Active NaCl absorption across split lamellae of posterior gills of the chinese crab Eriocheir sinensis: stimulation by eyestalk extract

2000 ◽  
Vol 203 (8) ◽  
pp. 1373-1381 ◽  
Author(s):  
H. Onken ◽  
A. Schobel ◽  
J. Kraft ◽  
M. Putzenlechner
Keyword(s):  
Electromotive Force ◽  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Eriocheir Sinensis ◽  
Dialysis Tubing ◽  
Channel Current ◽  
Dose Dependent ◽  
Cl Conductance ◽  
Stimulation Of

Split lamellae of the posterior gills of freshwater-adapted Chinese crabs (Eriocheir sinensis) were mounted in a modified Ussing-type chamber, and active and electrogenic absorption of Na(+) and Cl(−) were measured as positive (I(Na)) or negative (I(Cl)) short-circuit currents. Haemolymph-side addition of eyestalk extract stimulated I(Cl) by increasing both the transcellular Cl(−) conductance and the electromotive force for Cl(−) absorption. The effect was dose-dependent. Boiling the eyestalk extract did not change its effectiveness. The stimulating factor passed through dialysis tubing, indicating that it has a molecular mass of less than 2 kDa. R(p)cAMPS, a blocker of protein kinase A, reduced the stimulated I(Cl). Eyestalk extract stimulated I(Na) by increasing the transcellular Na(+) conductance at constant electromotive force. Amiloride-induced current-noise analysis revealed that stimulation of I(Na) was accompanied by an increase in the apparent number of open apical Na(+) channels at a slightly reduced single-channel current. In addition to the electrophysiological experiments, whole gills were perfused in the presence and in the absence of putative transport stimulators, and the specific activities of the V-ATPase and the Na(+)/K(+)-ATPase were measured. Eyestalk extract, theophylline or dibutyryl-cyclic AMP stimulated the activity of the V-ATPase, whereas the activity of the Na(+)/K(+)-ATPase was unaffected. The simultaneous presence of R(p)cAMPS prevented the stimulation of V-ATPase by eyestalk extract or theophylline.

CYCLIC AMP STIMULATION OF ELECTROGENIC UPTAKE OF Na+ AND Cl- ACROSS THE GILL EPITHELIUM OF THE CHINESE CRAB ERIOCHEIR SINENSIS

1994 ◽  
Vol 188 (1) ◽  
pp. 159-174 ◽  
Author(s):  
S Riestenpatt ◽  
W Zeiske ◽  
H Onken
Keyword(s):  
Cyclic Amp ◽  
Electromotive Force ◽  
Single Channel ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Eriocheir Sinensis ◽  
Na Channel ◽  
Before And After ◽  
Gill Lamellae ◽  
Stimulation Of

Split gill lamellae (epithelium plus cuticle) of hyperregulating Chinese crabs acclimated to fresh water were mounted in a modified Ussing chamber. Active and electrogenic absorption of sodium and chloride were measured as positive amiloride-sensitive and negative Cl--dependent short-circuit currents (INa, ICl), respectively. Both currents were characterized before and after treatment of the tissue with theophylline or dibutyryl cyclic AMP. Both drugs increased INa and ICl. A simple circuit analysis showed that INa stimulation reflected a marked increase in the transcellular Na+ conductance, whereas the respective electromotive force was unchanged. The Michaelis constant (KNa) for Na+ current saturation was decreased after INa stimulation, indicating an increased affinity of the transport mechanism for its substrate. Consequently, the affinity for the Na+ channel blocker amiloride decreased as expected for a competitive interaction between substrate and inhibitor. Analysis of the amiloride-induced current-noise revealed a marked increase in the number of apical Na+ channels after INa stimulation with theophylline, whereas there was little change in the single-channel current. Stimulation of Cl- absorption was accompanied by a substantial increase in both transcellular conductance and electromotive force, indicating an activation of the apical H+ pump that provides the driving force for active Cl- uptake via apical Cl-/HCO3- exchange and basolateral Cl- channels.

Crystal accumulation and very high short-circuit currents in rabbit urinary bladder

1985 ◽  
Vol 248 (1) ◽  
pp. F70-F77
Author(s):  
D. D. Loo ◽  
J. M. Diamond
Keyword(s):  
Urinary Bladder ◽  
Kidney Stones ◽  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Short Circuit Current ◽  
Channel Current ◽  
Unidentified Component ◽  
Crystal Accumulation ◽  
Very High

