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1.
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
2.
Dicyclohexylcarbodiimide (DCCD) is a carboxyl group modifier and it is an inhibitor of various ATPases. Present experiments,
using an in vitro preparation, were designed to study whether DCCD affected the transporters of the bullfrog cornea epithelium,
specifically, the Na+/K+ ATPase pump located in the basolateral membrane. For this purpose, corneas were impaled with microelectrodes and experiments
were done under short-circuit current (I
sc
) conditions. Addition of DCCD to a concentration of 10−4
m to the tear solution gave a marked decrease in I
sc
; a marked depolarization of the intracellular potential, V
o
; and a significant decrease in the apical membrane fractional resistance, fR
o
. There were small and variable although significant changes in the transepithelial conductance, g
t
. The effects may be explained by a decrease in the basolateral membrane K+ conductance, in combination with a partial inhibition of the Na+/K+-ATPase pump located in the basolateral membrane. There is also evidence for an increase in the apical membrane Cl− conductance.
Received: 12 August 1999/Revised: 16 November 1999 相似文献
3.
H.S. Cheng P.Y. Leung S.B. Cheng Chew P.S. Leung S.Y. Lam W.S. Wong Z.D. Wang H.C. Chan 《The Journal of membrane biology》1998,164(2):155-167
The present study investigated both HCO−
3 and Cl− secretions in a human pancreatic duct cell line, CAPAN-1, using the short-circuit current (I
sc
) technique. In Cl−/HCO−
3-containing solution, secretin (1 μm) or forskolin (10 μm) stimulated a biphasic rise in the I
sc
which initially reached a peak level at about 3 min and then decayed to a plateau level after 7 min. Removal of external
Cl− abolished the initial transient phase in the forskolin-induced I
sc
while the plateau remained. In HCO−
3/CO2-free solution, on the contrary, only the initial transient increase in I
sc
was prominent. Summation of the current magnitudes observed in Cl−-free and HCO−
3-free solutions over a time course of 10 min gave rise to a curve which was similar, both in magnitude and kinetics, to the
current observed in Cl−/HCO−
3-containing solution. Removal of external Na+ greatly reduced the initial transient rise in the forskolin-induced I
sc
response, and the plateau level observed under this condition was similar to that obtained in Cl−-free solution, suggesting that Cl−-dependent I
sc
was also Na+-dependent. Bumetanide (50 μm), an inhibitor of the Na+-K+-2Cl− cotransporter, and Ba2+ (1 mm), a K+ channel blocker, could reduce the forskolin-induced I
sc
obtained in Cl−/HCO−
3-containing or HCO−
3-free solution. However, they were found to be ineffective when external Cl− was removed, indicating the involvement of these mechanisms in Cl− secretion. On the contrary, the HCO−
3-dependent (in the absence of external Cl−) forskolin-induced I
sc
could be significantly reduced by carbonic anhydrase inhibitor, acetazolamide (45 μm). Basolateral application of amiloride (100 μm) inhibited the I
sc
; however, a specific Na+-H+ exchanger blocker, 5-N-methyl-N-isobutylamiloride (MIA, 5–10 μm) was found to be ineffective, excluding the involvement of
the Na+-H+ exchanger. However, an inhibitor of H+-ATPase, N-ethylmaleimide did suppress the I
sc
(IC50= 22 μm). Immunohistochemical studies also confirmed the presence of a vacuolar type of H+-ATPase in these cells. H2DIDS (100 μm), an inhibitor of Na+-HCO−
3 cotransporter, was without effect. Apical addition of Cl− channel blocker, diphenylamine-2,2′-dicarboxylic acid (DPC, 1 mm), but not disulfonic acids, DIDS (100 μm) or SITS (100 μm), exerted an inhibitory effect on both Cl− and HCO−
3-dependent forskolin-induced I
sc
responses. Histochemical studies showed discrete stainings of carbonic anhydrase in the monolayer of CAPAN-1 cells, suggesting
that HCO−
3 secretion may be specialized to a certain population of cells. The present results suggest that both HCO−
3 and Cl− secretion by the human pancreatic duct cells may occur concurrently and independently.
Received: 17 October 1997/Revised: 3 April 1998 相似文献
4.
