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1.
Summary The vacuolar H+ ATPase is inhibited by N-ethylmaleimide (NEM), a sulfhydryl compound, suggesting the involvement of a sulfhydryl group in this transport process. We have examined the effects of several sulfhydryl-containing compounds on the vacuolar H+ ATPase of rabbit renal cortical endosomes. A number of such compounds were effective inhibitors of endosomal H+ transport at 10–5–10–6
m, including NEM, mersalyl, aldrithiol, 5,5 dithiobis (2-nitrobenzoic acid),p-chloromercuribenzoic acid (PCMB) andp-chloromercuriphenyl sulfonic acid (PCMBS). NEM, mersalyl, aldrithiol and PCMBS had no effect on pH-gradient dissipation, whereas PCMB decreased the pH gradient faster than control. In the absence of ATP, PCMB (10–4
m) stimulated endosomal36Cl– uptake, particularly in the presence of an inside-alkaline pH gradient (pHin=7.6/pHout=5.5.). This result was not an effect of PCMB on the Cl–-conductive pathway. The less permeable PCMBS did not stimulate36Cl– uptake. The effects of PCMB were concentration dependent and were prevented by dithioerithritol,. ATP-dependent36Cl– uptake was decreased by addition of PCMB. Finally, PCMB had no effect on45Ca2+ uptake. These results support the presence of two functionally important sulfhydryl groups in this endosomal preparation. One such group is involved with ATP-driven H+ transport and must be located on the cytoplasmic surface of the endosomal membrane. The second sulfhydryl group must reside on the internal surface of the endosomal membrane and relates to a PCMB-activated Cl–/OH– exchanger that is functional both in the presence and absence of ATP. This endosomal transporter is similar to the PCMB-activated Cl–/OH– exchanger recently described in rabbit renal brush-border membranes. 相似文献
2.
Summary The effect of the K-sparing diuretic amiloride was assessed electrophysiologically in the isolated cortical collecting tubule of the rabbit, a segment which absorbs Na and secretes K. Low concentrations of amiloride in the perfusate caused a rapid, reversible, decrease in the magnitude of the lumen negative transepithelial potential difference,V
te, transepithelial conductanceG
te, and equivalent short-circuit current,I
sc, with an apparentK
1/2 of approximately 7×10–8
m. The effects of a maximum inhibitory concentration of amiloride (10–5
m) were identical to those observed upon Na removal from lumen and bath (Na removal from the bath alone has no effect). Removal of Na in the presence of 10–5
m amiloride had no affect onV
te,G
te, orI
sc, and is consistent with the view that amiloride blocks the Na conductive pathways of the apical cell membrane. Further, in the absence of Na, the subsequent addition of amiloride had no influence. In tubules where active Na absorption was either spontaneously low, or abolished by removal of Na from lumen and bath, the elevation of K from 5 to 155 meq/liter in the perfusate caused a marked change of theV
te in the negative direction and an increase in theG
te. These effects could be attributed to a high K permeability of the apical cell membrane and not of the tight junctions. Amiloride (10–5
m) had no effect on these responses to K. It is concluded that amiloride selectively blocks the apical cell membrane Na channels but has no effect on the K conductive pathways(s). This selective nature of amiloride may indicate that Na and K are transported across the apical cell membrane via separate conductive pathways. 相似文献
3.
Lawrence G. Palmer Isidore S. Edelman Bernd Lindemann 《The Journal of membrane biology》1980,57(1):59-71
Summary The basal-lateral surface of the epithelium of the urinary bladder of the toad (Bufo marinus) was depolarized by exposure of the serosal surface to 85mm KCL and 50mm sucrose. The extent of depolarization appeared to be virtually complete, as evaluated by the invariance in the transepithelial electrical potential difference and conductance on addition of nystatin (a monovalent cation ionophore) to the serosal medium. The Na-specific current (I
Na) was defined as the current sensitive to the removal of Na from the mucosal medium or inhibitable by addition of amiloride to this medium. In the presence of the high K-sucrose serosal medium, rapid, serial, stepwise clamping of the transepithelial voltage (V) yielded a curvilinear dependence ofI
Na onV; which is taken to represent theI–V curve of the apical Na channels. The constant field equation (Goldman, D.E. 1943;J. Gen. Physiol.
