共查询到20条相似文献,搜索用时 15 毫秒
1.
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
2.
C. Bookstein M.W. Musch Y. Xie M.C. Rao E.B. Chang 《The Journal of membrane biology》1999,171(1):87-95
Until recently, studies to characterize the intestinal epithelial Na+/H+ exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na+/H+ exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure
NHE activity. Because changes in pH
i
only approximate Na+/H+ exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation,
we have used 22[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these
measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in
the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated
by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition
by cAMP and Ca++ involves changes to both K
Na
and V
max
. In contrast, the same two second messengers inhibit NHE3 by a decrease in V
max
exclusively. Phorbol ester activation of protein kinase C alters both V
max
and K
Na
of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active
and are differentially regulated by signal transduction pathways.
Received: 28 January 1999/Revised: 18 May 1999 相似文献
3.
We have previously shown that epithelial Na+ channels in mouse mandibular gland duct cells are controlled by cytosolic Na+ and Cl−, acting, respectively, via G
o
and G
i
proteins. Since we found no evidence for control of epithelial Na+ channels by extracellular Na+ ([Na+]
o
), our findings conflicted with the long-held belief that Na+ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues,
like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel
site which otherwise inhibits channel activity in response to increasing [Na+]
o
. Instead, we now show that PCMPS acts by rendering epithelial Na+ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na+ and Cl− on Na+ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane,
block feedback inhibition of Na+ channels by cytosolic Na+, while leaving inhibition by cytosolic Cl− unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated α-subunit of
G
o
in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na+ receptor.
Received: 1 October 1997/Revised: 24 December 1997 相似文献
4.
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
5.
6.
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
7.
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
8.
Renal A6 cells have been reported in which hyposmolality stimulates Na+ transport by increasing the number of conducting amiloride-sensitive 4-pS Na+ channels at the apical membrane. To study a possible role of protein tyrosine kinase (PTK) in the hyposmolality-induced signaling,
we investigated effects of PTK inhibitors on the hyposmolality-induced Na+ transport in A6 cells. Tyrphostin A23 (a PTK inhibitor) blocked the stimulatory action of hyposmolality on a number of the
conducting Na+ channels. Tyrphostin A23 also abolished macroscopic Na+ currents (amiloride-sensitive short-circuit current, I
Na
) by decreasing the elevating rate of the hyposmolality-increased I
Na
. Genistein (another type of PTK inhibitor) also showed an effect similar to tyrphostin A23. Brefeldin A (BFA), which is an
inhibitor of intracellular translocation of protein, blocked the action of hyposmolality on I
Na
by diminishing the elevating rate of the hyposmolality-increased I
Na
, mimicking the inhibitory action of PTK inhibitor. Further, hyposmolality increased the activity of PTK. These observations
suggest that hyposmolality would stimulate Na+ transport by translocating the Na+ channel protein (or regulatory protein) to the apical membrane via a PTK-dependent pathway. Further, hyposmolality also caused
an increase in the plasma (apical) membrane capacitance, which was remarkably blocked by treatment with tyrphostin A23 or
BFA. These observations also suggest that a PTK-dependent pathway would be involved in the hyposmolality-stimulated membrane
fusion in A6 cells.
Received: 6 October 1999/Revised: 4 February 2000 相似文献
9.
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
10.
J. Scott Gens Lixuan Tackett Shaoyou Chu Marshall H. Montrose 《生物化学与生物物理学报:生物膜》2007,1768(5):1023-1035
We tested whether NHE3 and NHE2 Na+/H+ exchanger isoforms were recruited to the plasma membrane (PM) in response to changes in ion homeostasis. NHE2-CFP or NHE3-CFP fusion proteins were functional Na+/H+ exchangers when transiently expressed in NHE-deficient PS120 fibroblasts. Confocal morphometry of cells whose PM was labeled with FM4-64 measured the fractional amount of fusion protein at the cell surface. In resting cells, 10-20% of CFP fluorescence was at PM and stable over time. A protocol commonly used to activate the Na+/H+ exchange function (NH4-prepulse acid load sustained in Na+-free medium), increased PM percentages of PM NHE3-CFP and NHE2-CFP. Separation of cellular acidification from Na+ removal revealed that only NHE3-CFP translocated when medium Na+ was removed, and only NHE2-CFP translocated when the cell was acidified. NHE2/NHE3 chimeric proteins demonstrate that the Na+-removal response element resides predominantly in the NHE3 cytoplasmic tail and is distinct from the acidification response sequence of NHE2. 相似文献
11.
