共查询到20条相似文献,搜索用时 15 毫秒
1.
McLaughlin CW Zellhuber-McMillan S Peart D Purves RD Macknight AD Civan MM 《The Journal of membrane biology》2001,182(3):213-222
Experiments were performed to determine whether the transport properties of the ciliary epithelium vary over different regions.
Rabbit iris-ciliary bodies were incubated under experimental or control conditions for 30 min before quick freezing, cryosectioning,
dehydration and electron probe X-ray microanalysis. Cryosections were cut from three regions along the major axis of the iris-ciliary
body, i.e., the anterior, middle and posterior (pars plicata) regions. In bicarbonate/CO2 solution, the epithelial cells of the anterior and middle regions contained more Cl and K than did those of the posterior
region. These higher levels of Cl and K were reduced by the carbonic anhydrase inhibitor acetazolamide. Application of bumetanide,
an inhibitor of the Na+-K+-2Cl− cotransporter, resulted in significant increases in Cl and K in the anterior and middle regions but not in the posterior
region. In bicarbonate-free solution, the ratio for K/Na contents was higher in the posterior than in the two more anterior
regions; Na, K and Cl contents of epithelial cells in the three regions were otherwise similar. Cell composition did not differ
significantly between the crests and valleys of the posterior region. The divergent responses to perturbation of epithelial
transport in the different regions provide the first demonstration of functional heterogeneity along the major axis of the
iris-ciliary body. The response to inhibition of carbonic anhydrase raises the possibility that the anterior aspect of the
ciliary epithelium may be the major site of aqueous humor secretion.
Received: 4 December 2000/Revised: 24 April 2001 相似文献
2.
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
3.
CFTR Activation Raises Extracellular pH of NIH/3T3 Mouse Fibroblasts and C127 Epithelial Cells 总被引:2,自引:0,他引:2
Cystic Fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel found in apical membranes
of wet epithelia. Since CFTR is permeable to HCO−
3 and changes in extracellular fluid composition may contribute to CF lung disease, we investigated possible differences in
extracellular pH (pHo) between CFTR-expressing and control cell lines. The Cytosensor™ Microphysiometer was used to study
forskolin-stimulated extracellular acidification rates in CFTR-expressing and control mouse mammary epithelial (C127) and
fibroblast (NIH/3T3) cell lines. Forskolin, which activates CFTR via raised cAMP, caused decreased extracellular acidification of CFTR-expressing NIH/3T3 and C127 cells by 15–35%. By contrast, forskolin caused increased extracellular acidification of control cells by 10–20%. Ionomycin, which may activate CFTR via PKC, also elicited this decreased
extracellular acidification signal only in cells expressing CFTR. In control experiments, dideoxyforskolin had no effect on
the acidification rates and osmotic stimuli were shown to equally stimulate all cell lines. These results suggest a role for
CFTR in controlling pHo and complement recent evidence that HCO−
3 dependent epithelial secretion may be reduced in amount and altered in composition in CF.
Received: 20 June 2000/Revised: 13 November 2000 相似文献
4.
Brush-border membrane vesicles (BBMV) were prepared from superficial rat renal cortex by a divalent2+-precipitation technique using either CaCl2 or MgCl2. The dependence of the initial [14C]-d-glucose (or [3H]-l-proline) uptake rate and the extent of the overshoot of d-glucose or l-proline uphill accumulation from solutions containing 100 mm Na+ salt, was found to be dependent upon the precipitating divalent cation. With Mg2+ precipitation the initial uptake and overshoot accumulation of either d-glucose or l-proline were enhanced compared to BBMV prepared by Ca2+ precipitation. When the anion composition of the media was varied (uptake in Cl− media in comparison to gluconate−-containing media) it was found that the Cl−-dependent component of the initial uptake was markedly depressed with Ca2+-prepared BBMV (104.99 ± 33.31 vs. 13.83 ± 1.44 pmoles/sec/mg protein for Mg2+ and Ca2+ prepared vesicles respectively). When Ca2+ was loaded into Mg2+ prepared BBMV using a freeze-thaw technique, it was found that the magnitude and Cl− enhancement of d-glucose transport was reduced in a dose-dependent manner. Neomycin, an inhibitor of phospholipase C, had no effect on the
reduction of d-glucose uptake by Ca2+ in Mg2+ prepared vesicles. In contrast, phosphatase inhibitors such as vanadate and fluoride were able to partially reverse the Ca2+ inhibition of d-glucose uptake and restore the enhancement due to Cl− media. In addition, inhibitors of protein phosphatase 2B, deltamethrin (50 nm) and trifluoperazine (10 μm), caused partial reversal of Ca2+-dependent inhibition of d-glucose uptake. Direct measurement of changes in the bi-ionic (Cl−
vs. gluconate−) transmembrane electrical potential differences using the cyanine dye, 3,3′-dipropylthiodicarbocyanine iodide DiSC3-(5) confirmed that Cl− conductance was reduced in Ca2+-prepared vesicles. We conclude that a Cl− conductance coexists with Na+ cotransport in rat renal BBMV and this may be subject to negative regulation by Ca2+ via stimulation of protein phosphatase (PP2B).