27:37) fits theI–V data points closely, allowing estimates to be made of the permeability to Na of the apical membrane (P
Na) and of the intracellular Na activity (Na
c
). Exposure of the apical surface to amiloride (5×10–7
m) decreasedP
Na in proportion to the decrease inI
Na (i.e., 70%) but decreased Na
c
only 25%. In contrast, an equivalent lent reduction inI
Na elicited by exposure of the basallateral surface to ouabain was accompanied by only a 20% decrease inP
Na and a sixfold increase in Na
c
. The effects of amiloride onP
Na and ouabain on Na
c
are consistent with the primary pharmacological actions of these drugs. In addition,P
Na appears to be under metabolic control, in that 2-deoxyglucose, a specific inhibitor of glycolysis, decreasedI
Na andP
Na proportionately, and lowered Na
c
marginally, effects indistinguishable from those obtained with amiloride. 相似文献
4.
Klaus Turnheim Raymond A. Frizzell Stanley G. Schultz 《The Journal of membrane biology》1978,39(2-3):233-256
Summary Ouabain abolishes the short-circuit current (I
sc
) and decreases the transepithelial conductance (G
t
) of rabbit colon. In contrast, amphotericin B elicits a maximumI
sc
and markedly increasesG
t
. However, inboth instances the amiloride-sensitive Na entry step is completely blocked, presumably due to an increase in cell Na. Conversely, when Na-depleted tissues are suddenly exposed to 140mm Na, the amiloride-sensitiveI
sc
and the amiloride-sensitive component ofG
t
(
a
G
Na
) increase abruptly to their maximum values and the decline to steady-state plateaus with a half time of 6 min; throughout the decline (I
sc/a
G
Na)=E
Na is constant at a value of 95 mV. In the presence of amphotericin B, theI
sc
abruptly rises to the same maximum but does not decline. These findings indicate that in the presence of 140mm Na the conductance of the amiloride-sensitive Na entry step can vary from a maximum value of approximately 1.6 mmhos/cm2 when cell Na is depleted, to zero when cell Na is abnormally elevated (e.g., in the presence of ouabain or amphotericin B). Our findings are consistent with a system in which the pathway responsible for transcellular Na transport parallels another cellular compartment with which it communicates. The Na capacity of the active transport pathway appears to be very small so that this compartment fills rapidly after exposure of Na-depleted cells to 140mm Na, and active transepithelial Na transport is initiated and reaches steady-state levels quickly. The Na capacity of the second compartment is much larger; the Na content of this compartment appears to be responsible for the negative feedback effect on the permeability of the amiloride-sensitive entry step. 相似文献
5.
Yuan-Heng Tai Jehan-F. Desjeux Giustina Danisi Peter F. Curran 《The Journal of membrane biology》1977,31(1):189-208
Summary Na and Cl fluxes and short-circuit current (I
sc) in rabbit ileum have been studied as a function of ionic concentrations in HCO3-free solutions. Both net Na flux (J
net
Na
) andI
sc show similar saturation functions of [Na] at fixed [Cl]. They show no significant difference between zero and 112mm Na but at 140mm NaI
sc is significantly greater than theJ
net
Na
. Net Cl transport, secretion, is observed only at 140mm Na and is approximately equivalent to the difference between theI
sc andJ
net
Na
. The transcellular mucosa-to-serosa Na fluxes measured at 140 and 70mm Na do not differ significantly from the correspondingI
sc. The net Cl flux varies with [Cl] at fixed [Na] whileI
sc is virtually not affected by [Cl]. These results suggest that the absorptive Na transport process is electrogenic and responsible for theI
sc and that the secretory fluxes of Na and Cl are coupled, require high [Na], vary with [Cl], and do not contribute toI
sc. K-free solution abolishes theI
sc after a prolonged lag. Finally, the effect of a low resistance shunt pathway on active Na absorption is examined with a four-compartment model.Deceased (October 16, 1974). 相似文献
6.