The aim of this study was to clarify the mechanism of isotonic fluid transport in frog skin glands. Stationary ion secretion
by the glands was studied by measuring unidirectional fluxes of 24Na+, 42K+, and carrier-free 134Cs+ in paired frog skins bathed on both sides with Ringer's solution, and with 10−5
m noradrenaline on the inside and 10−4
m amiloride on the outside. At transepithelial thermodynamic equilibrium conditions, the 134Cs+ flux ratio, J
out
Cs/J
in
Cs, varied in seven pairs of preparations from 6 to 36. Since carrier-free 134Cs+ entering the cells is irreversibly trapped in the cellular compartment (Ussing & Lind, 1996), the transepithelial net flux
of 134Cs+ indicates that a paracellular flow of water is dragging 134Cs+ in the direction from the serosal- to outside solution. From the measured flux ratios it was calculated that the force driving
the secretory flux of Cs+ varied from 30 to 61 mV among preparations. In the same experiments unidirectional Na+ fluxes were measured as well, and it was found that also Na+ was subjected to secretion. The ratio of unidirectional Na+ fluxes, however, was significantly smaller than would be predicted if the two ions were both flowing along the paracellular
route dragged by the flow of water. This result indicates that Na+ and Cs+ do not take the same pathway through the glands. The flux ratio of unidirectional K+ fluxes indicated active secretion of K+. The time it takes for steady-state K+ fluxes to be established was significantly longer than that of the simultaneously measured Cs+ fluxes. These results allow the conclusion that — in addition to being transported between cells — K+ is submitted to active transport along a cellular pathway.Based on the recirculation theory, we propose a new model which
accounts for stationary Na+, K+, Cl− and water secretion under thermodynamic equilibrium conditions. The new features of the model, as compared to the classical
Silva-model for the shark-rectal gland, are: (i) the sodium pumps in the activated gland transport Na+ into the lateral intercellular space only. (ii) A barrier at the level of the basement membrane prevents the major fraction
of Na+ entering the lateral space from returning to the serosal bath. Thus, Na+ is secreted into the outside bath. It has to be assumed then that the Na+ permeability of the basement membrane barrier (P
BM
Na) is smaller than the Na+ permeability of the junctional membrane (P
JM
Na), i.e., P
JM
Na/P
BM
Na > 1. The secretory paracellular flow of water further requires that the Na+ reflection coefficients (σNa) of the two barriers are governed by the conditions, σBM
Na > 0, and σBM
Na > σJM
Na. (iii) Na+ channels are located in the apical membrane of the activated gland cells, so that a fraction of the Na+ outflux appearing downstream the lateral intercellular space is recirculated by the gland cells. Based on measured unidirectional
fluxes, a set of equations is developed from which we estimate the ion fluxes flowing through major pathways during stationary
secretion. It is shown that 80% of the sodium ions flowing downstream the lateral intercellular space is recycled by the gland
cells. Our calculations also indicate that under the conditions prevailing in the present experiments 1.8 ATP molecule would
be hydrolyzed for every Na+ secreted to the outside bath.
Received: 30 January 1996/Revised: 12 March 1996 相似文献
12.
Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells 总被引:5,自引:0,他引:5
The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings
of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of
channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K
Ca
) channel and (iii) a small K+ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear
current-voltage (I-V) relationship (27 pS) and was equally conductive for Na+ and K+, but not permeable to Cl− or N-methyl-d-glucamine. Channel activity required the presence of Ca2+ at the cytosolic face, but was detected at Ca2+ concentrations as low as 10−7
m (open probability (P
o
) = 0.11 ± 0.03, n= 8). Gadolinium decreased P
o
of the NSC channel from both the external and cytosolic side (IC50∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relationship in asymmetrical K+ and Na+ solutions. The channel was impermeable to Cl− and N-methyl-d-glucamine. P
o
of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca2+. TEA (20 mm), charybdotoxin (100 nm) and Ba2+ (1 mm) but not amiloride (1 mm) reduced P
o
from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P
o
and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward
current. We conclude that the NSC channel is Ca2+ activated, voltage-insensitive and involved in both constitutive K+ and Na+ reabsorption from endolymph while the BK channel might participate in the K+ pathway under stimulated conditions that produce an elevated intracellular Ca2+ or depolarized membrane potential.
Received: 14 October 1999/Revised: 10 December 1999 相似文献
13.
Na+/H+ exchanger (NHE) in the basolateral membrane is encoded by NHE-1 mRNA in LLC-PK1 clone 4 cells
Jun-Ichi Hanai Kenmei Takaichi Yoshihiro Miyajima Toshiro Fujita Kiyoshi Kurokawa 《Journal of cellular physiology》1997,171(3):318-324
Several isoforms of Na+/H+ exchanger (NHE-1–5) have been identified. LLC-PK1 clone 4 (CL4) expresses the amiloride-sensitive type of NHE predominantly in the basolateral membrane, which is believed to be NHE-1. It is not clear whether CL4 expresses NHE in the apical membrane and which side of NHE is encoded by the NHE-1 mRNA. Using acidified CL4 cells on the filter membrane, we examined Na+-dependent pH recovery of the apical and basolateral membranes separately. Na+ applied to the apical membrane recovered cell pH. Na+-dependent pH recovery in the apical membrane was not inhibited by SITS, DIDS, or contralateral amiloride. Li+ but not K+, chol+, or NMG+ could replace Na+. These data are consistent with the presence of NHE in the apical membrane. Transfection with an antisense oligonucleotide corresponding to the 5′ terminal site of NHE-1 cDNA of CL4 decreased NHE activity in the basolateral membrane but not in the apical membrane. We conclude that CL4 expresses NHE activities in both apical and basolateralmembranes and that NHE-1 mRNA encodes NHE only in the basolateral membrane. J. Cell. Physiol. 171:318–324, 1997. © 1997 Wiley-Liss, Inc. 相似文献
14.
