Nystatin studies of the skin of larval Rana catesbeiana

1983 ◽  
Vol 244 (1) ◽  
pp. R58-R65
Author(s):  
T. C. Cox ◽  
R. H. Alvarado
Keyword(s):  
Rana Catesbeiana ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Electrical Characteristics ◽  
Shunt Resistance ◽  
Selective Permeability ◽  
Ion Substitution

Transport and electrical characteristics of the isolated skin of larval Rana catesbeiana were analyzed using ion substitution and nystatin. When the inner (IBS) and outer (OBS) bathing solutions contained Na Ringer solution the electrical potential (TEP), short-circuit current (SCC), and resistance (R2) were 23.5 +/- 7.0 mV, 2.8 +/- 0.7 microA . cm-2, and 8.00 +/- 0.74 k omega. cm2, respectively (n = 4). When K was substituted for Na in the OBS these values were not changed significantly. When nystatin (120 U.cm-3), a drug that increases the permeability of membranes to small cations, was added to the OBS (Na Ringer) there was a striking increase in the TEP to 52.8 +/- 3.1 mV, SCC to 14.8 +/- 2.0 microA . cm-2, and drop in R2 to 3.75 +/- 0.52 k omega . cm2. The response to nystatin was similar with Na or K Ringer solution in the OBS (Na Ringer in the IBS). With Na Ringer in the OBS and IBS, the increase in SCC induced by low doses of nystatin equaled net Na flux measured isotopically. Plots of transepithelial conductance against SCC after nystatin were linear and provided estimates of shunt resistance (R*sh = 14.6 +/- 1.3 k omega . cm2) and electromotive driving force for ions (E*A = 76 +/- 3 mV). Similar curves were obtained with K Ringer in the OBS. In the presence of nystatin, characteristics of the basolateral membrane were evaluated. It displayed selective permeability to K relative to Na or Tris.

Electrical and transport characteristics of skin of larval Rana catesbeiana

1979 ◽  
Vol 237 (1) ◽  
pp. R74-R79 ◽  
Author(s):  
T. C. Cox ◽  
R. H. Alvarado
Keyword(s):  
Rana Catesbeiana ◽  
Short Circuit ◽  
Electrical Potential ◽  
Flux Ratio ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Open Circuit ◽  
Shunt Resistance ◽  
Ion Substitution ◽  
Adult Skin

Carefully dissected, mounted, and bathed with Ringer solution, the larval bullfrog skin has a resistance of about 9,000 omega.cm2 and a stable transepithelial electrical potential of about 20 mV (inside +). A short-circuit current of about 2 microA.cm-2 is generated that is comparable in magnitude to the net inward flux of Na+. At open circuit the flux ratio equation for Na+ is not satisfied. Larval skin is less sensitive to ouabain, amiloride, and ADH than adult skin. The current-voltage (C-V) relationship across the preparation is not linear; there are distinct breaks in both the hyperpolarizing and hypopolarizing regions. The former break, at about +130 mV, corresponds with a break observed in adult skin that corresponds with ENa. The shunt resistance (RS) and active pathway resistance (RA) were estimated by C-V curve analysis and by ion substitution. The two methods yielded comparable values with RS about 11 k omega.cm2 and RA about 62 k omega.cm2. It is suggested that transport is limited by the number of entry sites for sodium at the apical border of transport cells.

Active electrolyte transport in mammalian buccal mucosa

1988 ◽  
Vol 255 (3) ◽  
pp. G286-G291 ◽  
Author(s):  
R. C. Orlando ◽  
N. A. Tobey ◽  
V. J. Schreiner ◽  
R. D. Readling
Keyword(s):  
Buccal Mucosa ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Electrolyte Transport ◽  
Electrical Potential Difference ◽  
Ussing Chambers ◽  
Net Fluxes

The transmural electrical potential difference (PD) was measured in vivo across the buccal mucosa of humans and experimental animals. Mean PD was -31 +/- 2 mV in humans, -34 +/- 2 mV in dogs, -39 +/- 2 mV in rabbits, and -18 +/- 1 mV in hamsters. The mechanisms responsible for this PD were explored in Ussing chambers using dog buccal mucosa. After equilibration, mean PD was -16 +/- 2 mV, short-circuit current (Isc) was 15 +/- 1 microA/cm2, and resistance was 1,090 +/- 100 omega.cm2, the latter indicating an electrically "tight" tissue. Fluxes of [14C]mannitol, a marker of paracellular permeability, varied directly with tissue conductance. The net fluxes of 22Na and 36Cl were +0.21 +/- 0.05 and -0.04 +/- 0.02 mueq/h.cm2, respectively, but only the Na+ flux differed significantly from zero. Isc was reduced by luminal amiloride, serosal ouabain, or by reducing luminal Na+ below 20 mM. This indicated that the Isc was determined primarily by active Na+ absorption and that Na+ traverses the apical membrane at least partly through amiloride-sensitive channels and exits across the basolateral membrane through Na+-K+-ATPase activity. We conclude that buccal mucosa is capable of active electrolyte transport and that this capacity contributes to generation of the buccal PD in vivo.