Summary Pulsed nuclear magnetic resonance spectroscopy is employed to determine the time dependence of the change in erythrocyte water permeability following exposure top-chloromercuribenzoate (PCMB) orp-chloromercuribenzene sulfonic acid (PCMBS). pH variation was used to examine the environment of the sulfhydryl groups reactive to these drugs. PCMB reacted with at least two sulfhydryl groups which affect water permeability. This was shown by the double exponential character of the change in erythrocyte diffusional permeability with time after PCMB addition. However, only one inhibition rate process could be distinguished following PCMBS exposure, suggesting that one site bound by PCMB is not accessible to PCMBS. This site is postulated to be located in a hydrophobic region of the membrane, whereas the site reached by both drugs is located in the normal anion permeation channel. The effect of pH on the degree of inhibition due to each component and the inhibition rates is explained in terms of its effect on solubility of the reagents in the membrane and variation of the dissociated-to-undissociated ratio of PCMB. 相似文献
7.
Localization of Erythrocyte Membrane Sulfhydryl Groups Essential for Glucose Transport 总被引:21,自引:5,他引:16
The reactions of three organic mercurial compounds, chlormerodrin, parachloromercuribenzoate (PCMB), and parachloromercuribenzenesulfonate (PCMBS) with intact red blood cells, hemolyzed red cells, hemoglobin solutions, and hemoglobin-free ghosts have been characterized. Both PCMB and PCMBS react with only 2 to 3 sulfhydryl groups per mole of hemoglobin in solution, whereas chlormerodrin reacts with 6 to 7. In hemoglobin-free ghosts, however, all three reagents react with a similar number of sulfhydryl groups, approximately 4 x 10-17 moles per cell, or about 25 per cent of the total stromal sulfhydryl groups, which react with inorganic mercuric chloride. In the intact cell the membrane imposes a diffusion barrier; chlormerodrin and PCMB penetrate slowly, whereas PCMBS does not. Kinetic studies of chlormerodrin binding to intact cells reveal that the majority of stromal sulfhydryl groups is located inside the diffusion barrier, with only 1 to 1.5 per cent (or 1 to 1,400,000 sites per cell) located outside of this barrier. Reaction of PCMBS with intact cells is limited to this small fraction on the outer membrane surface. All three reagents are capable of inhibiting glucose transport in the red cell. With chlormerodrin and PCMBS it was demonstrated that the inhibition results from interactions with the sulfhydryl groups located on the outer surface of the membrane. 相似文献
8.
Jeffrey L. Garvin Sidney A. Simon Edward J. Cragoe Jr. Lazaro J. Mandel 《The Journal of membrane biology》1985,87(1):45-54
Summary Several new amiloride analogues and two reported photoaffinity analogues were tested for irreversible inhibition of short-circuit current,I
sc, in toad bladder. Bromoamiloride, a photoaffinity analogue, induced 40% irreversible inhibition at 500 m after irradiation with ultraviolet light 320 nm. Iodoamiloride caused no irreversible inhibition. Of the new analogues tested, only 3,5-diamino-6-chloro-N-[(phenylamino) aminomethylene] pyrazinecarboxamide,phenamil, irreversibly inhibitedI
sc at concentrations of 0.05 to 5 m when added to the mucosal solution. Irreversible inhibition ofI
sc by phenamil may be attributed to specific blockage of the mucosal sodium channels, which depended on: 1) time of exposure; 2) mucosal pH: 3) mucosal sodium concentration. For example, 5 m phenamil irreversibly inhibitedI
sc by 38% in 103mm Na at pH 8.6 and nearly 75% in 30mm Na at pH 6.4 after a 40-min exposure. Irreversible inhibition occurred in two phases with time constants of 10 min and approximately 140 min. Due to its irreversible nature, phenamil may be used to measure channel density. 相似文献
9.