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
15.
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
16.
R. Nielsen 《The Journal of membrane biology》1997,159(1):61-69
In the present work the coupling under short-circuited conditions between the net Na+-influx across isolated frog skin and the transepithelial transport of water was examined i.e., the short-circuit current
(I
sc
) and the transepithelial water movement (TEWM) were measured simultaneously. It has been shown repeatedly that the I
sc
across isolated frog skin is equal to the net transepithelial Na+ transport. Furthermore the coupling between transepithelial uptake of NaCl under open-circuit conditions and TEWM was also
measured.
The addition of antidiuretic hormone (AVT) to skins incubated under short-circuited conditions resulted in an increase in
the I
sc
and TEWM. Under control conditions I
sc
was 9.14 ± 2.43 and in the presence of AVT 45.9 ± 7.3 neq cm−2 min−1 (n= 9) and TEWM changed from 12.45 ± 4.46 to 132.8 ± 15.8 nL cm−2 min−1. The addition of the Na+ channel blocking agent amiloride resulted in a reduction both in I
sc
and TEWM, and a linear correlation between I
sc
and TEWM was found. The correlation corresponds to that 160 ± 15 (n= 7) molecules of water follow each Na+ across the skin. In another series of experiments it was found that there was a linear correlation between I
sc
and the increase in apical osmolarity needed to stop the TEWM.
The data presented indicate that the observed coupling between the net transepithelial Na+ transport and TEWM is caused by local osmosis.
Received: 16 October 1996/Revised: 6 March 1997 相似文献
17.
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
18.
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
19.
The gating and conduction properties of a channel activated by intracellular Na+ were studied by recording unitary currents in inside-out patches excised from lobster olfactory receptor neurons. Channel
openings to a single conductance level of 104 pS occurred in bursts. The open probability of the channel increased with increasing
concentrations of Na+. At 210 mm Na+, membrane depolarization increased the open probability e-fold per 36.6 mV. The distribution of channel open times could
be fit by a single exponential with a time constant of 4.09 msec at −60 mV and 90 mm Na+. The open time constant was not affected by the concentration of Na+, but was increased by membrane depolarization. At 180 mm Na+ and −60 mV, the distribution of channel closed times could be fit by the sum of four exponentials with time constants of
0.20, 1.46, 8.92 and 69.9 msec, respectively. The three longer time constants decreased, while the shortest time constant
did not vary with the concentration of Na+. Membrane depolarization decreased all four closed time constants. Burst duration was unaffected by the concentration of
Na+, but was increased by membrane depolarization. Permeability for monovalent cations relative to that of Na+ (P
X
/P
Na
), calculated from the reversal potential, was: Li+ (1.11) > Na+ (1.0) > K+ (0.54) > Rb+ (0.36) > Cs+ (0.20). Extracellular divalent cations (10 mm) blocked the inward Na+ current at −60 mV according to the following sequence: Mn2+ > Ca2+ > Sr2+ > Mg2+ > Ba2+. Relative permeabilities for divalent cations (P
Y
/P
Na
) were Ca2+ (39.0) > Mg2+ (34.1) > Mn2+ (15.5) > Ba2+ (13.8) > Na+ (1.0). Both the reversal potential and the conductance determined in divalent cation-free mixtures of Na+ and Cs+ or Li+ were monotonic functions of the mole fraction, suggesting that the channel is a single-ion pore that behaves as a multi-ion
pore when the current is carried exclusively by divalent cations. The properties of the channel are consistent with the channel
playing a role in odor activation of these primary receptor neurons.
Received: 17 September 1996/Revised: 15 November 1996 相似文献
20.
I.I. Ismailov B.K. Berdiev A.L. Bradford M.S. Awayda C.M. Fuller D.J. Benos 《The Journal of membrane biology》1996,149(2):123-132
The hypothesis that amiloride-sensitive Na+ channel complexes immunopurified from bovine renal papillary collecting tubules contain, as their core conduction component,
an ENaC subunit, was tested by functional and immunological criteria. Disulfide bond reduction with dithiothreitol (DTT) of
renal Na+ channels incorporated into planar lipid bilayers caused a reduction of single channel conductance from 40 pS to 13 pS, and
uncoupled PKA regulation of this channel. The cation permeability sequence, as assessed from bi-ionic reversal potential measurements,
and apparent amiloride equilibrium dissociation constant (K
amil
i
) of the Na+ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed
a substantial decrease in K
amil
i
following stretch (0.44 ± 0.12 μm versus 6.9 ± 1.0 μm). Moreover, stretch activation induced a loss in the channel's ability to discriminate between monovalent cations, and even
allowed Ca2+ to permeate. Polyclonal antibodies generated against a fusion protein of αbENaC recognized a 70 kDa polypeptide component
of the renal Na+ channel complex. These data suggest that ENaC is present in the immunopurified renal Na+ channel protein complex, and that PKA sensitivity is conferred by other associated proteins.
Received: 5 June 1995/Revised: 29 September 1995 相似文献