Further Observations on the Physiology of Salinity Adaptation in the Crab-Eating Frog (Rana Cancrivora)

1968 ◽  
Vol 49 (1) ◽  
pp. 185-193
Author(s):  
MALCOLM S. GORDON ◽  
VANCE A. TUCKER
Keyword(s):  
Sea Water ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Osmotic Concentration ◽  
Green Toad ◽  
The Face ◽  
Solution Bathing ◽  
Toad Bufo

1. Total rates of urea loss from adult euryhaline crab-eating frogs (Rana cancrivora) adapted to various environmental salinities between fresh water and 80 % sea water increase as salinity increases above 40% sea water. Oxygen consumption is constant in rate in all salinities studied. 2. The presence of urea in the Ringer solution bathing isolated pieces of skin of frogs adapted to 60% sea water increases both the electrical potential and the inwardly directed short-circuit current across the skin. 3. In skeletal muscle cells addition of intracellular solutes maintains tissue hydration in the face of large increases in plasma osmotic concentration in high-salinity media. Changes in the intracellular urea and free amino acid concentrations are primarily responsible for increases in intracellular osmotic concentration. 4. Some implications of these observations are discussed and comparisons made with the euryhaline green toad, Bufo viridis.

Mechanisms of sodium and chloride transport across equine tracheal epithelium

1990 ◽  
Vol 259 (6) ◽  
pp. L459-L467 ◽  
Author(s):  
G. J. Tessier ◽  
T. R. Traynor ◽  
M. S. Kannan ◽  
S. M. O3'Grady
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Tracheal Epithelium ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Cotransport System ◽  
Ethanesulfonic Acid

Equine tracheal epithelium, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasmalike Ringer solution generates a serosa-positive transepithelial potential of 10–22 mV and a short-circuit current (Isc) of 70–200 microA/cm2. Mucosal amiloride (10 microM) causes a 40–60% decrease in Isc and inhibits the net transepithelial Na flux by 95%. Substitution of Cl with gluconate resulted in a 30% decrease in basal Isc. Bicarbonate substitution with 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid decreased the Isc by 21%. The Cl-dependent Isc was inhibited by serosal addition of 1 mM amiloride. Bicarbonate replacement or serosal amiloride (1 mM) inhibits the net Cl flux by 72 and 69%, respectively. Bicarbonate replacement significantly reduces the effects of serosal amiloride (1 mM) on Isc, indicating its effect is HCO3 dependent. Addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 100 microM) causes a 40% increase in Isc. This effect is inhibited by subsequent addition of 10 microM serosal bumetanide. Bumetanide (10 microM) reduces net Cl secretion following stimulation with 8-BrcAMP (100 microM). Serosal addition of BaCl2 (1 mM) causes a reduction in Isc equal to that following Cl replacement in the presence or absence of 100 microM cAMP. These results suggest that 1) Na absorption depends on amiloride-inhibitable Na channels in the apical membrane, 2) Cl influx across the basolateral membrane occurs by both a Na-H/Cl-HCO3 parallel exchange mechanism under basal conditions and by a bumetanide-sensitive Na-(K?)-Cl cotransport system under cAMP-stimulated conditions, and 3) basal and cAMP-stimulated Cl secretion depends on Ba-sensitive K channels in the basolateral membrane.

Barium inhibition of basolateral membrane potassium conductance in tracheal epithelium

1983 ◽  
Vol 244 (6) ◽  
pp. F639-F645 ◽  
Author(s):  
M. J. Welsh
Keyword(s):  
Driving Force ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Potassium Conductance ◽  
Short Circuit Current ◽  
Electrical Circuit ◽  
Tracheal Epithelium ◽  
Bathing Solution ◽  
K Permeability

Addition of barium ion, Ba2+, to the submucosal bathing solution of canine tracheal epithelium reversibly decreased the short-circuit current and increased transepithelial resistance. The decrease in short-circuit current represented a decrease in the net rate of Cl secretion with no change in the rate of Na absorption. Intracellular microelectrode techniques and an equivalent electrical circuit analysis were used to localize the effect of Ba2+ to an inhibition of the permeability of the basolateral membrane to K. Ba2+ (2 mM) doubled basolateral membrane resistance, decreased the equivalent electromotive force at the basolateral membrane, and decreased the magnitude of the depolarization of basolateral membrane voltage produced by increasing the submucosal K concentration. The inhibition of the basolateral K permeability depolarized the negative intracellular voltage, resulting in both a decrease in the driving force for Cl exit and an estimated increase in intracellular Cl concentration. These studies indicate that there is a Ba2+-inhibitable K conductance at the basolateral membrane of tracheal epithelial cells and that the K permeability plays an important role in the generation of the negative intracellular electrical potential that provides the driving force for Cl exit from the cell.

