Cholinergic modulation of apical Na+ channels in turtle colon: analysis of CDPC-induced fluctuations

1990 ◽  
Vol 259 (4) ◽  
pp. C668-C674 ◽  
Author(s):  
D. J. Wilkinson ◽  
D. C. Dawson
Keyword(s):  
Single Channel ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Rate Coefficients ◽  
Corner Frequency ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Na Channels ◽  
Na Current

Current fluctuation analysis was used to investigate the properties of apical Na+ channels during muscarinic inhibition of active Na+ absorption. A reversible Na+ channel blocker, 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), was used to induce fluctuations in the short-circuit current (I(sc)). Power density spectra of the CDPC-induced fluctuations exhibited a clearly discernible Lorentzian component, characterized by a corner frequency that was linearly related to CDPC concentration between 20 and 100 microM. The on (k'on) and off (k(off)) rate coefficients for the CDPC blocking reaction were k'on = 11.1 +/- 0.8 rad.s-1.microM-1 and k(off) = 744 +/- 53 rad/s, and the microscopic inhibition constant was 67 microM (n = 11). CDPC blocking kinetics were not significantly different after inhibition of Isc by 5 microM serosal carbachol. Single-channel Na+ current (iNa) and the density of open and blocked Na+ channels (N(ob)) were estimated from the fluctuations induced by 40 microM CDPC. Under control conditions, iNa was 0.43 +/- 0.05 pA and N(ob) was 251 +/- 42 X 10(6)/cm2 (n = 10). After exposure to serosal carbachol (2-10 microM) for 60 min, Na+ current and N(ob) were reduced by approximately 50%, but iNa was not changed significantly. These results indicate that muscarinic inhibition of electrogenic Na+ absorption was associated with a reduction in the number of open Na+ channels in the apical membrane. They also suggest that this downregulation of transport involved a coordinated decrease in both apical and basolateral membrane conductances.

scholarly journals Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium.

1986 ◽  
Vol 87 (3) ◽  
pp. 443-466 ◽  
Author(s):  
J W Hanrahan ◽  
N K Wills ◽  
J E Phillips ◽  
S A Lewis
Keyword(s):  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Short Circuit Current ◽  
Corner Frequency ◽  
Fluctuation Analysis ◽  
K Channels ◽  
K Current

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.

Verapamil blocks basolateral K+ channels in the larval frog skin

1992 ◽  
Vol 262 (5) ◽  
pp. C1161-C1166 ◽  
Author(s):  
S. D. Hillyard ◽  
W. Van Driessche
Keyword(s):  
Single Channel ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Power Spectra ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Corner Frequency ◽  
Fluctuation Analysis ◽  
K Channels ◽  
Single Channel Currents

The short-circuit current (Isc) across isolated skin from larval frogs (Rana catesbeiana) was measured when the tissue was bathed with Na2SO4 Ringer solution on the serosal side and with a Ringer solution containing K+ as the primary cation on the mucosal side. When 150 U/ml nystatin was added to the mucosal solution, the Isc increased from 1.4 +/- 0.1 to 35.4 +/- 4.8 microA/cm2. When verapamil was added to the mucosal and serosal Ringer solutions in concentrations between 2.5 and 80 microM, Isc was inhibited in a stepwise manner. At 80 microM, Isc was reduced by 75.3% to 8.74 +/- 1.14 microA/cm2. Analysis of the inhibition of Isc with the direct linear plot method showed that the blockage of Isc could be described by pseudo-first-order kinetics with a Michaelis constant (Km) of 9.59 +/- 2.20 microM. Fluctuation analysis revealed a Lorentzian component in power spectra obtained from preparations treated with 10-80 microM verapamil. The corner frequency of these Lorentzian components increased in a linear manner over this range of verapamil concentrations. The Km calculated from the ratio of the dissociation and association rate constants (k10/k'01) was 39.5 microM. The single-channel currents (i) calculated from the fluctuation analysis parameters decreased significantly between verapamil concentrations of 10 and 80 microM. It appears that the inhibition of K+ channels in the basolateral membrane of this tissue has at least two components.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiology and noise analysis of K+-depolarized epithelia of frog skin

1985 ◽  
Vol 249 (5) ◽  
pp. C421-C429 ◽  
Author(s):  
J. Tang ◽  
F. J. Abramcheck ◽  
W. Van Driessche ◽  
S. I. Helman
Keyword(s):  
Frog Skin ◽  
Single Channel ◽  
Apical Membrane ◽  
Short Circuit ◽  
Noise Analysis ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Na Channel ◽  
Channel Density