We describe a condition in rabbits characterized by CaCO3 crystal accumulation and very high short-circuit current (Isc) in the urinary bladder. The incidence of the condition was high in rabbits kept for 2 mo or more in the vivarium. The crystal mass in the bladder increased with age or time until it occupied up to one-third of the bladder volume and occasionally aggregated as stones, and the urine attained a sludgy consistency. In animals with sludgy urine, the urine excreted had a crystal content 20 times lower than that of urine contained in the bladder at time of death, implying retention of crystals in the bladder. Kidney stones were not detected in rabbits with bladder crystals, and bladder crystals were absent in rabbits with kidney stones induced experimentally by uremia. In old rabbits with sludgy urine the Isc, a measure of Na+ transport, was up to 46 microA/cm2 and averaged 12 microA/cm2, seven times the Isc of rabbits with nonsludgy urine. The increased Isc was entirely amiloride sensitive. Noise analysis showed it to arise entirely from increased channel density, without change in single-channel current. With one possible exception, we could not find bacterial infection or abnormalities in plasma aldosterone, GFR, or urinary [Na+], [Ca2+], pH, or osmolality that could explain the condition. The exception is that some unidentified component accounting for half the osmolality of nonsludgy urine is absent or at low concentration in sludgy urine. It remains unknown why the condition develops and whether CaCO3 crystals cause high Isc or vice versa or whether both result from a third factor.

scholarly journals Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia

1998 ◽  
Vol 274 (4) ◽  
pp. C947-C957 ◽  
Author(s):  
Sandy I. Helman ◽  
Xuehong Liu ◽  
Kieron Baldwin ◽  
Bonnie L. Blazer-Yost ◽  
Willem J. Els
Keyword(s):  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Early Time ◽  
Fold Increase ◽  
Time Dependent ◽  
Open Probability ◽  
Modified Method ◽  
A6 Epithelia ◽  
Stimulation Of

To study and define the early time-dependent response (≤6 h) of blocker-sensitive epithelial Na+channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current ( i Na) channel open probability ( P o), and channel density ( N T) under transient conditions of transport as measured by macroscopic short-circuit currents ( I sc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 μA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. N T varied linearly over a 70-fold range of transport (0.5–35 μA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of P o occurred during control (10–20% over 2 h) and aldosterone experimental periods (10–30% over 6 h). When the P o of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, P o was observed to vary inversely with I sc, falling from ∼0.5 to ∼0.15 at the highest rates of Na+ transport or ∼25% per 3-fold increase of transport. Because decreases of P o from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane N T.

Current-noise analysis of Na absorption in the embryonic coprodeum: stimulation by aldosterone and thyroxine

1993 ◽  
Vol 265 (5) ◽  
pp. R1100-R1108 ◽  
Author(s):  
W. Clauss ◽  
B. Hoffmann ◽  
R. Krattenmacher ◽  
W. Van Driessche
Keyword(s):  
Sodium Transport ◽  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Current Noise ◽  
Na Channel ◽  
Channel Density ◽  
Channel Current ◽  
Single Channel Current

The mechanism and regulation of sodium transport in the embryonic coprodeum of chicken were investigated with isolated epithelia in vitro by electrophysiological techniques. Electrogenic sodium transport (INa) was measured in Ussing chambers by the short-circuit current (Isc) technique and identified by the diuretic amiloride or by removal of sodium from the apical medium. Apical sodium channels and the kinetics of amiloride binding were investigated by current-noise analysis. Isc and INa were measured under control conditions and under the influence of in vitro incubation with aldosterone and thyroxine. At 20 days the embryonic coprodeum has an Isc of 12.6 +/- 1.4 microA/cm2 and a transepithelial resistance of 519 +/- 40 omega.cm2. Amiloride blocks 9.0 +/- 1.3 microA/cm2 of the Isc, which represents electrogenic Na+ absorption and can be inhibited by serosal ouabain. Aldosterone does not stimulate Isc or INa, whereas thyroxine increases Isc and INa about threefold. Aldosterone in combination with thyroxine increases Isc and INa further to about five- to sixfold. In both cases the hormonal stimulation can be totally blocked by spironolactone. Current-noise analysis of the apical Na+ entry step reveals amiloride-sensitive Na+ channels with a single-channel current of approximately 2.3 pA and a channel density of 9-16 million/cm2 under stimulated conditions. Half-maximal amiloride block occurs at 0.8-1 microM. The hormones stimulate Na+ absorption by increasing the Na+ channel density and not the single-channel current.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Steroid hormone-dependent expression of blockersensitive ENaCs in apical membranes of A6 epithelia

1997 ◽  
Vol 273 (5) ◽  
pp. C1650-C1656 ◽  
Author(s):  
Lynn M. Baxendale-Cox ◽  
Randall L. Duncan ◽  
Xuehong Liu ◽  
Kieron Baldwin ◽  
Willem J. Els ◽  
...  
Keyword(s):  
Growth Medium ◽  
Single Channel ◽  
Apical Membrane ◽  
Short Circuit ◽  
Noise Analysis ◽  
Short Circuit Current ◽  
Growth Media ◽  
Open Probability ◽  
A6 Epithelia ◽  
Single Channel Currents