Summary The unidirectional influx of Na from the mucosal solution into the epithelium ofin vitro descending rabbit colon (J
me
Na
) determined under short-circuit conditions, is comprised of two components: one represents entry of Na into transporting epithelial cells and is abolished by amiloride which also abolishes Na absorption (J
net
Na
). The other represents diffusional Na entry into paracellular pathways traversing the epithelium. In all instances, exposure of the mucosal surface to amphotericin B increased tissue conductance andJ
me
Na
and elicited K secretion. Tissues showing a spontaneousI
sc of approximately 4 eq/cm2hr did not respond to amphotericin B with increasedI
sc andJ
net
Na
. However, in tissues characterized by a lowerI
sc under control conditions, amphotericin B increasedI
sc andJ
net
Na
to approximately 4eq/cm2hr. These findings suggest that amphotericin increasesJ
net
Na
and elicits K secretion by disrupting the normal permselectivity of the mucosal membrane. Under these conditions the extrusion of Na from cell-to-serosal solution becomes the rate limiting step in transepithelial Na transport. Finally, a close correlation betweenJ
me
Na
andJ
net
Na
was observed when the rate of Na absorption varied either spontaneously or experimentally with amiloride, suggesting that the backflux of Na from cell-to-mucosal solution is undetectably small. 相似文献
10.
Summary Segments of descending colon obtained from rabbits, that had been maintained on drinking water containing 25mm NaCl and an artificial diet which contains 1% Na and is nominally K-free, respond to aldosterone in vitro (after a 30 to 60-min lag period) with a marked increase in the short-circuit current (I
sc
), an equivalent increase in the rate of active Na absorption (J
net
Na
) and a decline in tissue resistance (R
t
). Aldosterone also brings about a marked increase in the unidirectional influx of Na into the cells across the mucosal membrane (zero-time rate of uptake) which does not differ significantly from the increase inI
sc
. Treatment of control tissues with amphotericin B brings about sustained increases inI
sc
andJ
net
Na
to levels observed in aldosterone-treated tissues. However, addition of amphotericin B to the mucosal solution of aldosteronetreated tissues does not result in a sustained increase inI
sc
orJ
net
Na
and these values do not differ markedly from those observed in control tissues treated with amphotericin B. These findings, together with other evidence that Na entry in the presence of amphotericin B is sufficiently rapid to saturate the active Na extrusion mechanism at the baso-lateral membrane, are consistent with the notion that the aldosterone-induced protein increases the permeability of the mucosal membrane to Na but does not increase the saturation level of the active Na pump within the time-frame of these studies (3 hr).Finally, aldosterone has no effect on the bidirectional or net transepithelial movements of K under short-circuit conditions, suggesting that the enhanced secretion of K observed in vivo is the result of increased diffusion of K from plasma to lumen via paracellular pathways in response to an increased transepithelial electrical potential difference (lumen negative). 相似文献
11.
Summary The renal cell line LLC-PK1 cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependentd-glucose coupled transport. The short-circuit current (I
sc) was acccounted for by net apical-to-basolaterald-glucose coupled Na flux, which was 0.53±0.09(8) eq cm–2hr–1, andI
sc, 0.50±0.50(8) eq cm–2hr–1. A linear plot of concurrent net Na vs. netd-glucose apical-to-basolateral fluxes gave a regression coefficient of 2.08. As support for a 21 transepithelial stoichiometry, sodium was added in the presence ofd-glucose and the response ofI
sc analyzed by a Hill plot. A slope of 2.08±0.06(5) was obtained confirming a requirement of 2 Na for 1d-glucose coupled transport. A Hill plot ofI
sc increase to addedd-glucose in the presence of Na gave a slope of 1.02±0.02(5). A direct determination of the initial rates of Na andd-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2 A coupling ratio of 2 for Na,d-glucose uptake, doubles the potential energy available for Na-gradient coupledd-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependentphlorizin binding was 1.1±0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of [Na]. Although occurring at the same site the process of Na-dependent binding of phlorizin differs from the binding and translocation ofd-glucose. Our results support a two-step, two-sodium model for Na-dependentd-glucose cotransport; the initial binding to the cotransporter requires a single Na andd-glucose, a second Na then binds to the ternary complex resulting in translocation. 相似文献
12.