Epithelia of frog skin bathed either symmetrically with a sulfate-Ringer solution or bathed asymmetrically and depolarized with a 112 mM K+ basolateral solution (Kb+) were studied with intracellular microelectrode techniques. Kb+ depolarization caused an initial decrease of the short-circuit current (Isc) with a subsequent return of the Isc toward control values in 60-90 min. Whereas basolateral membrane resistance (Rb) and voltage were decreased markedly by high [Kb+], apical membrane electrical resistance (Ra) was decreased also. After 60 min, intracellular voltage averaged -27.3 mV, transcellular fractional resistance (fRa) was 86.8%, and Ra and Rb were decreased to 36.1 and 13.0%, of their control values, respectively. Amiloride-induced noise analysis of the apical membrane Na+ channels revealed that Na+ channel density was increased approximately 72% while single-channel Na+ current was decreased to 39.9% of control, roughly proportional to the decrease of apical membrane voltage (34.0% of control). In control and Kb+-depolarized epithelia, the Na+ channel density exhibited a phenomenon of autoregulation. Inhibition of Na+ entry (by amiloride) caused large increases of Na+ channel density toward saturating values of approximately 520 X 10(6) channels/cm2 in Kb+-depolarized tissues.

Apical membrane properties and amiloride binding kinetics of the human descending colon

1984 ◽  
Vol 247 (6) ◽  
pp. G749-G757 ◽  
Author(s):  
N. K. Wills ◽  
W. P. Alles ◽  
G. I. Sandle ◽  
H. J. Binder
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Short Circuit ◽  
Membrane Properties ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Bathing Solution ◽  
Na Channels ◽  
Descending Colon ◽  
Single Channel Currents

The apical membrane properties of the isolated human descending colon were characterized by use of current fluctuation analysis methods and microelectrode techniques. The Na+ channel blocker amiloride was used to evaluate apical membrane conductance and the transepithelial short-circuit current (Isc). Amiloride significantly reduced Isc and increased the membrane resistance ratio. At submaximal doses of amiloride in the mucosal bathing solution, fluctuation analysis of the Isc revealed a Lorentzian component in the power-density spectra. The dose-response relationship between amiloride and current noise parameters was consistent with a two-state mechanism of blocker interaction with the channel. The on and off rate constants for the blocker-receptor reactions, the single-channel currents, and the Na+ channel densitywere estimated and were similar to those from Na+ channels from other so-called tight epithelia. In addition, these studies revealed an amiloride-insensitive conductance in the apical membrane in parallel to the amiloride-blockable Na+ channels. This conductance may be due to potassium ions. If so, the apical membrane properties of the human descending colon may closely resemble those of the rabbit descending colon and rat distal colon.

Effect of amiloride on the poorly selective cation channel of larval bullfrog skin

1989 ◽  
Vol 256 (1) ◽  
pp. C168-C174 ◽  
Author(s):  
S. D. Hillyard ◽  
W. Van Driessche
Keyword(s):  
Single Channel ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Ringer Solution ◽  
Cation Channel ◽  
Power Density Spectrum ◽  
Corner Frequency ◽  
Open Probability ◽  
Density Spectrum

A small, inward-directed, short-circuit current (SCC) was measured across the isolated skin of larval bullfrogs (Rana catesbeiana) when either NaCl or KCl Ringer solution bathed the mucosal surface. The addition of amiloride, in concentrations of 1-100 microM, produced a stepwise increase in SCC. As SCC values became maximally elevated by amiloride, the plateau value (So) of the Lorentzian component in the power-density spectrum increased, whereas the corner frequency (fc) decreased. This agonist effect of amiloride can be explained by an increase in the open probability and possibly the single-channel current of the larval channel. When the amiloride concentration was increased above 100 microM, the SCC values declined progressively but usually remained above pretreatment values. This suggests an antagonist effect of amiloride that is concurrent with the agonist effect. The removal of Ca2+ from the mucosal Ringers increased SCC in conjunction with an increase in So and a decrease in fc. Under these conditions, the maximal agonist effect of amiloride was observed at concentrations of 10-20 microM. Ca2+ thus exerts an inhibitory effect on the larval cation channel that interferes with the agonist effect of amiloride. The addition of Ba2+ to Ca2+-free preparations lowered SCC and reduced the agonist effect of amiloride.