T.-H. Hwang D.-J. Suh H.-R. Bae S.-H. Lee J.-S. Jung 《The Journal of membrane biology》1996,154(3):251-257
To study K+ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on
permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml)
in the symmetrical high K+ (145 mm) Ringer solution. During measurement of the macroscopic K+ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types
of K+ currents: one is an inwardly rectifying K+ current and the other is a slowly activating outwardly rectifying K+ current. The inwardly rectifying K+ current is inhibited by Ba2+. The slowly activating K+ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature.
This is consistent with the biophysical characteristics of I
SK channel. RT-PCR analysis revealed the presence of I
SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba2+ only had minimal affect on short-circuit current (I
sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I
SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia.
Received: 1 March 1996/Revised: 5 August 1996 相似文献
5.
The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K+ channels was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with double-barrelled microelectrodes and the K+ current was monitored under voltage clamp in 0.1–30 mm K+ and in equivalent concentrations of Rb+, Cs+ and Na+. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K+ activated current through outward-rectifying K+ channels (I
K,
out) at the plasma membrane in a voltage-dependent fashion. Increasing [K+]
o
shifted the voltage-sensitivity of I
K,
out in parallel with the equilibrium potential for K+ across the membrane. A similar effect of [K+]
o
was evident in the kinetics of I
K,
out activation and deactivation, as well as the steady-state conductance- (g
K
−) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K+, −80 mV in 1.0 mm K+, and −20 mV in 10 mm K+. Similar data were obtained with Rb+ and Cs+, but not with Na+, consistent with the relative efficacy of cation binding under equilibrium conditions (K+≥ Rb+ > Cs+ > > Na+). Changing Ca2+ or Mg2+ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of g
K
or on I
K,
out activation kinetics, although 10 mm [Ca2+]
o
accelerated current deactivation at voltages negative of −75 mV. At any one voltage, increasing [K+]
o
suppressed g
K
completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K+ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively. These, and additional data indicate that extracellular K+ acts as a ligand and alters the voltage-dependence of I
K,
out gating; the results implicate K+-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the
channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of
two K+ ions outside affects the distribution between closed states of the channel.
Received: 27 November 1996/Revised: 4 March 1997 相似文献
6.
M. Heinz R. Krattenmacher B. Hoffmann W. Clauss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1991,161(4):363-370
Summary Transepithelial electrogenic Na+ transport (INa) was investigated in the coprodeum of 20-days-old chicken embryos in Ussing chambers. Short circuit current (Isc) and transepithelial resistance (Rt) were 14.7±4.8 A · cm-2 (n=12) and 0.53±0.09 k · cm-2 (n=12), respectively. INa was calculated from changes in Isc by substitution of mucosal Na+ by (N-methyl-d-glucamine) (NMDG). Isc inversed during Na+ removal, and INa was found to be 27.8±4.7 A · cm-2 (n=12). Amiloride (100 mol · l-1) inhibited only about 60% of INa. Analysis of Isc fluctuations revealed a Lorentzian component in the power density spectrum with a corner frequency of about 57 Hz. This component was not correlated to INa, and its origin is still unclear. Removal of mucosal Ca2+ increased INa about 2.5-fold due to an increase of the amiloride-insensitive component of INa in additionally investigated adult tissues. The results clearly show that this is due to a non-selective cation channel with an apparent order of selectivity Cs+>Na+=K+>Rb+>Li+. The Ca2+ concentration required to block 50% of the Isc was about 18 mol · l-1. The I
sc
Ca
could also be supressed by other divalent cations such as Mg2+ and Ba2+. Additionally, an INa-linked Lorentzian component occurred which dominated the control spectrum with a significantly higher corner frequency (about 88 Hz). The results indicate that Na+ absorption in the coprodeum of the chicken embryo is more complex than in adult hens. However, the Ca2+ sensitivity of INa is similar to comparable effects described for other epithelia. This possibly reflects the existence of two types of amiloride-insensitive apical cation channels as pathways for Na+ absorption, which may be involved to differing degrees in ontogenetic developments of nonselective channels to Na+-specific ion channels.Abbreviations
DPL
direct-linear-plot method
-
slope of the back-ground noise component
-
EGTA
ethylene glycol-bi(2-amino-ethylether)-N,N,N,N-tetraacetic acid
-
f
frequency
-
f
c
corner frequency of the Lorentzian noise component
-
G
t
transepithelial conductance
-
HEPES
N-hydroxyethylpiperazine-N-ethanesulfonic acid
-
I
sc
short-circuit current
-
I
Na
transepithelial sodium current
-
I
sc
Ca
Ca2+-sensitive short-circuit current
-
K
m
Ca
Michaelis-Menten constant for Ca2+
-
K
B
power density of the background noise component at f=1Hz
-
m
mucosal
-
NMDG
N-methyl-D-glucamine
-
R
t
transepithelial resistance
-
s
serosal
-
SEM
standard error of mean
-
S(f)
power density of the Lorentzian noise component
-
S
o
plateau value of the Lorentzian noise component 相似文献
7.
Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na+-K+-ATPase activity from the dihydroouabain-sensitive current (I
DHO) in the presence of increasing concentrations of tetraethylammonium (TEA+; 0, 5, 10, 20, 40 mm), a well-known blocker of K+ channels. The effects of TEA+ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V
M) and a saturable inhibitory component affecting an outward current easily detectable at positive V
M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA+ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA+ on K+ channels. Interestingly, this latter component disappears when the Na+-K+-ATPase is inhibited by 10 m DHO. Conversely, TEA+ inhibits a component of I
DHO with a k
d of 25±4 mm at +50 mV. As the TEA+-sensitive current present in I
DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K+ channels whose activity varies in parallel with the Na+-K+-ATPase activity. Supporting this hypothesis, the inward portion of this TEA+-sensitive current can be completely abolished by the addition of 1 mm Ba2+ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA+-sensitive K+ currents and indicates that, in the absence of effective K+ channel inhibitors, I
DHO does not exclusively represent the Na+-K+-ATPase-generated current. 相似文献
8.
Summary The ionic dependencies of the transepithelial and intracellular electrical parameters were measured in the isolated frog cornea. In NaCl Ringer's the intracellular potential differenceV
sc measured under short-circuit conditions depolarized by nearly the same amount after either increasing the stromal-side KCl concentration from 2.5 to 25mm or exposure to 2mm BaCl2 (K+ channel blocker). With Ba2+ the depolarization of theV
sc by 25mm K+ was reduced to one-quarter of the control change. If the Cl-permselective apical membrane resistanceR
o remained unchanged, the relative basolateral membrane resistanceR
i, which includes the lateral intercellular space, increased at the most by less than twofold after Ba2+. These effects in conjunction with the depolarization of theV
sc by 62 mV after increasing the stromal-side K+ from 2.5 to 100mm in Cl-free Ringer's as well as the increase of the apparent ratio of membrane resistances (a=R
o/Ri) from 13 to 32 are all indicative of an appreciable basolateral membrane K+ conductance. This ratio decreased significantly after exposure to either 25mm K+ or Ba2+. The decline ofR
o/Ri with 25mm K+ appears to be anomalous since this decrease is not consistent with just an increase of basolateral membrane conductance by 25mm K+, but rather perhaps a larger decrease ofR
o thanR
iAlso an increase of lateral space resistance may offset the effect of decreasingR
i with 25mm K+. In contrast,R
o/Ri did transiently increase during voltage clamping of the apical membrane potential differenceV
o and exposure to 25mm K+ on the stromal side. This increase and subsequent decrease ofR
o/Ri supports the idea that increases in stromal K+ concentration may produce secondary membrane resistance changes. These effects onR
o/Ri show that the presence of asymmetric ionic conductance properties in the apical and basolateral membranes can limit the interpretative value of this parameter. The complete substitution of Na+ withn-methyl-glucamine in Cl-free Ringer's on the stromal side hyperpolarized theV
sc by 6 mV whereas 10–4
m ouabain depolarized theV
sc by 7 mV. Thus the basolateral membrane contains K+, Na+ and perhaps Cl– pathways in parallel with the Na/K pump component. 相似文献
9.
