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1.
(1) The effects of the Na+-channel blocker, amiloride, on the short-circuit current carried by Na+ was studied with fluctuation analysis, in rabbit descending colon epithelium. (2) In the presence of mucosal amiloride, the power spectrum of the Na+-current noise showed a Lorentzian component. When the Na+ current was reduced by increasing the blocker concentrations, the Lorentzian plateau decreased and corner frequency increased. Macroscopic short-circuit current and current-noise data are evidence for a two-state mechanism of the blocker interaction with the Na+ channel. (3) On- and off-rate constants for the blocker-receptor reaction, single-channel currents and Na+-channel density were calculated at room temperature and at 37 degrees C. Also, the activation energy for the amiloride-receptor reaction was estimated. The microscopic parameters obtained for the Na+ channel in the colon were similar to those found for Na+ channels in other tight epithelia. 相似文献
2.
In the presence of the Na+-channel blocker amiloride, the short-circuit current across the skins of bullfrog tadpoles in metamorphic stages XIX–XXIV was subjected to fluctuation analysis. The resulting power spectra contained a Lorentzian component of which the plateau value () decreased while the corner frequency () increased as the mucosal amiloride concentration was increased from 0.5 to 24 μM. From the linear relationship between the values and the amiloride concentrations it was possible to determine the binding () and unbinding () constants for amiloride to its receptor on the Na+ channel. With these parameters as well as short-circuit current and values, the current through the individual Na+ channels () was calculated (average 0.58 pA). It did not increase significantly during late metamorphosis. The density of Na+ channels () in the apical membrane, on the other hand, increased significantly. It would appear that the increase in short-circuit current which occurs at this time is due primarily to an increase in amiloride-blockable Na+ channels. Unexpectedly, a Lorentzian component could be fitted to power spectra in amiloride-treated skins (stages XIX–XXI) which showed no amiloride-sensitive short-circuit current. Moreover, the typical increase in with the amiloride concentration did not occur in these animals. 相似文献
3.
Summary In this paper we describe current fluctuations in the mammalian epithelium, rabbit descending colon. Pieces of isolated colon epithelium bathed in Na+ or K+ Ringer's solutions were studied under short-circuit conditions with the current noise spectra recorded over the range of 1–200 Hz. When the epithelium was bathed on both sides with Na+ Ringer's solution (the mucosal solution contained 50 m amiloride), no Lorentzian components were found in the power spectrum. After imposition of a potassium gradient across the epithelium by replacement of the mucosal solution by K+ Ringer's (containing 50 m amiloride), a Lorentzian component appeared with an average corner frequency,f
c=15.6±0.91 Hz and a mean plateau valueS
o=(7.04±2.94)×10–20 A2 sec/cm2. The Lorentzian component was enhanced by voltage clamping the colon in a direction favorable for K+ entry across the apical membrane. Elimination of the K+ gradient by bathing the colon on both sides with K+ Ringer's solutions abolished the noise signal. The Lorentzian component was also depressed by mucosal addition of Cs+ or tetraethylammonium (TEA) and by serosal addition of Ba2+. The one-sided action of these K+ channel blockers suggests a cellular location for the fluctuating channels. Addition of nystatin to the mucosal solution abolished the Lorentzian component. Serosal nystatin did not affect the Lorentzian noise. This finding indicates an apical membrane location for the fluctuating channels. The data were similar in some respects to K+ channel fluctuations recorded from the apical membranes of amphibian epithelia such as the frog skin and toad gallbladder. The results are relevant to recent reports concerning transcellular potassium secretion in the colon and indicate that the colon possesses spontaneously fluctuating potassium channels in its apical membranes in parallel to the Na+ transport pathway. 相似文献
4.
