Transepithelial transport kinetics and Na entry in frog skin: effects of novobiocin

1976 ◽  
Vol 231 (6) ◽  
pp. 1866-1874 ◽  
Author(s):  
LJ Cruz ◽  
TU Biber
Keyword(s):  
Kinetic Parameters ◽  
Frog Skin ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Close Correlation ◽  
Transport Kinetics ◽  
Transepithelial Transport ◽  
Linear Component ◽  
Na Transport ◽  
Transepithelial Na Transport

Na+ entry across the outer surface of frog skin and transepithelial Na transport were studied simultaneously at different [Na] in either the presence or absence of novobiocin by direct measurements of J12 (unidirectional uptake) and Io (short-circuit current). J12 consisted of two components: one linear, the other saturable. The kinetic parameters of the saturating components in controls were close to the kinetic parameters of overall transepithelial transport (Jm12 = 1.68+/-0.13 mleq cm-2h-1; Io =1.80+/-0.14 mueq cm-2h-1. K12 = 6.02+/-1.27 mM;Kio=6.12+/-1.33 mM). Novobiocin significantly augmented net transepithelial Na transport by increasing J13. J31 remained unaffected. A 1:1 relationship between the saturating component of J12 and Io was observed in both treated and untreated skins at all [Na] tested. (Jm12Iom, k12, and Kio were significantly larger in treated skins, but despite very drastic changes in transport rates, a close correlation between kinetic parameters of entry step and transepithelial transport was maintained. This suggests that the kinetics of transepithelial transport may simply reflect those of the rate-limiting step: the Na entry across the outer barrier of the skin. The results indicate that the linear component of J12 is not involved in transepithelial transport kinetics.

scholarly journals Effect of Changes in Transepithelial Transport on the Uptake of Sodium across the Outer Surface of the Frog Skin

1971 ◽  
Vol 58 (2) ◽  
pp. 131-144 ◽  
Author(s):  
Thomas U. L. Biber
Keyword(s):  
Outer Surface ◽  
Frog Skin ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Sodium Concentration ◽  
Transepithelial Transport ◽  
Linear Component ◽  
Sodium Uptake

The unidirectional sodium, uptake at the outer surface of the frog skin was measured by the method described by Biber and Curran (8). With bathing solutions containing 6 mM NaCl there is a good correlation between sodium uptake and short-circuit current (SCC) measured simultaneously except that the average uptake is about 40% higher than the average SCC. The discrepancy between uptake and SCC increases approximately in proportion to an increase in sodium concentration of the bathing solutions. Amiloride inhibits the unidirectional sodium uptake by 21 and 69% at a sodium concentration of 115 and 6 mM, respectively. This indicates that amiloride acts on the entry step of sodium but additional effects cannot be excluded. The sodium, uptake is not affected by 10-4 M ouabain at a sodium concentration of 115 mM but is inhibited by 40% at a sodium concentration of 6 mM. Replacement of air by nitrogen leads to a 40% decrease of sodium uptake at a sodium concentration of 6 mM. The results support the view proposed previously (8) that the sodium uptake is made up of two components, a linear component which is, essentially, not involved in transepithelial movement of sodium and a saturating component which reflects changes in transepithelial transport. Amiloride, seems largely to affect the saturating component.

scholarly journals Ouabain on active transepithelial sodium transport in frog skin: studies with microelectrodes.

1979 ◽  
Vol 74 (1) ◽  
pp. 105-127 ◽  
Author(s):  
S I Helman ◽  
W Nagel ◽  
R S Fisher
Keyword(s):  
Frog Skin ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Equilibrium Potential ◽  
Electrical Model ◽  
Na Transport ◽  
Rapid Phase ◽  
Transepithelial Na Transport ◽  
Transepithelial Voltage ◽  
Transepithelial Sodium Transport