Acidification stimulates chloride and fluid absorption across frog retinal pigment epithelium

1994 ◽  
Vol 266 (4) ◽  
pp. C946-C956 ◽  
Author(s):  
J. L. Edelman ◽  
H. Lin ◽  
S. S. Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Short Circuit ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Open Circuit ◽  
Disulfonic Acid ◽  
Fluid Absorption

Radioactive tracers and a modified capacitance-probe technique were used to characterize the mechanisms that mediate Cl and fluid absorption across the bullfrog retinal pigment epithelium (RPE)-choroid. In control (HCO3/CO2) Ringer solution, 36Cl was actively absorbed (retina to choroid) at a mean rate of 0.34 mu eq.cm-2.h-1 (n = 34) and accounted for approximately 25% of the short-circuit current. Apical bumetanide (100 microM) or basal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 1 mM) inhibited active Cl transport by 70 and 62%, respectively. Active Cl absorption was doubled, either by removing HCO3 from the bathing media or by elevating CO2 from 5 to 13%, and the increased flux was inhibited by apical bumetanide or basal DIDS. Open-circuit measurements of fluid absorption rate (Jv) and the net fluxes of 36Cl, 22Na, and 86Rb (K substitute) indicated that CO2-induced acidification stimulated NaCl and fluid absorption across the RPE. During acidification, bumetanide produced a twofold larger inhibition of Jv compared with control. Stimulation of net Cl absorption was most likely caused by inhibition of the the basolateral membrane intracellular pH-dependent Cl-HCO3 exchanger.

scholarly journals Na+ and K+ transport at basolateral membranes of epithelial cells. I. Stoichiometry of the Na,K-ATPase.

1986 ◽  
Vol 87 (3) ◽  
pp. 467-483 ◽  
Author(s):  
T C Cox ◽  
S I Helman
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Na Transport ◽  
Basolateral Membranes ◽  
Dependent Component ◽  
Epithelial Sheets ◽  
Apical Membranes ◽  
K Transport

The stoichiometry of pump-mediated Na/K exchange was studied in isolated epithelial sheets of frog skin. 42K influx across basolateral membranes was measured with tissues in a steady state and incubated in either beakers or in chambers. The short-circuit current provided estimates of Na+ influx at the apical membranes of the cells. 42K influx of tissues bathed in Cl- or SO4-Ringer solution averaged approximately 8 microA/cm2. Ouabain inhibited 94% of the 42K influx. Furosemide was without effect on pre-ouabain-treated tissues but inhibited a ouabain-induced and Cl--dependent component of 42K influx. After taking into account the contribution of the Na+ load to the pump by way of basolateral membrane recycling of Na+, the stoichiometry was found to increase from approximately 2 to 6 as the pump-mediated Na+ transport rate increased from 10 to 70 microA/cm2. Extrapolation of the data to low rates of Na+ transport (less than 10 microA/cm2) indicated that the stoichiometry would be in the vicinity of 3:2. As pump-mediated K+ influx saturates with increasing rates of Na+ transport, Na+ efflux cannot be obligatorily coupled to K+ influx at all rates of transepithelial Na+ transport. These results are similar to those of Mullins and Brinley (1969. Journal of General Physiology. 53:504-740) in studies of the squid axon.

scholarly journals Amiloride analog stimulation of short-circuit current in larval frog skin epithelium.

1997 ◽  
Vol 200 (23) ◽  
pp. 3055-3065
Author(s):  
T Cox
Keyword(s):  
Rana Catesbeiana ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Similar Time ◽  
Peak Response ◽  
Ussing Chambers ◽  
Apical Side ◽  
Basolateral Side

