309, 9 ± 2 nm for EMD 96785 and 8 ± 2 nm for HOE 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC50 values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach
is a fast and simple method to measure the activity of the Na+/H+ antiporter and allows a quantitative kinetic analysis of the proton excretion rate.
Received: 3 September 1998/Revised: 20 November 1998 相似文献
13.
A partially purified preparation of the lobster muscle Na+/Ca2+ exchanger was reconstituted with, presumably, random orientation in liposomes. Ca2+ efflux from 45Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular
(ev) medium. Extravesicular Na+ (Na
ev
)-dependent Ca2+ efflux depended directly upon the extravesicular Ca2+ concentration ([Ca2+]
ev
) with a half-maximal activation at [Ca2+]
ev
= 0.6 μm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca2+, as in most other species. In contrast, at low [Na+]
ev
, the Ca
ev
-binding site (i.e., on the cytoplasmic surface) for Ca2+ transported via Ca2+/Ca2+ exchange was half-maximally activated by about 7.5 μm Ca2+. Mild proteolysis of the Na+/Ca2+ exchanger by α-chymotrypsin also upregulated the Na
ev
-dependent Ca2+ efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca2+. Proteolytic digestion in the presence of 1.9 μm free ev Ca2+, however, induced only a 1.6-fold augmentation of Ca2+ efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca2+ also inhibited proteolytic degradation of the Na+/Ca2+ exchanger measured by immunoblotting. These data suggest that Ca2+, bound to a high affinity binding site, protects against the activation of the Na+/Ca2+ exchanger by α-chymotrypsin. Additionally, we observed a 6-fold increase in the Na+/Ca2+ exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster
body fluids.
Received: 20 October 1999/Revised: 13 January 2000 相似文献
14.
The Na+/HCO3
− cotransporter is the main system that mediates bicarbonate removal out of the proximal tubule cell into the blood. We have
previously partially purified this protein and showed that chemical modification of the α-amino groups by fluorescein isothiocyanate
(FITC) inhibited the activity of the Na+/HCO3
− cotransporter. The inhibition was prevented by the presence of Na and bicarbonate suggesting that this compound binds at
or near the substrate transport sites of the cotransporter. We examined the effect of agents that modify the sulfhydryl group
(dithiothreitol), carboxyl groups (n-n′dicyclohexyl carbodiimide) and tyrosine residues (p-nitrobenzene sulfonyl fluoride, n-acetyl imidazole and tetranitromethane) on the activity of the cotransporter to gain insight into the chemical residues which
may be important for transport function. The sulfhydryl residues modifier, carboxyl group modifier, and tyrosine modifier
significantly inhibited bicarbonate dependent 22Na uptake in basolateral membranes by 50–70% without altering the 22Na uptake in the presence of gluconate indicating that these agents directly affected the cotransporter without affecting
diffusive sodium uptake. The effect of the tyrosine modifier n-acetylimidazole was not prevented by the presence of Na and bicarbonate suggesting that the tyrosine residues are not at
the substrate binding sites. To determine the presence and role of glycosylation on the Na+/HCO3
− cotransporter protein, we examined the effects of different glycosidases (endoglycosidase F and H, N-glycosidase F, O-glycanase) on the cotransporter activity. All glycosidases caused a significant 50–80% inhibition of cotransporter
activity. These data demonstrate that N-glycosylation as well as O-glycosylation are important for the function of the Na+/HCO3
− cotransporter protein. Taken together, these results suggest that chemical modifiers of tyrosine, carboxyl and sulfhydryl
groups as well as glycosylation are important for expression of full functional activity of the cotransporter.
Received: 8 October 1996/Revised: 23 January 1997 相似文献
15.
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
16.
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
17.
The uptake of 3H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium.
Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1
μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected:
(i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when
Na+ was replaced by Li+, K+ or Mg2+, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na+ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na+ concentration. Kinetic properties of the uptake of 0.1 μm
3H-choline in the presence and absence of medium Na+ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na+; a Na+-dependent portion (a mean of 0.52 of the total) had a K
m
for choline of 1.5 μm while K
m
in the absence of Na+ (Li+ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave Ki values of 1.7 μm and 5.0 μm HC-3 for the Na+-dependent and -independent fractions. The apparent K
m
of the Na+-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K
m
values reported for high-affinity, Na+-dependent choline uptake by neuronal cells.
Received: 18 February 1997/Revised: 7 December 1997 相似文献
18.
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
19.
Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells
into spermatozoa. Cytoplasmic pH (pH
i
) has been shown to be an important parameter in cell physiology, regulating namely cell metabolism and differentiation. However,
membrane transport mechanisms involved in pH
i
regulation mechanisms of Sertoli cells have not yet been elucidated. In this work, pH
i
was determined using the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF).
Addition of weak acids resulted in rapid acidification of the intracellular milieu. Sertoli cells then recovered pH
i
by a mechanism that was shown to be sensitive to external Na+. pH
i
recovery was also greatly reduced in the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and amiloride.
These results point toward the action of an Na+-driven HCO3−/Cl− exchanger and/or an Na+/HCO3− cotransporter and the action of the Na+/H+ exchanger on pH
i
regulation in the experimental conditions used. pH
i
recovery was only slightly affected by ouabain, suggesting that the inhibition of Na+/K+-ATPase affects recovery indirectly, possibly via the shift on the Na+ gradient. On the other hand, recovery from the acid load was independent of the presence of concanamycin A, a specific inhibitor
of the V-type ATPases, suggesting that these pumps do not have a relevant action on pH
i
regulation in bovine Sertoli cells. 相似文献
20.
D. Gillis L.D. Shrode E. Krump C.M. Howard E.A. Rubie L.A. Tibbles J. Woodgett S. Grinstein 《The Journal of membrane biology》2001,181(3):205-214
The Na+/H+ exchanger (NHE) becomes activated by hyperosmolar stress, thereby contributing to cell volume regulation. The signaling pathway(s)
responsible for the shrinkage-induced activation of NHE, however, remain unknown. A family of mitogen-activated protein kinases
(MAPK), encompassing p42/p44 Erk, p38 MAPK and SAPK, has been implicated in a variety of cellular responses to changes in
osmolarity. We therefore investigated whether these kinases similarly signal the hyperosmotic activation of NHE. The time
course and osmolyte concentration dependence of hypertonic activation of NHE and of the three sub-families of MAPK were compared
in U937 cells. The temporal course and dependence on osmolarity of Erk and p38 MAPK activation were found to be similar to
that of NHE stimulation. However, while pretreatment of U937 cells with the kinase inhibitors PD98059 and SB203580 abrogated
the osmotic activation of Erk and p38 MAPK, respectively, it did not prevent the associated stimulation of NHE. Thus, Erk1/2
and/or p38 MAPK are unlikely to mediate the osmotic regulation of NHE. The kinetics of NHE activation by hyperosmolarity appeared
to precede SAPK activation. In addition, hyperosmotic activation of NHE persisted in mouse embryonic fibroblasts lacking SEK1/MKK4,
an upstream activator of SAPK. Moreover, shrinkage-induced activation of NHE still occurred in COS-7 cells that were transiently
transfected with a dominant-negative form of SEK1/MKK4 (SEK1/MKK4-A/L) that is expected to inhibit other isoforms of SEK as
well. Together, these results demonstrate that the stimulation of NHE and the activation of Erk, p38 MAPK and SAPK are parallel
but independent events.
Received: 27 November 2000/Revised: 20 March 2001 相似文献