Studies were done with isolated frog skin to determine the effects of 10(-4) M ouabain on the electrophysiological parameters of outer and inner barriers of the Na-transporting cells. Microelectrodes were used to impale the skins from the outer surface to determine the intracellular voltages (Vsco) under conditions of short-circuiting and under conditions where a voltage clamp was used to vary the transepithelial voltage, VT. From this, the electrical resistances of outer (Rfo) and inner (RI) barriers were estimated. In addition, the driving force for active transepithelial Na transport (ENa = E'1) was estimated from the values of VT when the Vo = 0 mV (Helman and Fisher. 1977. J. Gen. Physiol. 69: 571-604). Studies were done with skins bathed with the usual 2.4 meq/liter [K]i in the inner solution as well as with reduced [K]i of 0.5 and 0 meq/liter. Characteristically, the responses to ouabain could be described by an initial rapid phase (5-10 min) during which time the Ri was increased markedly and the E'1 was decreased from control values. Thereafter, during the slow phases of the response, the resistances of both outer and inner barriers increased continuously and markedly with time leading ultimately to essentially complete inhibition of the short-circuit current. Similar studies were done with skins exposed to 10(-4) M amiloride in the outer solution. Although estimates of Ri could not be obtained under these conditions, the effects on the Vsco and E'1 were similar to those observed for the Na-transporting skins. However, the magnitudes of the effects were less and relatively slower than observed for the Na-transporting skins. The results of these studies were analyzed within the context of a proposed electrical model that takes into account the observation that the magnitude of the voltage at the inner barrier appears to exceed the equilibrium potential for K especially when transepithelial Na transport is inhibited at the apical barrier of the cells.

scholarly journals Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder.

1987 ◽  
Vol 89 (4) ◽  
pp. 563-580 ◽  
Author(s):  
J R Demarest ◽  
A L Finn
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
K Channel ◽  
Channel Blockers ◽  
Na Transport ◽  
Transepithelial Na Transport ◽  
Pump Activity ◽  
Relationship Of

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.

Na+ transport and pH in principal cells of frog skin: effect of antidiuretic hormone

1994 ◽  
Vol 267 (1) ◽  
pp. R107-R114
Author(s):  
V. Lyall ◽  
T. S. Belcher ◽  
J. H. Miller ◽  
T. U. Biber
Keyword(s):  
Antidiuretic Hormone ◽  
Frog Skin ◽  
Apical Membrane ◽  
Short Circuit ◽  
Apical Cell ◽  
Short Circuit Current ◽  
Arginine Vasotocin ◽  
Na Transport ◽  
Apical Cell Membrane ◽  
Principal Cells

Intracellular pH (pHi), apical membrane potential (Va), and fractional apical membrane resistance (FRa) were measured in principal cells of isolated frog skin (Rana pipiens) with double-barreled microelectrodes under short-circuit conditions. Basolateral exposure to 10 mU/ml arginine vasotocin (AVT) depolarized Va by 30 mV, decreased FRa by 33%, increased short-circuit current (Isc) by 17 microA, and increased pHi by 0.17 pH units. The response of Va, Isc, and pHi occurred concurrently. Forskolin, theophylline, and 8-(4-chlorophenyl-thio)-adenosine 3',5'-cyclic monophosphate caused similar changes in Va, Isc, and pHi. The enhanced response of Isc, Va, and FRa to short pulses of apical amiloride applied during AVT or cAMP exposure suggests an increase in apical Na+ conductance. The presence of cAMP agonists also enhanced the response of pHi to amiloride. We conclude that the AVT- and cAMP-induced increase in Na+ transport across the apical cell membrane is associated with a change in pHi. These data are consistent with the hypothesis that changes in pHi may play a role in the second messenger cascade initiated by the antidiuretic hormone.

Ca-sensitive Na transport in sheep omasum

1999 ◽  
Vol 276 (6) ◽  
pp. G1331-G1344 ◽  
Author(s):  
Gerhard Schultheiss ◽  
Holger Martens
Keyword(s):  
Divalent Cations ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Voltage Divider ◽  
Close Correlation ◽  
Na Transport ◽  
Luminal Side ◽  
Flux Measurements ◽  
Voltage Divider Ratio ◽  
Peak Value