Brefeldin A inhibition of apical Na+ channels in epithelia

1996 ◽  
Vol 270 (1) ◽  
pp. C138-C147 ◽  
Author(s):  
R. S. Fisher ◽  
F. G. Grillo ◽  
S. Sariban-Sohraby
Keyword(s):  
Cultured Cells ◽  
Single Channel ◽  
Noise Analysis ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Na Channel ◽  
Na Channels ◽  
Na Transport ◽  
Channel Current ◽  
Vacuolar System

Brefeldin A (BFA) is used to probe trafficking of proteins through the central vacuolar system (CVS) in a variety of cells. Transepithelial Na+ transport by high-resistance epithelia, such as A6 cultured cells, is inhibited by BFA. Apical Na+ channels, as well as basolateral pumps and K+ channels, are complex proteins that probably traverse the CVS for routing to the plasma membrane. BFA (5 micrograms/ml) decreases transepithelial Na+ current near zero and increases resistance reversibly after 4 h. Longer exposures are toxic. When tissues were treated for 20 h with 0.2 microgram/ml BFA, Na+ transport also was reversibly inhibited. Using noise analysis, we found that BFA drastically reduced apical Na+ channel density. The increase in single channel current was consistent with cell hyperpolarization. After apical permeabilization with nystatin, changes in transepithelial current reflect changes in basolateral membrane transport. Transport at this membrane was inhibited by ouabain and cycloheximide, but not by BFA. After BFA, aldosterone was ineffective, suggesting that an intact CVS is required for stimulation by this hormone. Thus BFA inhibition of Na+ transport is localized at the apical membrane. Implications for channel turnover as a mechanism for regulating the Na+ transport rate are discussed.

Chronic regulation of transepithelial Na+ transport by the rate of apical Na+ entry

1996 ◽  
Vol 270 (2) ◽  
pp. C600-C607 ◽  
Author(s):  
M. D. Rokaw ◽  
E. Sarac ◽  
E. Lechman ◽  
M. West ◽  
J. Angeski ◽  
...  
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Maximal Activity ◽  
Na Channels ◽  
Na Transport ◽  
Bottom Structures ◽  
Stimulation Of

In several settings in vivo, prolonged inhibition of apical Na+ entry reduces and prolonged stimulation of apical entry enhances the ability of renal epithelial cells to reabsorb Na+, an important feature of the load-dependent regulation of renal tubular Na+ transport. To model this load dependency, apical Na+ entry was inhibited or stimulated for 18 h in A6 cells and vectorial transport was measured as short-circuit current (Isc) across monolayers on filter-bottom structures. Basal amiloride-sensitive Isc represents the activity of apical Na+ channels, whereas Isc after permeabilization of the apical membrane to cations with nystatin represents maximal activity of the basolateral Na(+)-K(+)-ATPase. Chronic inhibition of apical Na+ entry by 18-h apical exposure to amiloride or replacement of apical Na+ with tetramethylammonium (TMA+), followed by washing and restoration of normal apical medium, revealed a persistent decrease in Isc that remained despite exposure to nystatin. Both basal and nystatin-stimulated Isc recovered progressively after restoration of normal apical medium. In contrast, chronic stimulation of apical Na+ entry by short circuiting the epithelium increased Isc in the absence and presence of nystatin, indicating upregulation of both apical Na+ channels and basolateral Na(+)-K(+)-ATPase. Basolateral equilibrium [3H]ouabain binding was reduced to 67 +/- 5% in TMA+ vs. control cells, whereas values in 18-h short-circuited cells increased by 42 +/- 19%. The results demonstrate that load dependency of tubular Na+ transport can be modeled in vitro and indicate that the regulation of Na(+)-K(+)-ATPase observed in these studies occurs in part by changes in the density of functional transporter proteins within the basolateral membrane.

Modification of carboxyl of Na+ channel inhibits aldosterone action on Na+ transport

1983 ◽  
Vol 245 (6) ◽  
pp. F726-F734 ◽  
Author(s):  
J. Kipnowski ◽  
C. S. Park ◽  
D. D. Fanestil
Keyword(s):  
Amphotericin B ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Channels ◽  
Na Transport ◽  
Maximal Increase ◽  
Osmotic Water ◽  
Stimulation Of