Received: 14 December 1994/Revised: 27 November 1995 相似文献
5.
High speed video imaging microscopy and the pH-sensitive fluorophore2′,7′,-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
were used to examine acid-base functions of beta-intercalated cells of the rabbit cortical collecting duct. The presence of
intercalated cells was established and the properties of apical and basolateral acid-base transporters assessed by monitoring
cell pH during acid loading and luminal and basolateral ion substitutions. We showed that treatment of beta-intercalated cells
with ammonium chloride (20 mm) induced a profound decrease of their intracellular pH from 6.98 ± 5.93 ± 0.08. pH recovery occurred after different lag
periods ranging between 2 to 15 min (0.22 ± 0.04 dpH/dt). We demonstrated that this pH recovery mechanism was independent
of basolateral Na+ and apical HCO−
3 and K+. It was also not affected by apical and basolateral addition of NEM, by basolateral DIDS and by apical application of the
H-KATPase inhibitor SCH28080. The process of pH recovery was however, critically dependent on basolateral HCO−
3. These results are best explained by acid-induced insertion and/or activation of chloride-bicarbonate exchangers that are
functional properties with their apical analogues.
Received: 11 January 1994/Revised: 13 June 1997 相似文献
6.
7.
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
8.
Fluid and electrolyte secretion from secretory epithelia is a highly regulated process. Chloride channel activity at the
apical membrane determines the rate and direction of salt and water secretion. Multiple classes of Cl− channels with distinct gating mechanisms are involved in moving ions and water. Secretory agonists that induce intracellular
increases in two second messenger systems, cAMP and [Ca2+]
i
, are generally associated with secretion. However, changes in cell volume and the membrane potential may also play a role
in regulating fluid and electrolyte secretion in some tissues. In this review we discuss the regulation of the different types
of Cl− channels found in secretory epithelia.
Received: 16 September 1997/Revised: 13 November 1997 相似文献
9.
Cremaschi D Porta C Bottà G Bazzini C Baroni MD Garavaglia M 《The Journal of membrane biology》2000,176(1):53-65
Cl− apically enters the epithelium of rabbit gallbladder by a Na+-Cl− symport, sensitive to hydrochlorothiazide (HCTZ). Since HCTZ also activates an apical SITS-sensitive Cl− conductance (G
Cl
), the symport inhibition might be merely due to a short circuit of the symport by G
Cl
rather than to a direct action of HCTZ on the symporter. To examine whether the symport is directly inhibited by HCTZ and
whether the symporter belongs to the family of thiazide-sensitive cotransporters (TSC), radiochemical measurements of the
apical Cl− uptake, electrophysiological determinations of intracellular Cl− and Na+ activities (a
i,Cl
and a
i,Na
) with selective theta microelectrodes and molecular biology methods were used. The 36Cl− uptake proved to be a measurement of the apical unidirectional Cl− influx (J
mc
) and of the symport only (without backflux components), with measuring times of 45 sec under all experiment conditions; its
inhibition by HCTZ was unaffected by G
Cl
activation or abolition. After HCTZ treatment the decrease in a
i,Cl
(measured as the initial rate or in 3 min) was larger than the decrease in a
i,Na
. The difference was reduced to one third in a group of epithelia in which the elicited G
Cl
was reduced to one third; moreover it was abolished in any case when G
Cl
was abolished with 10−4
m SITS. The SITS-insensitive rate of a
i,Cl
decrease was equal to that of the a
i,Na
decrease in any case. Thus the a
i,Cl
decrease displays a component dependent on G
Cl
activation and a second component dependent on symport inhibition. Using the RT-PCR technique a cDNA fragment was obtained
that was 99% identical to the corresponding region of the rabbit renal TSC isoform. The results indicate that in rabbit gallbladder
epithelium HCTZ displays a dual action, namely G
Cl
activation and Na+-Cl− symport inhibition. This Na+-Cl− symporter is the first TSC found to be functionally expressed in a nonrenal or nonrenal-like epithelium.
Received: 29 July 1999/Revised: 23 March 2000 相似文献
10.