W. -M. Weber B. Dannenmaier W. Clauss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(2):153-159
The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm-2) with an initial short-circuit current of 29.0±2.9 A·cm-2. Removal of Na+ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l-1) added to the basolateral solution, depressed the short-circuit current completely. The Na+ current saturated at a concentration of 90 mmol Na+·l-1 in the apical solution (K
M=11.2±1.8 mmol·l-1). Amiloride (100 mol·l-1) on the apical side inhibited ca. 40% of the Na+ current and indicated the presence of Na+ channels. The dependence of Na+ current on the amiloride concentration followed Michaclis-Menten kinetics (K
i=2.9±0.4 mol·l-1). The amiloride analogue benzamil had a higher affinity to the Na+ channel (K
i=0.7±0.2 mol·l-1). Thus, Na+ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na+·l-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na+ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l-1) and isobutylmethylxanthine (1 mmol·l-1) nearly doubled short-circuit current and increased amiloride-sensitive Na+ currents by 50%. By current fluctuation analysis we estimated single Na+ channel current (2.7±0.9 pA) and Na+ channel density (3.6±0.6 channels·m-2) under control conditions. After cyclic adenosine monophosphate stimulation Na+ channel density increased to 5.4±1.1 channels·m-2, whereas single Na+ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na+ channel, and show the similarities and differences to vertebrate Na+ channels. Whereas the channel properties are different from the classical vertebrate Na+ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na+ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na+ channels.Abbreviations
slope of the background noise component
- ADH
antidiuretic hormone
- cAMP
cyclic adenosine monophosphate
-
f
frequency
-
f
c
coner frequency of the Lorentzian noise component
- Hepes
N-hydroxyethylpiperazine-N-ethanesulphonic acid
- BMX
isobutyl-methylxanthine
-
i
Na
single Na+ channel current
-
I
Na
max, maximal inhibitable Na+ current
-
I
SC
short circuit current
-
K
i
half maximal blocker concentration
-
K
M
Michaelis constandard error of the mean
-
S
(f)
power density of the Lorentzian noise component
-
S
0
plateau value of the Lorentzian noise component
- TMA
tetramethylammonium
- Trizma
TRIS-hydroxymethyl-amino-methane
-
V
max
maximal reaction velocity
-
V
T
transepithelial potential
-
K
half maximal blocker concentration 相似文献
5.
In the presence of the Na+ -channel blocker amiloride, the short-circuit current across the skins of bullfrog tadpoles in metamorphic stages XIX-XXIV was subjected to fluctuation analysis. The resulting power spectra contained a Lorentzian component of which the plateau value (S0) decreased while the corner frequency (fc) increased as the mucosal amiloride concentration was increased from 0.5 to 24 microM. From the linear relationship between the fc values and the amiloride concentrations it was possible to determine the binding (k'01) and unbinding (k10) constants for amiloride to its receptor on the Na+ channel. With these parameters as well as short-circuit current and S0 values, the current through the individual Na+ channels (i) was calculated (average 0.58 pA). It did not increase significantly during late metamorphosis. The density of Na+ channels (M) in the apical membrane, on the other hand, increased significantly. It would appear that the increase in short-circuit current which occurs at this time is due primarily to an increase in amiloride-blockable Na+ channels. Unexpectedly, a Lorentzian component could be fitted to power spectra in amiloride-treated skins (stages XIX-XXI) which showed no amiloride-sensitive short-circuit current. Moreover, the typical increase in fc with the amiloride concentration did not occur in these animals. 相似文献
6.
J. Pácha 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(6):495-498
To evaluate the developmental changes in colonic Na+ transport, Na, K-ATPase activity and the sensitivity of the short-circuit current to amiloride were investigated. The amiloride-sensitive short-circuit current which represents the electrogenic, amiloride-sensitive Na+ transport through Na+ channels, was not present in chicken embryos but rose significantly after hatching in chicks which were kept on a low-salt diet. Amiloride-sensitive short-circuit current increased gradually but the plateau was not reached during the first 15 days of life. Drinking of 0.9% NaCl totally inhibited the induction of amiloride-sensitive Na+ transport. Na+, K+-ATPase activity increased during development but was not influenced by changes in salt intake. Na+ transport in chicken colon therefore undergoes profound developmental changes. The increase of Na+ transport refleets not only the adaptation of colonocytes to low salt intake but also the maturation of Na+ absorption in colon. The possible role of aldosterone in the adaptation to low-salt intake is discussed.Abbreviations LS
low-salt
- HS
high-salt
-
I
sc
short-circuit current 相似文献
7.