Na transport across a preparation of sheep omasum was studied. All tissues exhibited a serosa-positive short-circuit current ( I sc), with a range of 1–4 μeq ⋅ h−1 ⋅ cm−2. A Michaelis-Menten-type kinetic was found between the Na concentration and the I sc(Michaelis-Menten constant for transport of Na = 6.7 mM; maximal transport capacity of Na = 4.16 μeq ⋅ h−1 ⋅ cm−2). Mucosal amiloride (1 mM), phenamil (1 or 10 μ), or serosal aldosterone (1 μM for 6 h) did not change I sc. Removal of divalent cations (Ca and Mg) enhanced I sc considerably from 2.61 ± 0.24 to a peak value of 11.18 ± 1.1 μeq ⋅ h−1 ⋅ cm−2. The peak I sc(overshoot) immediately declined to a plateau I sc of ∼6–7 μeq ⋅ h−1 ⋅ cm−2. Na flux measurements showed a close correlation between changes in I sc and Na transport. Transepithelial studies demonstrated that K, Cs, Rb, and Li are transported, indicating putative nonselective cation channels, which are inhibited by divalent cations (including Ca, Mg, Sr, Ba) and by (trivalent) La. Intracellular microelectrode recordings from the luminal side clearly showed changes of voltage divider ratio when mucosal divalent cations were removed. The obtained data support the assumption of a distinct electrogenic Na transport mechanism in sheep omasum.

scholarly journals Na+ and K+ transport at basolateral membranes of epithelial cells. II. K+ efflux and stoichiometry of the Na,K-ATPase.

1986 ◽  
Vol 87 (3) ◽  
pp. 485-502 ◽  
Author(s):  
T C Cox ◽  
S I Helman
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Pump Current ◽  
Ringer Solution ◽  
K Channel ◽  
Na Transport ◽  
Transepithelial Na Transport ◽  
Dependent Component ◽  
Na Efflux ◽  
K Transport

Changes of 42K efflux (J23K) caused by ouabain and/or furosemide were measured in isolated epithelia of frog skin. From the kinetics of 42K influx (J32K) studied first over 8-9 h, K+ appeared to be distributed into readily and poorly exchangeable cellular pools of K+. The readily exchangeable pool of K+ was increased by amiloride and decreased by ouabain and/or K+-free extracellular Ringer solution. 42K efflux studies were carried out with tissues shortcircuited in chambers. Ouabain caused an immediate (less than 1 min) increase of the 42K efflux to approximately 174% of control in tissues incubated either in SO4-Ringer solution or in Cl-Ringer solution containing furosemide. Whereas furosemide had no effect on J23K in control tissues bathed in Cl-rich or Cl-free solutions, ouabain induced a furosemide-inhibitable and time-dependent increase of a neutral Cl-dependent component of the J23K. Electroconductive K+ transport occurred via a single-filing K+ channel with an n' of 2.9 K+ efflux before ouabain, normalized to post-ouabain (+/- furosemide) values of short-circuit current, averaged 8-10 microA/cm2. In agreement with the conclusions of the preceding article, the macroscopic stoichiometry of ouabain-inhibitable Na+/K+ exchange by the pump was variable, ranging between 1.7 and 7.2. With increasing rates of transepithelial Na+ transport, pump-mediated K+ influx saturated, whereas Na+ efflux continued to increase with increases of pump current. In the usual range of transepithelial Na+ transport, regulation of Na+ transport occurs via changes of pump-mediated Na+ efflux, with no obligatory coupling to pump-mediated K+ influx.

scholarly journals Na Transport across Frog Skin at Low External Na Concentrations

1966 ◽  
Vol 49 (6) ◽  
pp. 1161-1176 ◽  
Author(s):  
THOMAS U. L. BIBER ◽  
RONALD A. CHEZ ◽  
PETER F. CURRAN
Keyword(s):  
Frog Skin ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Open Circuit ◽  
Potential Profile ◽  
Na Transport ◽  
Ringer’S Solution ◽  
Electrical Potentials ◽  
Single Compartment

Isolated frog skin was bathed with a dilute solution containing 1 mm NaCl on the outside and with normal Ringer’s solution on the inner surface. Net Na flux was determined by simultaneous measurement of unidirectional fluxes with Na22 and Na24 and intracellular electrical potentials were examined with microelectrodes. There was a net inward transport of Na under both open-circuit and short-circuit conditions. The short-circuit current was approximately 15% greater than the net Na flux; the discrepancy could be accounted for by a small outward flux of Cl. The electrical potential profile did not differ greatly from that observed in skins bathed on the outside with normal Ringer’s solution. Under open-circuit conditions, there were usually several potential steps and under short-circuit conditions the cells were negative relative to the bathing solutions. Estimates of epithelial Na concentrations utilizing radioactive Na suggested that if all epithelial Na were in a single compartment, an active entry step would be necessary to allow a net inward Na transport. The results could also be explained by a series arrangement of Na compartments without necessarily postulating an active Na entry. The behavior of the potential profile suggested that this latter alternative was more likely.