The ectoderm of the one-day chick embryo generates dorsoventrally oriented short-circuit current (I
sc) entirely dependent on extracellular sodium.At the dorsal cell membrane, the I
sc was modified reversibly and in a concentration-dependent manner by: amiloride (60% decrease at 1 mm, with 2 apparent IC50s: 0.13 and 48 m), phlorizin (0.1 mm) or removal of glucose (30% decrease, additive to that of amiloride), SITS (1 mm, 13% decrease). Acidification or alkalinization of the dorsal (but not ventral) superfusate produced, respectively, decrease or increase of I
sc with a pH50 of 7.64.Ba2+ (0.1–1 mm) from either side of the ectoderm decreased the I
sc by 30%. Anthracene-9-carboxylic acid, furosemide and inducers of cAMP had no effect on electrophysiological properties of the blastoderm.The chick ectoderm is therefore a highly polarized epithelium containing, at the dorsal membrane, the high and low affinity amiloride-sensitive Na+ channels, Na+-glucose cotransporter, K+ channels and pH sensitivity, and, at the ventral membrane, the Na+, K+-ATPase and K+ channels. The Na+ transport reacts to pH, but lacks the cAMP regulatory system, well known in many epithelia.The active Na+ transport drives glucose and fluid into the intraembryonic space, across and around the blastoderm which, in the absence of blood circulation, could secure renewal of extracellular fluid and disposal of wastes and thus maintain the cell homeostasis.This work was supported by the Swiss National Research Foundation (grant 3.418-0.86 to P.K.), by the Roche Research Foundation (grant to U.K.), the Fond du 450ème anniversaire de l'Université de Lausanne and the Société Académique Vaudoise (grants to H.A.). We thank C. Bareyre, G. de Torrenté and R. Ksontini for excellent technical assistance and Drs. E. Raddatz, Y. de Ribaupierre and B. Prod'hom for helpful discussions. 相似文献
13.
Sodium permeability of dog red blood cell membranes. I. Identification of regulatory sites
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Divalent cations and group-specific chemical modifiers were used to modify sodium efflux in order to probe the molecular structure of sodium channels in dog red blood cells. Hg++, Ni++, Co++, and PCMBS (parachloromercuribenzene sulfonic acid), a sulfhydryl reactive reagent, induce large increases in Na+ permeability and their effects can be described by a curve which assumes 2:1 binding with the sodium channel. The sequence of affinities, as measured by the dissociation constants, reflects the reactivity of these divalent cations with sulfhydryl groups. In addition, the effects of Hg++ and PCMBS can be reversed by the addition of dithiothreitol, an SH-containing compound, to the medium. Much smaller increases in Na+ permeability are produced by Zn++ and the amino-specific reagents, TNBS (2,4,6-trinitrobenzene sulfonic acid) and SITS (4-acetamido-4'-isothiocyano-stilbene-2-2'-disulfonic acid). The Zn++ effect can be described by a curve which assumes bimolecular binding with the channel, and its effect on Na+ permeability can be reversed by the addition of glycine to the medium. The effects of Ni++ and SITS can be completely reversed by washing the cells in 0.16 M NaCl while TNBS binding is partially irreversible. Measurements of mean cell volumes (MCV) indicate that the modifier-induced increases in Na+ permeability are not caused by shrinkage of the cells. It is concluded that the movement of sodium ions through ionic channels in dog red blood cells can be enhanced by modification of amino and sulfhydryl groups. Zn++, TNBS, and SITS increase Na+ permeability by modifying amino groups in the channel while Hg++, Ni++, Co++, and PCMBS act on sulfhydryl groups. 相似文献
14.
15.
Kinetics of the effect of amiloride on the permeability of the apical membrane of rabbit descending colon to sodium 总被引:2,自引:0,他引:2
William M. Moran Randall L. Hudson Stanley G. Schultz 《The Journal of membrane biology》1985,87(1):55-65
Summary The effects of the addition of graded concentrations of amiloride, (A)
m
, to the mucosal bathing solution on the permeability of the apical membrane of rabbit descending colon to Na (P
Na
m
) were determined when the Na activity in the mucosal bathing solution, (Na)
m
, was 18, 32 or 100mm.P
Na
m
was obtained from current-voltage relations determined on tissues bathed with a high-K serosal solution before and after the addition of a maximally inhibitory concentration of amiloride to the mucosal solution as described by Turnheim et al. (Turnheim, K., Thompson, S.M., Schultz. S.G. 1983.J. Membrane Biol.