In cultured A6 monolayers from distal Xenopus kidney, external Ni2+ stimulated active Na+ uptake via the epithelial Na+ channel, ENaC. Transepithelial capacitance measurements ruled out exocytosis of ENaC-containing vesicles underlying the Ni2+ effect. Na+ current noise analysis was performed using the neutral Na+-channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide (CDPC) and amiloride. The analysis of CDPC-induced noise in terms of a three-state channel model revealed that Ni2+ elicits an increase in the number of open channels as well as in the spontaneous open probability. While Ni2+ had no influence on CDPC-blocker kinetics, the macroscopic and microscopic blocking kinetics of amiloride were affected. Ni2+ turned out to compete with amiloride for a putative binding site but not with CDPC. Moreover, external Na+—known to compete with amiloride and so producing the self-inhibition phenomenon—and Ni2+ exerted mutually exclusive analogous effects on amiloride kinetics. Na+ current kinetics revealed that Ni2+ prevents ENaC to be downregulated by self-inhibition. Co2+ behaved similarly to Ni2+, whereas Zn2+ did not. Attempts to disclose the chemical nature of the site reacting with Ni2+ suggested cysteine but not histidine as reaction partner. 相似文献
8.
B. Illek H. Fischer W. Clauss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(7):556-562
Basolateral K+ channels and their regulation during aldosterone- and thyroxine-stimulated Na+ transport were studied in the lower intestinal epithelium (coprodeum) of embryonic chicken in vitro. Isolated tissues of the coprodeum were mounted in Ussing chambers and investigated under voltage-clamped conditions. Simultaneous stimulation with aldosterone (1 mol·l-1) and thyroxine (1 mol·l-1) raised short-circuit current after a 1- to 2-h latent period. Maximal values were reached after 6–7 h of hormonal treatment, at which time transepithelial Na+ absorption was more than tripled (77±11 A·cm-2) compared to control (24±8 A·cm-2). K+ currents across the basolateral membrane with the pore-forming antibiotic amphotericin B and application of a mucosal-to-serosal K+ gradient. This K+ current could be dose dependently depressed by the K+ channel blocker quinidine. Fluctuation analysis of the short-circuit current revealed a spontaneous and a blocker-induced Lorentzian noise component in the power density spectra. The Lorentzian corner frequencies increased linearly with the applied blocker concentration. This enabled the calculation of single K+ channel current and K+ channel density. Single K+ channel current was not affected by stimulation, whereas the number of quinidine-sensitive K+ channels in the basolateral membrane increased from 11 to 26·106·cm-2 in parallel to the hormonal stimulation transepithelial Na+ transport. This suggests that the basolateral membrane is a physiological target during synergistic aldosterone and thyroxine regulation of transepithelial Na+ transport for maintaining intracellular K+ homeostasis.Abbreviations
f
frequency
-
f
c
Lorentzian corner frequency
-
g
K
single K+ channel conductance
- HEPES
N-2-hydroxyethylpiperazin-N'-2-ethansulfonic acid
-
i
K
single K+ channel current
- IAmpho
amphotericin B induced K+ current
-
I
sc
short-circuit current
-
I
K
quinidine blockable K+ current
-
I
max
maximally blocked current by quinidine
-
IC
50
half-maximal blocker concentration
-
k
on, k
off
on- and off-rate coefficients of reversible single channel block by quinidine
-
M
K
number of conducting K+ channels
- [Q]
quinidine concentration
-
R
t
transepithelial resistance
-
S
spectral density
-
S
o
Lorentzian plateau
- TBM cells
toad urinary bladder cell line
Present address: University of California at Berkeley, Dept. of Molecular and Cell Biology Berkeley, CA 94720, USA 相似文献
9.