A.A. Bernardo F.T. Kear J.A. Stim O.S. Ruiz J.A.L. Arruda 《The Journal of membrane biology》1996,154(2):155-162
We have previously partially purified the basolateral Na+/HCO−
3 cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement
of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na+/HCO−
3 cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity.
Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that
the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against
the Na+/HCO−
3 cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na+/HCO−
3 cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes.
The purified antibody fraction caused a concentration-dependent inhibition of the Na+/HCO−
3 cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of
the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence
of the substrates (NaHCO3 or Na2CO3) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared
with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies
recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in
the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and
small intestine but was not detected in red blood cell membranes. Localization of the Na+/HCO−
3 cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes
of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against
the 56 kDa basolateral protein inhibit the activity of the Na+/HCO−
3 cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact
at or near the substrate binding sites. The Na+/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues.
Received: 27 January 1996/Revised: 23 July 1996 相似文献
11.
R.Z. Sabirov R.R. Azimov Y. Ando-Akatsuka T. Miyoshi Y. Okada 《The Journal of membrane biology》1999,172(1):67-76
To examine the extracellular Na+ sensitivity of a renal inwardly rectifying K+ channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K+ channel, ROMK1 (Kir1.1). When extracellular Na+ was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel
ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na+ from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na+ removal from the bath solution. A blocker of Na+/H+ antiporters, amiloride, largely inhibited the Na+ removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much
less sensitive to Na+ removal. Na+ removal was found to induce a significant decrease in intracellular pH in the oocytes using H+-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na+/H+ antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na+ in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular
Na+, and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na+ sensitivity of ROMK1 channel via regulating intracellular pH.
Received: 13 April 1999/Revised: 15 July 1999 相似文献
12.
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
13.
D. Claveau I. Pellerin M. Leclerc M.G. Brunette 《The Journal of membrane biology》1998,165(3):265-274
In the rabbit as well as the rat, a Na+/H+ exchanger is expressed in the apical membrane of both the proximal and distal tubules of the renal cortex. Whereas the isoform
derived from the proximal tubule has been extensively studied, little information is available concerning the distal luminal
membrane isoform. To better characterize the latter isoform, we purified rabbit proximal and distal tubules, and examined
the ethylpropylamiloride (EIPA)-sensitive 22Na uptake by the luminal membrane vesicles from the two segments. The presence of 100 μm EIPA in the membrane suspension decreased the 15 sec Na+ uptake to 75.70 ± 4.70% and 50.30 ± 2.23% of the control values in vesicles from proximal and distal tubules, respectively.
The effect of EIPA on 35 mm Na+ uptake was concentration dependent, with a IC50 of 700 μm and 75 μm for the proximal and distal luminal membranes. Whereas the proximal tubule membrane isoform was insensitive to cimetidine
and clonidine up to a concentration of 2 mm, the 35 mm Na+ uptake by the distal membrane was strongly inhibited by cimetidine (IC50 700 μm) and modestly inhibited by clonidine (IC50 1.6 mm).
The incubation of proximal tubule suspensions with 1 mm (Bu2) cAMP decreased the 15-sec EIPA-sensitive Na+ uptake by the brush border membranes to 24.1 ± 2.38% of the control values. Unexpectedly, the same treatment of distal tubules
enhanced this uptake by 46.5 ± 10.3%. Finally, incubation of tubule suspensions with 100 nm phorbol 12-myristate 13-acetate (PMA) decreased the exchanger activity to 58.6 ± 3.04% and 79.7 ± 3.21% of the control values
in the proximal and distal luminal membranes, respectively. In conclusion, the high sensitivity of the distal luminal membrane
exchanger to various inhibitors, and its stimulation by cAMP-dependent protein kinase A, indicate that this isoform differs
from that of the proximal tubule and probably corresponds to isoform 1.
Received: 6 March 1998/Revised: 6 July 1998 相似文献
14.
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
15.
C. Bookstein M.W. Musch P.K. Dudeja R.L. McSwine Y. Xie T.A. Brasitus M.C. Rao E.B. Chang 《The Journal of membrane biology》1997,160(3):183-192
This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants
of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease
of ≈50% in the specific activities of both NHE1 and NHE3. Only V
max was affected; K
Na was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride,
5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely
unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues
in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular
transport by incubation at 4°C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect
does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity)
influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.
Received: 23 January 1997/Revised: 1 August 1997 相似文献
16.
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
17.