We investigated the effect of the carboxyl-selective reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on aldosterone stimulation of Na+ transport in the urinary bladder of the toad. Na+ transport, measured as the short-circuit current (SCC), was irreversibly inhibited by EEDQ in a dose- and time-dependent manner prior to addition of aldosterone. The greater the percentage inhibition by EEDQ (X), the smaller was the maximal increase of SCC after aldosterone (Y). This relationship gave the regression equation Y = 128.41 - 1.73X, r = -0.99 (n = 35). Evidence that the inhibition of SCC produced by EEDQ was limited to effects at the mucosal membrane was attested by the following: 1) EEDQ did not alter the stimulation by aldosterone of the osmotic water flow response to antidiuretic hormone; 2) whereas inhibition of protein synthesis by cycloheximide prevented this effect of aldosterone; 3) amphotericin B fully restored SCC previously inhibited by EEDQ to the level produced in tissues not inhibited by EEDQ; 4) comparison of the effects of amiloride vs. EEDQ pretreatment on the SCC response to aldosterone and amphotericin B revealed nearly identical characteristics; 5) in contrast, amphotericin B stimulation of SCC was limited when Na+ transport was limited by antimycin A (an inhibitor of energy production) or by ouabain. The findings fail to provide positive evidence for the hypothesis that aldosterone induces the synthesis of new Na+ channels but are consistent with hormonal activation of previously existing but nonfunctioning Na+ channels.

Possible role of aldosterone and T3 in development of amiloride-blockable SCC across frog skin in vivo

1999 ◽  
Vol 277 (5) ◽  
pp. R1305-R1312 ◽  
Author(s):  
Makoto Takada ◽  
Michio Shiibashi ◽  
Miyoko Kasai
Keyword(s):  
Thyroid Hormone ◽  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Short Circuit Current ◽  
Rate Coefficients ◽  
Adult Type ◽  
Apparent Dissociation Constant

There are inconsistencies between the in vitro and in vivo effects of thyroid hormone and aldosterone (Aldo) on the development of an amiloride-blockable short-circuit current (SCC) across bullfrog skin [Takada, M., H. Yai, and K. Takayama-Arita. Am. J. Physiol. 268 ( Cell Physiol. 37): C218–C226, 1995]. To address this issue, tadpoles were raised in Aldo + T3. An amiloride-blockable SCC developed across the skin before forelimbs appeared. Noise analysis of the characteristics (single-channel current, blocking and unblocking rate coefficients, and apparent dissociation constant) of this amiloride-blockable Na+ channel showed that it really was of the adult type. A similar SCC developed at stage XIX in the skin of tadpoles raised with Aldo alone. These results strongly support our hypothesis that the crucial hormone in the development of this SCC is Aldo but that a suppression mechanism attenuates its effect on SCC development until it is removed by the increase in the serum concentration of thyroid hormone (which starts at stages XVIII–XIX in vivo).

Aldosterone regulation of basolateral potassium channels in alveolar epithelium

1990 ◽  
Vol 259 (4) ◽  
pp. L230-L237 ◽  
Author(s):  
B. Illek ◽  
H. Fischer ◽  
W. Clauss
Keyword(s):  
Short Circuit ◽  
Alveolar Epithelium ◽  
Short Circuit Current ◽  
Corner Frequency ◽  
K Channel ◽  
Fluctuation Analysis ◽  
K Channels ◽  
Ussing Chambers ◽  
Channel Current ◽  
K Current

To reveal the regulatory mechanism of the mineralocorticoid aldosterone on basolateral K+ channels, the aldosterone-sensitive lung epithelium of Xenopus laevis was investigated in Ussing chambers under voltage-clamp conditions. Transepithelial measurements were supplemented by current fluctuation analysis of short-circuit current noise in nonstimulated and aldosterone-stimulated lung tissues. The addition of 10(-6) M aldosterone stimulated short-circuit current from 11.3 +/- 2.0 to 27.8 +/- 4.8 microA/cm2 (n = 11) within 4–5 h. In the presence of an alveolar-to-pleural K+ gradient, transepithelial K+ currents were induced by permeabilizing the apical membrane with the pore-forming antibiotic amphotericin B. When the local anesthetic lidocaine (25-1,000 microM) was added to the pleural solution, macroscopic K+ current was dose dependently depressed. Lidocaine induced a Lorentzian component in the power density spectra, and the corner frequency increased linearly with blocker concentration. Aldosterone treatment did not affect mean single K+ channel current, which was 1.5 +/- 0.12 pA corresponding to a 15-pS channel conductance, whereas the number of basolateral K+ channels doubled. We conclude that the basolateral K+ channels in alveolar epithelia are a target site of aldosterone action.

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