10.
R. Krattenmacher Rosita Voigt W. Clauss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1990,160(2):161-165
Summary Transepithelial electrogenic Na transport (INa) was investigated in the colon of the frog Xenopus laevis with electrophysiological methods in vitro. The short circuit current (Isc) of the voltage-clamped tissue was 24.2±1.8 A·cm-2 (n=10). About 60% of this current was generated by electrogenic Na transport. Removal of Ca2+ from the mucosal Ringer solution stimulated INa by about 120%. INa was not blockable by amiloride (0.1 mmol·l-1), a specific Na-channel blocker in epithelia, but a fully and reversible inhibition was achieved by mucosal application of 1 mmol·l-1 lanthanum (La3-). No Na-self-inhibition was found, because INa increased linearly with the mucosal Na concentration. A stimulation of INa by antidiuretic hormones was not possible. The analysis of fluctuations in the short circuit current (noise analysis) indicated that Na ions pass the apical cell membrane via a Ca-sensitive ion channel. The results clearly demonstrate that in the colon of Xenopus laevis Na ions are absorbed through Ca-sensitive apical ion channels. They differ considerably in their properties and regulation from the amiloride-sensitive Na channel which is typically found in the colon of vertebrates.Abbreviations
G
T
transepithelial conductance
-
I
sc
short circuit current
-
I
Na
transepithelial Na-current
-
m
mucosal
-
s
serosal
-
PDS
power density spectrum
-
f
frequency
-
f
c
corner frequency of the Lorentzian component of the PDS
-
S(f)
power density of the Lorentzian component of the PDS
- So
plateau value of the Lorentzian component of the PDS 相似文献
11.
12.
Stanley D. Hillyard Willy Van Driessche 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1991,161(3):257-263
Summary The short-circuit current (SCC) across isolated skin from bullfrog larvae in developmental stage XXI was small and insensitive to amiloride. Overnight incubation of this tissue with 10-6
M aldosterone stimulated the SCC from 1.35±0.55 to 14.55±4.12 A·cm-2 with 11.18±4.46 A·cm-2 being blocked by 100 M amiloride. Histologic examination of aldosterone-treated skins revealed a separation of the apical cell layer from the underlying epidermis that was not seen in untreated preparations. The onset of amiloride-sensitive Na+ transport thus coincided with the exposure of the apical surface of newly differentiated epithelial cells. Similar results were obtained with skin from stage XXI larvae whose rate of metamorphosis had been stimulated by 10 g·l-1 thyroxine (T4) but not with skin from T4-treated larvae in stages XIX and XX. Fluctuation analysis of the amiloride-sensitive SCC of the above preparations failed to show a consistent Lorentzian component in the power-density spectrum. Fluctuation analysis was possible on skins from larvae whose development had been accelerated by 7–9 days treatment with 10 g·l-1 triiodothyronine (T3). Aldosterone treatment of these tissues resulted in a significant increase in Na+ channel density.Abbreviations
ASCC
component of the short-circuit current (A·cm-2) that is blocked by amiloride
-
fc
frequency (Hz) at which the magnitude of the Lorenzian component of the power spectra is reduced by half
-
i
current (pA) through individual amiloride-sensitive Na+ channels
-
I
Na+
amiloride-sensitive short-circuit current (A·cm-2) that remains after treatment with a given amiloride concentration
-
k
01
the rate constant (s-1·M-1) for the association of amiloride with Na+ channels
-
k
10
rate constant (s-1) for the dissociation of amiloride from Na+ channels
-
K
b
magnitude of the power spectrum (A2·s·cm-2) at a frequency of 1 Hz
-
KSCC
short-circuit (A·cm-2) current with K+ as the primary mucosal cation
-
M
density of amiloride-sensitive Na+ channels in the apical cell membrane
-
SCC
short-circuit current (A·cm-2)
-
S
(f)
magnitude of the power spectra (A2·s·cm-2) at a given frequency
-
S
0
the magnitude of the plateau region of the Lorentzian component of the power spectra (A2·s·cm-2)
-
T
3
Triiodothyronine
-
T
4
Thyroxine 相似文献
13.