Weak channel blocker-induced noise analysis was used to determine the way in which the steroids aldosterone and corticosterone stimulated apical membrane Na+ entry into the cells of tissue-cultured A6 epithelia. Among groups of tissues grown on a variety of substrates, in a variety of growth media, and with cells at passages 73–112, the steroids stimulated both amiloride-sensitive and amiloride-insensitive Na+ transport as measured by short-circuit currents in chambers perfused with either growth medium or a Ringer solution. From baseline rates of blocker-sensitive short-circuit current between 2 and 7 μA/cm2, transport was stimulated about threefold in all groups of experiments. Single channel currents averaged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and were decreased 6–20% from controls by steroid due to the expected decreases of fractional transcellular resistance. Irrespective of baseline transport rates, the steroids in all groups of tissues stimulated transport by increase of the density of blocker-sensitive epithelial Na+ channels (ENaCs). Channel open probability was the same in control and stimulated tissues, averaging ∼0.3 in all groups of tissues. Accordingly, steroid-mediated increases of open channel density responsible for stimulation of Na+ transport are due to increases of the apical membrane pool of functional channels and not their open probability.

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.

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).

Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine

2002 ◽  
Vol 205 (23) ◽  
pp. 3729-3737 ◽  
Author(s):  
Fatima Atia ◽  
Irina Mountian ◽  
Jeannine Simaels ◽  
Etienne Waelkens ◽  
Willy Van Driessche
Keyword(s):  
Liquid Chromatography ◽  
System Analysis ◽  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Gel Filtration ◽  
Maximal Effect ◽  
Na Transport ◽  
Open Probability ◽  
Stimulatory Action

SUMMARY Effects of the extract of Nigella arvensis (NA) seeds on transepithelial Na+ transport were studied in cultured A6 toad kidney cells by recording short-circuit current (Isc),transepithelial conductance (GT), transepithelial capacitance (CT) and fluctuation in Isc. Apical application of NA extract had merely a small stimulatory effect on Na+ transport, whereas basolateral administration markedly increased Isc, GT and CT. A maximal effect was obtained at 500 μl l-1 of lyophilized NA extract. The increase in CT suggests that the activation of Isc occurs through the insertion of transport sites in the apical membrane. In experiments performed in the absence of Na+transport [apical Na+ was replaced by N-methyl-D-glucamine(NMDG+)], basolateral NA extract did not affect Isc and GT, indicating that Cl- conductance was not influenced. Noise analysis of Isc using 6-chloro-3,5-diaminopyrazine-2-carboxamide(CDPC) showed that NA extract reduced single-channel current(iNa) and decreased channel open probability(Po) but evoked a threefold increase in channel density(NT), which confirms the insertion of Na+channels. The separation of the compounds in the crude extract of NAwas performed by fast protein liquid chromatography (FPLC) on a Superdex 200 gel-filtration column and by reverse-phase high-pressure liquid chromatography(RPHPLC) on an μRPC C2/C18 SC2.1/10 column connected to a SMART system. Analysis of the purified active fraction by mass spectrometry demonstrated the presence of adenosine as the single organic compound in the extract that had a stimulatory effect on Na+ transport. In a separate series of experiments, we confirmed that 1 μmol l-1 adenosine had similar effects on the parameters of Na+ transport as did the NAextract. The action of adenosine was further identified by experiments in which NA extract was added after adenosine. In these experiments, NA extract did not affect Isc, GT or CT. These results clearly demonstrate an essential role of adenosine in the stimulatory action of NA extract.

Active and electrogenic absorption of Na + and Cl- across posterior gills of Eriocheir sinensis: influence of short-term osmotic variations

1996 ◽  
Vol 199 (4) ◽  
pp. 901-910 ◽  
Author(s):  
H Onken
Keyword(s):  
Electromotive Force ◽  
Marked Decrease ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Eriocheir Sinensis ◽  
Induced Current ◽  
Na Current ◽  
Short Term ◽  
External Barrier ◽  
Short Circuit Currents

Split lamellae of posterior gills of Chinese crabs (Eriocheir sinensis) acclimated to fresh water were mounted in a modified Ussing-type chamber, and the transepithelial short-circuit current and conductance were measured. The epithelium shows independent active and electrogenic absorption mechanisms for Na+ and Cl- that can be measured as positive and negative short-circuit currents, respectively, in the absence of the counter ion. Increasing the osmolarity of the haemolymph-side saline by addition of sucrose resulted in a marked decrease in active uptake of both Na+ and Cl-. In contrast, increasing the internal osmolarity by addition of urea or moderately decreasing the haemolymph-side osmolarity resulted in a marked increase in Na+ as well as Cl- transport. Circuit analysis revealed that Na+ current changes are mostly due to alterations in the apical amiloride-sensitive Na+ conductance, while Cl- current changes are caused not only by alterations in the transcellular conductance but also by changes in the electromotive force for Cl- absorption. Osmotic perturbations in the external bath induced current changes in the same directions, but the magnitudes of the effects were smaller than those after internal osmotic variations, indicating that the external barrier has a lower water permeability than the internal barrier. Short-term osmotic perturbations did not significantly affect the leak conductance, which is not associated with active transport and which may mostly reflect the paracellular conductance.

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