76:299–309).The results indicate that: (1) As demonstrated previously (Turnheim et al., 1983),P
Na
m
decreases with increasing (Na)
m
. (2)P
Na
m
also decreases hyperbolically with increasing (A)
m
. Kinetic analyses of the effect of amiloride onP
Na
m
are consistent with the conclusions that: (i) the stoichiometry between the interaction of amiloride with apical membrane receptors that results in a decrease inP
Na
m
is one-for-one; (ii) there is no evidence for cooperativity between amiloride and these binding sites; (iii) the value of (A)
m
needed to halveP
Na
m
at a fixed (Na)
m
is 0.6–1.0 m; and, (iv) this value is independent of (Na)
m
over the fivefold range studied.These findings are consistent with the notion that the sites with which amiloride interacts to bring about closure of the channels through which Na crosses the apical membrane arekinetically distinct from the sites with which (Na)
m
interacts to bring about closure (i.e., self-inhibition). In short, the effects of (Na)
m
and (A)
m
onP
Na
m
in this tissue appear to be independent and additive. 相似文献
16.
Summary Na, K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na+ transport during early postnatal development. The maximum Na+-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (I
sc
N
) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats.I
sc
N
was entirely inhibited by ouabain; the inhibitory constant was 0.1mm in 10-day-old (young) and 0.4mm in 90-day-old (adult) rats. The affinity of the Na, K pump for Na+ was higher in young (11mm) than in adult animals (19mm). The Na, K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (K
I
) was not changed during development (0.1–0.3mm). Specific [3H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (K
D
) was 8 and 21 m in young and adult rats, respectively. The Na+ turnover rate per single Na, K pump, which was calculated fromI
sc
N
and estimated density of binding sites per cm2 of tissue was 500 in adult and 6400 Na+/min·site in young rats. These data indicate that the very high Na+ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na+ of a single isoform of the Na, K pump. The development of Na+ extrusion across the basolateral membrane is not directly regulated by corticosteroids. 相似文献
17.
J. F. García-Díaz G. Klemperer L. M. Baxendale A. Essig 《The Journal of membrane biology》1986,92(1):37-46
Summary Cell Na activity,a
Na
c
, was measured in the short-circuited frog skin by simulaneous cell punctures from the apical surface with open-tip and Na-selective microelectrodes. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular conductance, with NaNO3 Ringer on the apical surface. Under control conditionsa
Na
c
averaged 8±2mm (n=9,sd). Apical addition of amiloride (20 m) or Na replacement reduceda
Na
c
to 3mm in 6–15 min. Sequential decreases in apical [Na] induced parallel reductions ina
Na
c
and cell current,I
c
. On restoring Na after several minutes of exposure to apical Na-free solutionI
c
rose rapidly
to a stable value whilea
Na
c
increased exponentially, with a time constant of 1.8±0.7 min (n=8). Analysis of the time course ofa
Na
c
indicates that the pump Na flux is linearly related toa
Na
c
in the range 2–12mm. These results indicate thata
Na
c
plays an important role in relating apical Na entry to basolateral active Na flux. 相似文献
18.
Summary The effect of amiloride on the sensitivity to Na of the mucosal border of toad urinary bladder was investigated by recording Na concentration-dependent transepithelial potential difference (V
t
) and the intracellular potential. When mucosal Na concentration was normal, amiloride added to the mucosal solution at 10–4
m markedly reduced the mucosal membrane potential (V
m
) and altered the potential profile from a two-step type to a well type. Similar changes were observed when Na was totally eliminated from the mucosal medium. The serosal membrane potential was insensitive to amiloride and elimination of mucosal Na. In the absence of amiloride, theV
t
could be described by the Goldman-Hodgkin-Katz equation in the range of mucosal Na concentration from 0 to 16mm, and amiloride extended this concentration range. By using the Goldman-Hodgkin-Katz equation, Na permeability was calculated from the data ofV
t
's obtained in the allowed ranges of Na concentration and compared before and after the addition of amiloride. The results show that Na permeability decreases to 1/600 of control when the maximum dose of amiloride (10–4
m) is applied. The relationship between Na permeability and amiloride concentration is well explained on the basis of assumptions that amiloride binds to the Na site of the mucosal border in one-to-one fashion and in a competitive manner with Na and that Na permeability reduces in proportion to increase in number of the sites bound with amiloride. 相似文献
19.