S. A. Lewis M. S. Ifshin D. D. F. Loo J. M. Diamond 《The Journal of membrane biology》1984,80(2):135-151
Summary Sodium channels in rabbit urinary bladder were studied by noise analysis. There are two components of short-circuit current (I
sc) and correspondingly two components of apical Na+ entry, one amiloride-sensitive (termedI
A and the A channel, respectively) and one amiloride-insensitive (I
L
and the leak pathway, respectively). The leak pathway gives rise tol/f noise, while the A channel in the presence of amiloride gives rise to Lorentzian noise. A two-state model of the A channel accounts well for how the corner frequency and plateau value of Lorentzian noise vary with amiloride concentration. The single-channel current is 0.64 pA, and the conducting channel density is on the order of 40 copies per cell. Triamterene blocks the A channel alone, and increasing external Na+ decreases the number but not the single-channel permeability of the A channel. Hydrostatic pressure pulses (punching) increase the number of both pathways. Repeated washing of the mucosal surface removes most of the leak pathway without affecting the A channel.Properties of the A channel revealed by noise analysis of various tight epithelia are compared, and the mechanism ofl/f noise is discussed. It is suggested that the A channel is synthesized intracellularly, stored in intracellular vesicles, transferred with or from vesicular membrane into apical membrane under the action of microfilaments, and degraded into the leak pathway, which is washed out into urine or destroyed. The A channel starts withP
Na/P
K30 and loses selectivity in stages untilP
Na/P
K reaches the free-solution mobility ratio (0.7) for the leak pathway. This turnover cycle functions as a mechanism of repair and regulation for Na+ channels, analogous to the repair and regulation of most intracellular proteins by turnover. Vesicular delivery of membrane channels may be operating in several other epithelia. 相似文献
14.
Na+ transport was evaluated in brush border membrane vesicles isolated from the human placental villous tissue. Na+ uptake was assayed by the rapid filtration technique in the presence and the absence of an uphill pH gradient. Amiloride strongly decreased Na+ uptake whether a pH gradient was present or not. In pH gradient conditions (pH 7.5 in and 9.0 out), 1 mM amiloride decreased the 10 mM Na+ uptake by 84%. In the absence of pH gradient (pH 7.5 in and out), Na+ uptake was lower but still sensitive to amiloride. The Lineweaver-Burk plot of Na+ uptake consistently showed a single kinetics. Increasing the pH gradient decreased Km values of the amiloride-sensitive Na+ uptake, leaving the Vmax unchanged. In the absence of a pH gradient, the amiloride sensitive Na+ transport was maximal at pH 7.5. Here again, a single kinetics was observed, and pH influenced exclusively the Km of Na+. Since ethylisopropylamiloride, the specific Na/H exchanger inhibitor mimicked the effects of amiloride, decreasing by 98% the 10 mM Na+ uptake, whereas benzamil, the Na+ channel blocker, had no effect, it was concluded that the amiloride sensitive Na+ uptake was predominantly or exclusively due to a Na+-H+ exchanger activity. K+ in trans-position significantly decreased the amiloride sensitive uptake. In contrast, the presence of the cation in cis-position had no effect. The amiloride resistant Na+ transport was neither influenced by pH, nor saturable. Incubation of the placental tissue with 100 μM or 1 mM dibutyryl cAMP, 0.1 or 1 μM phorbol myristate acetate, 10−7 M insulin, 10−10 M angiotensin II, or 10−8 M human parathyroid hormone (PTH) did not influence Na+ transport by subsequently prepared brush border membranes. Finally, we failed to demonstrate any Na+-H+ exchange activity in the basal plasma membrane. These results indicate that (1) in the absence of co-substrates such as phosphate and aminoacids, the Na+-H+ exchange is probably the unique mechanism of Na+ transport by the placental brush border membrane, (2) the placental isoform of the exchanger is not regulated by PTH, angiotensin, nor insulin and, therefore, is different from the isoform present in the renal brush border membrane, and (3) there is no exchanger activity in the basal plasma membrane. © 1996 Wiley-Liss, Inc. 相似文献
15.