The skin of the bullfrog Rana catesbeiana tadpole contains an apical non-selective cation channel that is activated by amiloride. This is in contrast to the adult skin, which has a highly Na+-selective channel that is blocked by amiloride. The purpose of the present study was to characterize further the nature of the tadpole channel using amiloride and its analogs benzamil, dimethyl amiloride (DMA), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and methyl isobutyl amiloride (MIBA). Tadpole skins were mounted in modified Ussing chambers with Ca2+-free KCl or NaCl Ringer on the apical side and standard NaCl Ringer (containing 2 mmol l-1 Ca2+) on the basolateral side. Drugs were added to the apical solution at concentrations between 0.1 and 1000 micromol l-1. Amiloride caused the short-circuit current (Isc) to increase rapidly from near zero to a peak of approximately 30-50 microA and then to decline back towards zero over several seconds. The peak response was largest at 100 micromol l-1. The rate of decline was noticeably faster at the higher concentrations. Benzamil and DMA had similar time courses to amiloride, but with smaller effects on Isc. The largest peak responses occurred at 5-50 micromol l-1. EIPA and MIBA gave small responses at 1-10 micromol l-1 and, at higher concentrations (50-500 micromol l-1), the responses consisted of rapid, small increases in Isc followed by rapid decreases. The largest peak response occurred at 10 micromol l-1 for both drugs. After apical membrane resistance had been reduced by nystatin, addition of analogs to the apical solution caused no change in Isc or transepithelial resistance. This suggests that the decline in Isc after amiloride analog treatment was not due to increases in the resistance of the basolateral membrane. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) blocked stimulation by all of the analogs. These data are consistent with amiloride analogs acting as both activators and inhibitors of short-circuit current in tadpole skin and extend the list of ligands that activate these channels.

Net basolateral potassium flux and short-circuit current in ouabain-treated frog skin

1990 ◽  
Vol 259 (5) ◽  
pp. R936-R942
Author(s):  
T. C. Cox ◽  
R. E. Woods
Keyword(s):  
Frog Skin ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Tight Coupling ◽  
Transport Pathway ◽  
Apical Side ◽  
Treatment Changes ◽  
A New Technique

A new technique has been developed to correlate K loss from cells (JK) across the basolateral membrane into a K-free ouabain Ringer solution and short-circuit current (Isc) for a model Na-transporting epithelium, the frog skin. Distinct differences were observed when the tissue was bathed in sulfate or chloride Ringer. In sulfate Ringer, K-free ouabain treatment caused both JK and Isc to decline in a nearly parallel fashion with time. JK-Isc was approximately 1 microA/cm2. In sulfate Ringer, isoproterenol caused parallel increases, whereas amiloride (apical side) caused parallel decreases in JK and Isc. In chloride Ringer, K-free ouabain treatment caused Isc to decline at a slightly faster rate than JK.JK-Isc was approximately 8 microA/cm2. Bumetanide decreased JK with very little effect on Isc. Barium caused small parallel changes in both Isc and JK. Amiloride decreased Isc with very little effect on JK. These experiments show that after ouabain treatment changes in JK from the cells across the basolateral membrane can largely account for changes in Isc. However, JK also occurs via neutral mechanisms and perhaps from cells not related to the transport pathway, demonstrating that there is not always a tight coupling of K loss at the basolateral membrane with Na entry across the apical membrane.

Sodium and chloride transport across the isolated porcine gallbladder

1989 ◽  
Vol 257 (1) ◽  
pp. C45-C51 ◽  
Author(s):  
S. M. O'Grady ◽  
P. J. Wolters
Keyword(s):  
Apical Membrane ◽  
Chloride Transport ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Disulfonic Acid ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Conductive Pathway

Porcine gallbladder, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasma-like Ringer solution generates a serosal positive transepithelial potential of 4-7 mV and a short-circuit current (Isc) of 50-120 microA/cm2. Substitution of Cl with gluconate or HCO3 with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) results in a 50% decrease in Isc. Treatment with 1 mM amiloride (mucosal side) or 0.1 mM acetazolamide (both sides) causes 25-27% inhibition of the Isc. Mucosal addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits the Isc by 17%. Serosal addition of 0.1 mM bumetanide inhibits the Isc by 28%. Amiloride (1 mM) inhibits the net transepithelial fluxes of Na and Cl by 55 and 41%, respectively. Substitution of Cl with gluconate inhibits the net Na flux by 50%, whereas substitution of HCO3 with HEPES inhibits 85-90% of the net Na flux and changes Cl absorption to net secretion. Based on these results, it is hypothesized that Na and Cl transport across the apical membrane is mediated by two pathways, Na-H/Cl-HCO3 exchange and Na-HCO3 cotransport. Partial recycling of Cl and HCO3 presumably occurs through a Cl conductive pathway and Cl-HCO3 exchange, respectively, in the apical membrane. This results in net Na absorption, which accounts for most of the Isc observed under basal conditions. The effect of bumetanide on the basolateral membrane and the fact that Cl secretion occurs when HCO3 is absent suggests that Cl secretion involves a basolateral NaCl or Na-K-Cl cotransport system arranged in series with a Cl conductive pathway in the apical membrane.

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