Adenosine 3′,5′-cyclic monophosphate (cAMP) is known to stimulate exogenous IsK channel current in the Xenopus oocyte expression system. The present study was performed to determine whether elevation of cytosolic cAMP in a native mammalian
epithelium known to secrete K+ through endogenously expressed IsK channels would stimulate K+ secretion through these channels. The equivalent short circuit current (I
sc
) across vestibular dark cell epithelium in gerbil was measured in a micro-Ussing chamber and the apical membrane current
(I
IsK
) and conductance (g
IsK
) of IsK channels was recorded with both the on-cell macro-patch and nystatin-perforated whole-cell patch-clamp techniques. It has
previously been shown that I
sc
can be accounted for by transepithelial K+ secretion and that the apical IsK channels constitute a significant pathway for K+ secretion. The identification of the voltage-dependent whole-cell currents in vestibular dark cells was strengthened by the
finding that a potent blocker of IsK channels, chromanol 293B, strongly reduced I
IsK
from 646 ± 200 to 154 ± 22 pA (71%) and g
IsK
from 7.5 ± 2.6 to 2.8 ± 0.4 nS (53%). Cytoplasmic cAMP was elevated by applying dibutyryl cyclic AMP (dbcAMP), or the phosphodiesterase
inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-20-1724. dbcAMP (1 mm) increased I
sc
and I
IsK
from 410 ± 38 to 534 ± 40 μA/cm2 and from 4.3 ± 0.8 to 11.4 ± 2.2 pA, respectively. IBMX (1 mm) caused transient increases of I
sc
from 415 ± 30 to 469 ± 38 μA/cm2 and Ro-20-1724 (0.1 mm) from 565 ± 43 to 773 ± 58 μA/cm2. IBMX increased I
IsK
from 5.5 ± 1.5 to 16.9 ± 5.8 pA in on-cell experiments and from 191 ± 31 to 426 ± 53 pA in whole-cell experiments. The leak
conductance due to all non-IsK channel sources did not change during dbcAMP and IBMX while 293B in the presence of dbcAMP reduced I
IsK
by 84% and g
IsK
by 62%, similar to unstimulated conditions. These results demonstrate that the cAMP pathway is constitutively active in vestibular
dark cells and that the cAMP pathway stimulates transepithelial K+ secretion by increasing IsK channel current rather than by altering another transport pathway.
Received: 9 June 1995/Revised: 17 October 1996 相似文献
10.
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
11.
Summary We have studied current (I
Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I
Str was measured as the strophanthidin-sensitive current in the presence of Ba2–, Cd2+ and gluconate (in place of external Cl–). In addition,I
Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK
0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na+-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI
Str depended directly onV
m
up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na+] markedly decreased the voltage sensitivity ofI
p
, while producing only a small change in the maximal outwardI
Str. In contrast, decreasing the external [K+] from 25 to 2.5mm reducedI
Str at 0 mV without substantially affecting its voltage dependence. At K+ concentrations of 1mm, both the absolute value ofI
Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI
Str by [K+]
o
over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K+ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K+]
o
>1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na+ and K+ are consistent with results obtained in other tissues and may reflect an ion-well effect. 相似文献
12.
F. Lemtiri-Chlieh 《The Journal of membrane biology》1996,153(2):105-116
One of the main effects of abscisic acid (ABA) is to induce net loss of potassium salts from guard cells enabling the stomata
to close. K+ is released from the vacuole into the cytosol and then to the extracellular space. The effects of increasing cytosolic K+ on the voltage- and time-dependence of the outwardly rectifying K+-current (I
K,out) in guard cell protoplasts (GCP) was examined in the whole-cell configuration of the patch-clamp technique. The same quantitative
analysis was performed in the presence of ABA at different internal K+ concentrations ([K+]
i
). Varying [K+]
i
in the patch pipette from 100 to 270 mm increased the magnitude of I
K,out in a nonlinear manner and caused a negative shift in the midpoint (V
0.5) of its steady-state activation curve. External addition of ABA (10–20 μm) also increased the magnitude of I
K,out at all [K+]
i
, but caused a shift in V
0.5 of the steady-state activation curve only in those GCP loaded with 150 mm internal K+ or less. Indeed, V
0.5 did not shift upon addition of ABA when the [K+]
i
was above 150 mm and up to 270 mm, i.e., the shift in V
0.5 caused by ABA depended on the [K+]
i
. Both increase in [K+]
i
and external addition of ABA, decreased (by ≈ 20%) the activation time constant (τ
n
) of I
K,out. The small decrease in τ
n
, in both cases, was found to be independent of the membrane voltage. The results indicate that ABA mimics the effect of increasing
cytoplasmic K+, and suggest that ABA may increase I
K,out and alter V
0.5 of its steady-state activation curve via an enhancement in cytosolic K+. This report describes for the first time the effects of [K+]
i
on the voltage- and time-dependence of I
K,out in guard cells. It also provides an explanation for the quantitative (total membrane current) and qualitative (current kinetics)
differences found between intact guard cells and their protoplasts.
Received: 1 December 1995/Revised: 8 May 1996 相似文献
13.