Horst Fischer Wolfgang Kromer Wolfgang Clauss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1991,161(4):333-338
Summary Freshly isolated epithelial cells from hen colon were investigated using the patch-clamp technique. The aim of this investigation was to characterise the cellular conducting site for Cl- secretion. In cell-attached mode two types of Cl--channels were found. Both showed distinct outward rectification. The channel types differed in single channel conductances and the marked voltage dependence of the open probabilities. A low conductance Cl--channel was observed with a mean conductance at negative holding potentials of g-=9 pS, and of g+=34 pS at positive potentials. This channel was predominantly open at negative potentials, corresponding to cell hyperpolarization. The second channel type observed had conductances of g-=35 pS and g+=77 pS, and showed increasing open probabilities with increasing holding potentials (cell depolarisation). Both channel types were blockable by the Cl--channel blocker NPPB. These data in combination with previously published transepithelial transport data on hen colon indicate that these channels are the Cl- secretory sites in colon epithelium.Abbreviations DNSO
dimethylsulfoxide
- EGTA
ethyleneglycol triacetic acid
- g+, g-
single channel conductance at positive and negative voltages
- HEPES
N-(2-hydroxy-ethyl)piperazine-N-(2-ethane-sulfonic acid)
- i
single channel current
- NMDG
N-methyl-d-glucosamine
- NPPB
5-hitro-2-(3-phenylpropylamino)-benzoate
- Po
open probability
- Vp
holding potential 相似文献
16.
BJ
RN EKMEHAG BERTIL PERSSON PATRIK RORSMAN HANS RORSMAN 《Pigment cell & melanoma research》1994,7(5):333-338
The electrophysiological properties of cultured human melanocytes were investigated using the whole-cell configuration of the patch-clamp technique. Depolarizations to membrane potentials more positive than -30 mV resulted in the rapid development (<1 ms to peak) of an inward current. The maximum peak current was observed at +10 mV and reached an average amplitude of about 270 pA. During the depolarizations, the current inactivated with a time constant of about 2 ms. The current was abolished by the addition of 0.3 μM tetrodotoxin, a blocker of voltage-gated Na+-channels, and disappeared when Na+ was omitted from the extracellular medium. In addition, the melanocytes contain at least two types of outward K+-current. The first type, observed in every cell, was highly sensitive (Ki 1 mM) to the K+-channel blocker TEA, required depolarizations beyond zero to be activated and did not inactivate. The second type was less regularly observed (10% of the cells). This current activated at more negative voltages (–20 mV), was resistant to TEA (20 mM) but was blocked by 2 mM 4-aminopyridine and inactivated rapidly during depolarizations. We conclude that human melanocytes are equipped with voltage-dependent Na+-channels, a delayed rectifying K+-current and a K+-current similar to the A-current in neurones. 相似文献
17.