Timothy R. Traynor Scott M. O'Grady 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1992,162(1):47-53
Summary Porcine distal colon epithelium was mounted in Ussing chambers and bathed in plasma-like Ringer solution. Tissue conductances ranged from 10 to 15 mS and the short-circuit current (Isc) ranged from-15 to 220 A·cm-2. Variations in basal Isc resulted from differences in the amount of amiloride (10M mucosal addition)-sensitive Na+ absorption. Ion substitution and transepithelial flux experiments showed that 10 M amiloride produced a decrease in the mucosal-to-serosal (M-S) and net Na flux, and that this effect on Isc was independent of Cl- and HCO
3
-
replacement. When the concentration of mucosal amiloride was increased from 10 to 100 M, little change in Isc was observed. However, increasing the concentration to 1 mM produced a further inhibition, which often reversed the polarity of the Isc. The decrease in Isc due to 1 mM amiloride was dependent on both Cl- and HCO
3
-
, and was attributed to reductions in the M-S and net Na+ fluxes as well as the M-S unidirectional Cl- flux. Ion replacement experiments demonstrated that Cl- substitution reduced the M-S and net Na fluxes, while replacement of HCO
3
-
with HEPES abolished net Cl- absorption by reducing the M-S unidirectional Cl- flux. From these data it can be concluded that: (1) Na+ absorption is mediated by two distinct amiloride-sensitive transport pathways, and (2) Cl- absorption is completely HCO
3
-
-dependent (presumably mediated by Cl-/HCO
3
-
exchange) and occurs independently of Na+ absorption.Abbreviations Gt
tissue conductance
- HEPES
tris (hydroxymethyl) aminomethane
- (tris)
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- Isc
short-circuit current
- Jr
residual flux
- M-S
mucosal-to-scrosal
- S-M
serosal-to-mucosal
- TTX
tetrodotoxin 相似文献
20.
Cristina Ibarra Pierre Ripoche Jacques Bourguet 《The Journal of membrane biology》1989,110(2):115-126
Summary It has been suggested that during the oxytocin-induced hydrosmotic response, water crosses the luminal membrane of urinary bladder epithelium cells through membranespanning proteins. Although specific inhibitors of osmotic water transport have not been found, certain sulfhydryl reagents such as mercurial compounds may help to identify the proteins involved in this permeation process. We tested the effects ofp-chloromercuribenzene sulfonate (PCMBS) and of fluoresceinmercuric acetate (FMA) on the net water flux, the microtubule and microfilament structures of the frog urinary bladder, and the distribution of intramembrane particle aggregates in the luminal membrane.We observed that: (i) 5mm PCMBS at pH 5 and 0.5mm FMA at pH 8 added to the mucosal bath at the maximum of the response to oxytocin partially inhibited the net water flux. Inhibition then increased progressively when the preparation was repeatedly or continuously stimulated, until it reached a maximal inhibition at 120 min. This inhibition was not reversed even when cystein was added in the mucosal bath. PCMBS and FMA effects were also observed when cyclic AMP (3,5 cyclic adenosine monophosphate) was used to increase water permeability. (ii) PCMBS mucosal pretreatment did not modify the basal water flux but potentiated the inhibitory effect of PCMBS or FMA on the hydrosmotic response to oxytocin. (iii) Microtubule and microfilament network, visualized in target cells by immunofluorescence, was not affected by PCMBS. (iv) The maximal PCMBS or FMA inhibition was not associated with a reduction of aggregate surface area in the apical membrane.The persistence of the intramembrane particle aggregates associated with the oxytocin-induced hydrosmotic response during the net water flux inhibition by PCMBS, suggests that the PCMBS effect occurs possibly at the level of sulfhydryl groups of the water channel itself. 相似文献