Complex response of epithelial cells to inhibition of Na+ transport by amiloride

1988 ◽  
Vol 254 (2) ◽  
pp. C297-C303 ◽  
Author(s):  
R. S. Fisher ◽  
J. W. Lockard
Keyword(s):  
Urinary Bladder ◽  
Frog Skin ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Second Phase ◽  
Toad Urinary Bladder ◽  
Apical Surface ◽  
Na Transport ◽  
Transport Inhibition

When toad urinary bladder or frog skin epithelia are treated with amiloride, short-circuit current (Isc), which represents the net active transepithelial Na+ transport rate from the apical to basolateral surface, decreases rapidly (2-5 s) to approximately 15-20% of control values and then slowly, over several minutes, continues falling toward zero. The contribution of this second phase of the decline is dependent on the transporting condition of the tissue before administration of amiloride. Attenuation of the second phase was observed if tissues were subjected to a period of transport inhibition. Tissues preincubated in 0 Na+ Ringer solution on the apical surface were returned to control Na+ Ringer, which caused an approximately 25% increase of Isc above control values. Immediate reapplication of amiloride caused Isc to decrease more rapidly than the previous exposure to values near zero, substantially reducing or eliminating the secondary slow decline. After long-term reincubation of tissues in control, 100 mM Na+ solution, another treatment with amiloride indicated that the magnitude of the secondary decline increased in frog skin but not in urinary bladder epithelia. We conclude that the effect of amiloride is complex and may cause additional effects besides simply blocking entry of Na+ into the apical membrane channel, and we suggest that regulatory mechanisms may be invoked in response to transport inhibition.

Insulin stimulation of Na+ transport and glucose metabolism in cultured kidney cells

1982 ◽  
Vol 242 (1) ◽  
pp. C121-C123 ◽  
Author(s):  
M. L. Fidelman ◽  
J. M. May ◽  
T. U. Biber ◽  
C. O. Watlington
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Metabolic Responses ◽  
Transepithelial Transport ◽  
Kidney Cells ◽  
Insulin Stimulation ◽  
Na Transport ◽  
Basolateral Side ◽  
Permeable Supports ◽  
Stimulation Of

A line of toad kidney cells (A6) in continuous culture was evaluated for ion transport and metabolic responses to insulin. The cells were grown on permeable supports to allow access of the medium to both basolateral and apical sides of the epithelium. Insulin, on the basolateral side only, produced an increase in short-circuit current (Isc) that was maximal at 40-60 min. A concentration-dependent increase in Isc and potential difference (PD) was found in the range of 10-3.2 X 10(3) microunits/ml insulin. The maximal stimulation of Isc and PD was approximately six- and twofold, respectively. After insulin exposure Isc was equivalent to net Na+ transport, indicating active Na+ transport stimulation. Insulin was also found to increase the incorporation of radiolabeled glucose into glycogen. Thus A6 cells exhibit both transepithelial transport and metabolic responses to insulin.

Vasopressin, theophylline, PGE2, and indomethacin on active Na transport in frog skin: studies with microelectrodes

1981 ◽  
Vol 241 (3) ◽  
pp. F279-F288 ◽  
Author(s):  
W. J. Els ◽  
S. I. Helman
Keyword(s):  
Frog Skin ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Membrane Voltage ◽  
Electrical Model ◽  
Na Transport ◽  
Basolateral Membranes ◽  
Transepithelial Voltage

Active transepithelial Na transport in frog skin is influenced by vasopressin, theophylline, indomethacin, and PGE2. During stimulation or inhibition of the short-circuit current, the transapical membrane voltage of short-circuited skins was recorded using an intracellular microelectrode. The microelectrode also permitted determination of the fractional resistance of the apical barrier of the cells (fRo) and the E'1 (transepithelial voltage at which the apical membrane voltage is zero). Analysis of the data according to an electrical model proposed previously indicated that changes of ISC were mediated primarily via changes of the slope resistance Rfo (Vo negative) of the apical barrier of the cells with little or no effect on the Thevenin emf or resistance of the basolateral membranes. These data are in accordance with previous observations that ADH had no effect on the ENa and are discussed in relation to the origin of the ENa at the basolateral membranes of the epithelial cells.

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