D. Merlin C. Augeron X. -Y. Tien X. Guo C. L. Laboisse U. Hopfer 《The Journal of membrane biology》1994,137(2):137-149
The response of confluent monolayers of HT29-Cl.16E cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin and electrolyte secretion. Mucin secretion was measured as release of glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels and electrolyte secretion as shortcircuit current (Isc). Luminal ATP stimulated a transient increase in the release of mucins and of I
sc corresponding to a secretory Cl– current. Both secretions peaked at 3 to 5 min after addition of ATP. Maximal ATP-stimulated mucin secretion over 15 min was up to 18-fold above control with an apparent ED50 of approximately 40 m. Maximal peak I
sc after stimulation with ATP was approximately 35 A/cm2 with an apparent ED50 of about 0.4 mm. ATP-dependent I
sc was at least in part due to Cl– secretion since removal of Cl– from the medium reduced the peak I
sc by 40% and the I
sc integrated over 40 min by 80%. The secretory responses were not associated with cell damage as assessed by failure of ethidium bromide to enter into the cells, absence of release of lactate dehydrogenase, maintenance of monolayer conductance, viability, and responses to repeated applications of ATP. The order of efficacy of nucleotide agonists was similar for both processes with ATP>ADP>AMPadenosine. Luminal ATP was much more effective than basolateral addition of this compound. These results suggest involvement of a luminal P2-type receptor which can initiate signaling pathways for granule fusion and mucin release as well as for activation of Cl– channels. P2-receptor-stimulated mucin and I
sc release was strongly inhibited by a 30 min preincubation with the classical K+ channel blockers quinine (1 mm), quinidine (1 mm), and Ba2+ (3 mm). Experiments with amphotericin B to measure separately the conductance changes of either luminal or basolateral plasma membrane revealed that quinidine did not directly block the ATP-induced basolateral K+ or the luminal anion channels. The quinidine inhibition after preincubation is therefore most easily explained by interference with granule fusion and location of anion channels in granule membranes. Luminal P2 receptors may play a role in intestinal defense mechanisms with both fluid and mucin secretion aiding in the removal of noxious agents from the mucosal surface.Supported by grants from the National Institutes of Health (DK 39658) to U.H., the Philippe Foundation to D.M., the French Cystic Fibrosis Foundation (AFLM) and L'Association Pour La Recherche Sur Le Cancer to C.L. The authors thank Mr. J. Polack for his efforts and skill with electron microscopy and Dr. George Dubyak for helpful discussions. We also acknowledge the Cystic Fibrosis Center Core grant (DK-27651) for its support of electron and light microscopy. 相似文献
14.
Ba2+-Induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria) 总被引:7,自引:0,他引:7
Summary We studied the influence of mucosal Ba2+ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol.
47:77) transepithelial, mucosa towards serosa directed K+ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),I
sc (short-circuit current, K+ current), as well as the noise ofI
sc were recorded. Addition of millimolar concentrations of Ba2+ to the mucosal K+-containing solution resulted in a sudden but quickly reversible drop inI
sc.G andI
sc decreased continuously with increasing Ba2+ concentration, (Ba2+)
o
. The apparent Michaelis constant of the inhibition by Ba2+ lies within the range 40–80 m. The apical membrane seems to remain permselective for K+ up to 500 m (Ba2+)
o
. Higher (Ba2+)
o
, however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K+-channel block by K+–Ba2+ competition. In the presence of Ba2+, the power spectrum of the K+ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K+-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K+ and can be depressed by addition of Cs+ ions, thus indicating that Ba2+ ions induce K+-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba2+)
o
, shows a rise in the plateau values to a maximum around 60 m (Ba2+)
o
, followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba2+-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba2+)
o
with a tendency to saturate at higher (Ba2+)
o
. Based on a three-state model for the K+ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba2+) computer calculations compared favorably with our results. The effect of Ba2+ could be explained by assuming reversible binding at the outer side of the apical K+ channel, thereby blocking the open channel in competition with K+. The association-dissociation of Ba2+ at its receptor site is thought to cause a chopping of the K+ current, resulting in modulated current fluctuations. 相似文献
15.