To study the role of intracellular pH (pHi) in catecholamine secretion and the regulation of pHi in bovine chromaffin cells, the pH-sensitive fluorescent indicator [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] was used to monitor the on-line changes in pHi. The pHi of chromaffin cells at resting state is approximately 7.2. The pHi was manipulated first by incubation of the cells with NH4+, and then the solution was replaced with a NH4(+)-free solution to induce acidification of the cytoplasm. The pHi returned toward the basal pH value after acidification within 5-10 min in the presence of Na+ or Li+, but the pHi stayed acidic when Na(+)-free buffers were used or in the presence of amiloride and its analogues. These results suggest that the pH recovery process after an acid load is due to the Na+/H+ exchange activity in the plasma membrane of the chromaffin cells. The catecholamine secretion evoked by carbachol and Na+ removal was enhanced after the cytoplasm had been made more acidic. It appears that acidic pH favors the occurrence of exocytosis. 相似文献
18.
The rabbit Na+/glucose cotransporter (SGLT1) exhibits a presteady-state current after step changes in membrane voltage in the absence of
sugar. These currents reflect voltage-dependent processes involved in cotransport, and provide insight on the partial reactions
of the transport cycle. SGLT1 presteady-state currents were studied as a function of external Na+, membrane voltage V
m
, phlorizin and temperature. Step changes in membrane voltage—from the holding V
h
to test values, elicited transient currents that rose rapidly to a peak (at 3–4 msec), before decaying to the steady state,
with time constants τ≈4–20 msec, and were blocked by phlorizin (K
i
≈30 μm). The total charge Q was equal for the application of the voltage pulse and the subsequent removal, and was a function of V
m
. The Q-V curves obeyed the Boltzmann relation: the maximal charge Q
max was 4–120 nC; V
0.5, the voltage for 50% Q
max was −5 to +30 mV; and z, the apparent valence of the moveable charge, was 1. Q
max and z were independent of V
h
(between 0 and −100 mV) and temperature (20–30°C), while increasing temperature shifted V
0.5 towards more negative values. Decreasing [Na+]
o
decreased Q
max, and shifted V
0.5 to more negative voltages 9by −100 mV per 10-fold decrease in [Na+]
o
). The time constant τ was voltage dependent: the τ-V relations were bell-shaped, with maximal τmax 8–20 msec. Decreasing [Na+]
o
decreased τmax, and shifted the τ-V curves towards more negative voltages. Increasing temperature also shifted the τ-V curves, but did not affect τmax. The maximum temperature coefficient Q
10 for τ was 3–4, and corresponds to an activation energy of 25 kcal/mole. Simulations of a 6-state ordered kinetic model for
rabbit Na+/glucose cotransport indicate that charge-movements are due to Na+-binding/dissociation and a conformational change of the empty transporter. The model predicts that (i) transient currents
rise to a peak before decay to steady-state; (ii) the τ-V relations are bell-shaped, and shift towards more negative voltages as [Na+]
o
is reduced; (iii) τmax is decreased with decreasing [Na+]
o
; and (iv) the Q-V relations are shifted towards negative voltages as [Na+]
o
is reduced. In general, the kinetic properties of the presteady-state currents are qualitatively predicted by the model.
Received: 12 August 1996/Revised: 30 September 1996 相似文献
19.
20.
Since the major mechanisms responsible for regulation of intracellular pH of enterocytes are located in the basolateral membrane,
respective effects may be expected on pH in the compartment near the basolateral membrane. A method was established to estimate
the pH at the basolateral membrane (pH
b
) of isolated caecal epithelia of guinea pig using pH-sensitive fluorescein attached to lectin (lens culinaris). In the presence of bicarbonate and a perfusion solution-pH of 7.4, pH
b
was 7.70 ± 0.15. In the absence of bicarbonate or chloride as well as by inhibition of the basolateral Cl−-HCO−
3 exchange with H2-DIDS, pH
b
was reduced near to solution-pH. Inhibition of the basolateral Na+-H+ exchanger by adding a sodium- and bicarbonate-free, low-buffered solution increased pH
b
. Decrease of pH of serosal perfusion solution to 6.4 provoked a similar decrease of pH
b
to solution pH. Short-chain fatty acids (SCFA) added to the mucosal solution caused a slight decrease of pH
b
. SCFA added to the serosal side alkalized pH
b
. However, in the presence of bicarbonate pH
b
returned quickly to the initial pH
b
, and after removal of SCFA a transient acidification of pH
b
was seen. These responses could not be inhibited by MIA or H2-DIDS. We conclude that no constant pH-microclimate exists at the basolateral side. The regulation of the intracellular pH
of enterocytes reflects pH
b
. The slightly alkaline pH
b
is due to the bicarbonate efflux. Data support the presence of an SCFA−-HCO−
3 exchange.
Received: 17 December 1998/Revised: 24 February 1999 相似文献