We have used the patch clamp technique to study the effects of inhibiting the apical Na+ transport on the basolateral small-conductance K+ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 μM amiloride decreased the activity of SK, defined as nP
o (a product of channel open probability and channel number), to 61% of the control value. Application of 1 μM benzamil, a specific Na+ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na+ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 μM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca2+ because removal of Ca2+ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca2+, we observed that amiloride and benzamil significantly decreased intracellular Ca2+ in the Ca2+-containing solution but had no effect in a Ca2+-free bath. Furthermore, raising intracellular Ca2+ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca2+ are responsible for the effects on SK activity of inhibition of the Na+ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca2+ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP–dependent pathways. Addition of 10 μM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 μM 8-bromoguanosine 3′:5′-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca2+-free bath solution. We conclude that Ca2+-dependent NO generation mediates the effect of inhibiting the apical Na+ transport on the basolateral SK in the rat CCD. 相似文献
18.
Robert Nielsen 《The Journal of membrane biology》1982,65(3):227-234
Summary Effect of amiloride, ouabain, and Ba++ on the nonsteady-state Na–K pump flux and short-circuit current in isolated frog skin epithelia.The active Na+ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na+–K+ pump at the basolateral membrane. In isolated epithelia with a very small Na+ efflux, the appearing Na+-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na+ transport. It was found that blocking the passive diffusion of Na+ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na+ transport, whereas the appearing flux (the rate of the Na+–K+ pump) decreased with a halftime of 1.9 min. Addition of the Na+–K+ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na+ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K+ channel inhibitor Ba++, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na–K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.It is argued that in the short period, where the Ba++-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (SCC) is equal to the net K+ flux via the Na+–K+ pump, and the coupling ratio () of the Na+–K+ pump can be calculated from the following equation =SCC
t=0/SCC where SCC
t=0 is the steady-state SCC before the addition of Ba++. 相似文献
19.
Ion channels of the degenerin/epithelial Na+ channel gene family are Na+ channels that are blocked by the diuretic amiloride and are implicated in several human diseases. The brain liver intestine Na+ channel (BLINaC) is an ion channel of the degenerin/epithelial Na+ channel gene family with unknown function. In rodents, it is expressed mainly in brain, liver, and intestine, and to a lesser extent in kidney and lung. Expression of rat BLINaC (rBLINaC) in Xenopus oocytes leads to small unselective currents that are only weakly sensitive to amiloride. Here, we show that rBLINaC is inhibited by micromolar concentrations of extracellular Ca2+. Removal of Ca2+ leads to robust currents and increases Na+ selectivity of the ion pore. Strikingly, the species ortholog from mouse (mBLINaC) has an almost 250-fold lower Ca2+ affinity than rBLINaC, rendering mBLINaC constitutively active at physiological concentrations of extracellular Ca2+. In addition, mBLINaC is more selective for Na+ and has a 700-fold higher amiloride affinity than rBLINaC. We show that a single amino acid in the extracellular domain determines these profound species differences. Collectively, our results suggest that rBLINaC is opened by an unknown ligand whereas mBLINaC is a constitutively open epithelial Na+ channel. 相似文献
20.
(1) Single myelinated nerve fibres of Rana esculenta were treated with the steroidal alkaloid batrachotoxin, and Na+ currents and Na+-current fluctuations were measured near the resting potential under voltage-clamp conditions. Between test pulses fibres were held at hyperpolarizing membrane potentials. (2) The spectral density of Na+-current fluctuations was fitted by the sum of a component and a Lorentzian function. The time constant obtained from the corner frequency ?c of the Lorentzian function approximately agreed with the activation time constant τm of the macroscopic currents. (3) The conductance γ of a single Na+ channel modified by batrachotoxin was calculated from the integral of the Lorentzian function and the steady-state Na+ current. At the resting potential V = O we obtained γ = 1.6 pS, higher γ-values of 3.2 and 3.45 pS were found at V = ?8 and ?16 mV, respectively. (4) The conductance of a modified Na+ channel is significantly lower than the values 6.4 to 8.85 pS reported in the literature for normal Na+ channels. Hence, our experiments are in agreement with the view that batrachotoxin acts in an ‘all-or-none’ manner on Na+ channels and creates a distinct population of modified channels. 相似文献