In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO3) has been shown to mediate a large fraction of the total K+ transport. In the present study, Cl−-dependent and Cl−-independent K+ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K+ ([K+]
e
and [K+]
i
) concentration. The dependence of ouabain-resistant Cl−-dependent K+ (86Rb) influx on [K+]
e
over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K
m
) of 8.2 ± 1.3 mm and maximal velocity (V
max
) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl−-dependent K+ influx increased both K
m
(12.8 ± 1.7 mm, P < 0.05) and V
max
(20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K+]
e
above 20 mm in isotonic media significantly reduced the Cl−-dependent K+ influx due to a reciprocal decrease of the external Na+ ([Na+]
e
) concentration below 50 mm. Replacing [Na+]
e
by NMDG+ markedly decreased V
max
(3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K
m
(15.7 ± 2.1 mm, P < 0.03) of Cl−-dependent K+ influx. Moreover, NMDG+ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb+ uptake and K+ loss from red cells. Cell swelling did not affect the Na+-dependent changes in Rb+ uptake and K+ loss. In a nominally K+(Rb+)-free medium, net K+ loss was reduced after lowering [Na+]
e
below 50 mm. These results indicate that over 50 mm [Na+]
e
is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K+, Cl−-dependent K+ loss in K+-free media was a linear function of [K+]
i
, with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr−1 (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry
with respect to transported K+ ions. The residual, ouabain-resistant K+ fluxes in NO3 were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were
not different from those for K+ effluxes in isotonic (∼0.014 hr−1) and hypotonic (∼0.022 hr−1) media, but cell swelling resulted in a significant increase in the rate constants.
Received: 19 November 1998/Revised: 23 August 1999 相似文献
16.
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
17.
Peter M. Spooner Isidore S. Edelman 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,444(3):663-673
The action of aldosterone on active Na+ transport was assessed under aerobic and anaerobic conditions in the isolated urinary bladder of the toad, BUfo marinus. Aldesterone augmented the short-circuit current (Isc) under rigorous anaerobiosis. Four lines of evidence indicate that the increase in anaerobic Isc does not represent an equivalent increase in active Na+ transport: 1. Net Na+ transport, determined by isotopic fluxes, was the same in the aldosterone-treated and control quarter-bladders, and significantly greater than the simultaneously measured Isc. 2. Amiloride, an inhibitor of the apiral entry of Na+, did not reduce the steroid-dependent increase in the anaerobic Isc. 3. Substitution of choline for Na+ in the mucosal medium reduced the magnitude of the anaerobic Isc values did not eliminate the effect of aldosterone. 4. Addition of ouabain, a potent inhibitor of the Na+ pump, partially inhibited the effect of aldosterone on the anerobic Isc but a significant hormonal increment remained. The source of the anaerobic Isc was not identified; an effort was made, however, to determine the dependence of this current on glycolysis. During anaerobics, aldosterone increased the integral Isc by 42% but did not alter lactate production. These results suggest that the steroid-dependent increase in the anaerobic Isc may involve effects on permeability properties of the epithelium rather than on active tranport systems. 相似文献
18.
Brian E. Peerce 《The Journal of membrane biology》1989,110(2):189-197
Summary The substrate stoichiometry of the intestinal Na+/phosphate cotransporter was examined using two measures of Na+-dependent phosphate uptake: initial rates of uptake with [32P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC3(5). Isotopic phosphate measures electrogenic and electroneutral Na+-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na+/phosphate cotransport was found to have similar Na+ activations (apparentK
0.5's of 28 and 25mm), apparentK
m
's for phosphate (100 and 410 m), andK
0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na+-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na+ with apparentK
0.5 for phosphate of 200 m; similar to the apparentK
m
for phosphate uptake, andK
0.5 for phosphate protection against FITC-PG inhibition of Na+-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na+/phosphate cotransporter is electrogenic at pH 7.4, that H2PO
4
–
is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na+/phosphate cotransporter. 相似文献
19.
Simon A. Lewis A. Grant Butt M. Joanne Bowler John P. Leader Anthony D. C. Macknight 《The Journal of membrane biology》1985,83(1-2):119-137
Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl– on transepithelial Na+ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V
T) decreased rapidly, transepithelial resistance (R
T) increased, and calculated short-circuit current (I
sc) decreased. CalculatedE
Na was unaffected, indicating that the inhibition of Na+ transport was a consequence of a decreased apical membrane Na+ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV
T decreased,R
T increased andI
sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE
Na fell substantially (from 130 to 68 mV on average). Ba2+ (3mm) reduced calculatedE
Na in Cl– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE
Na did not fall. Replacement of serosal isosmotic Cl– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na+ transport. Thus the inhibition of Na+ transport can be correlated with changes in cell volume rather than with the change in Cl– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K+ gluconate mucosa, Na+ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na+/K+ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl– results in a loss of highly specific Ba2+-sensitive K+ conductance channels from the basolateral plasma membrane. It is possible that the number of Na+ pump sites in this membrane is also decreased. 相似文献