Apical and basolateral membrane conductances in the TBM cell line

1991 ◽  
Vol 260 (6) ◽  
pp. C1172-C1181 ◽  
Author(s):  
J. D. Horisberger
Keyword(s):  
Cell Line ◽  
Epithelial Transport ◽  
Apical Membrane ◽  
Ex Vivo ◽  
Biochemical Study ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Cultured Cell ◽  
Membrane Conductances ◽  
Collagen Membranes

Cultured cell lines present several advantages over whole organ or ex vivo isolated epithelium for the physiological and biochemical study of epithelial transport. We have developed a new technique allowing for simultaneous intracellular and transepithelial electrophysiological measurements in the epithelium formed by a cultured cell line grown on thin collagen membranes. This technique was applied to the TBM 18/23 (toad bladder origin) cell line. The transepithelial and basolateral membrane potentials were -30 +/- 11 and -72 +/- 8 (SD) mV (n = 36), respectively. With the use of the effect of amiloride, which partially blocked the apical membrane conductance, and circuit analysis, the apical and basolateral membrane conductances were estimated to 0.7 +/- 0.1 and 2.8 +/- 0.4 mS/cm2, respectively. A sodium-selective conductive pathway was demonstrated in the apical membrane, and a barium-sensitive K(+)-selective conductance was shown to be present in the basolateral membrane. The basolateral membrane conductance was not modified by sudden inhibition of sodium transport by amiloride, but it was significantly reduced after a long-term decrease of Na+ transport. The cultured TBM cell line appears to be a convenient model to investigate the regulation of membrane ionic conductances in tight epithelia.

EGF and PGE2 inhibit rabbit CCD Na+ transport by different mechanisms: PGE2 inhibits Na(+)-K+ pump

1993 ◽  
Vol 264 (4) ◽  
pp. F670-F677 ◽  
Author(s):  
D. H. Warden ◽  
J. B. Stokes
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Membrane Voltage ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Na Transport ◽  
Flux Measurements ◽  
Epidermal Growth

The rabbit cortical collecting duct absorbs Na+ by a transport system comprised of an apical membrane Na+ channel and a basolateral membrane Na(+)-K(+)-adenosinetriphosphatase. The rate of Na+ absorption across this epithelium is acutely inhibited by several hormones and autacoids including epidermal growth factor (EGF) and prostaglandin E2 (PGE2). We used electrophysiological analysis to determine which Na+ transport mechanism is primarily regulated in response to EGF and PGE2. We used concentrations of EGF and PGE2 that inhibited Na+ absorption to a comparable degree. We assessed the effects of these agents on Na+ transport primarily by the calculated equivalent current; the validity of this indicator was verified using simultaneous tracer flux measurements. EGF and PGE2 had different effects on the intracellular electrophysiological parameters. EGF (in the presence of a cyclooxygenase inhibitor) hyperpolarized the apical membrane voltage in a manner analogous to the Na(+)-channel blocker amiloride, reduced the transepithelial conductance, and increased the fractional resistance of the apical membrane. In comparison, PGE2 depolarized the apical membrane voltage in a manner analogous to the Na(+)-K+ pump inhibitor ouabain, and caused no significant changes in transepithelial conductance or apical membrane conductance. The finding that EGF hyperpolarized the apical membrane indicates that this agent attenuates Na+ absorption by reducing apical Na+ entry due to a decrease in the magnitude of the apical membrane Na+ conductance. In contrast, the electrophysiological changes produced by PGE2 indicate primary inhibition of the basolateral Na(+)-K+ pump following PGE2 treatment.

scholarly journals Nystatin as a probe for investigating the electrical properties of a tight epithelium.

1977 ◽  
Vol 70 (4) ◽  
pp. 427-440 ◽  
Author(s):  
S A Lewis ◽  
D C Eaton ◽  
C Clausen ◽  
J M Diamond
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Voltage Divider ◽  
Junctional Conductance ◽  
Transepithelial Voltage ◽  
Almost All ◽  
Voltage Divider Ratio ◽  
Rabbit Urinary Bladder ◽  
Tight Epithelium

We show how the antibiotic nystatin may be used in conjunction with microelectrodes to resolve transepithelial conductance Gt into its components: Ga, apical membrane conductance; Gbl, basolateral membrane conductance; and Gj, junctional conductance. Mucosal addition of nystatin to rabbit urinary bladder in Na+-containing solutions caused Gt to increase severalfold to ca. 460 micrometerho/muF, and caused the transepithelial voltage Vt to approach +50 mV regardless of its initial value. From measurements of Gt and the voltage-divider ratio as a function of time after addition or removal of nystatin, values for Ga, Gbl, and Gj of untreated bladder could be obtained. Nystatin proved to have no direct effect on Gbl or Gj but to increase Ga by about two orders of magnitude, so that the basolateral membrane then provided almost all of the electrical resistance in the transcellular pathway. The nystatin channel in the apical membrane was more permeable to cations than to anions. The dose-response curve for nystatin had a slope of 4.6. Use of nystatin permitted assessment of whether microelectrode impalement introduced a significant shunt conductance into the untreated apical membrane, with the conclusion that such a shunt was negligible in the present experiments. Nystatin caused a hyperpolarization of the basolateral membrane potential in Na+-containing solutions. This may indicate that the Na+ pump in this membrane is electrogenic.

ADH increases apical Na+, K+, 2Cl- entry in mouse medullary thick ascending limbs of Henle

1987 ◽  
Vol 252 (1) ◽  
pp. F177-F187 ◽  
Author(s):  
D. A. Molony ◽  
W. B. Reeves ◽  
S. C. Hebert ◽  
T. E. Andreoli
Keyword(s):  
Antidiuretic Hormone ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Resistance ◽  
Mouse Kidney ◽  
Membrane Conductances ◽  
Transepithelial Voltage

These studies were designed to evaluate the mechanism for the ADH-dependent increase in transcellular conductance (Gc, mS X cm-2), which accompanies hormone-dependent increases in the spontaneous transepithelial voltage (Ve, mV) and in the net rate of Cl- absorption in single medullary thick ascending limbs of Henle (mTALH) isolated from mouse kidney. The total transepithelial conductance (Ge, mS X cm-2) was measured with perfusing solutions containing 5 mM K+, zero Ba2+; Gc was that component of Ge blocked by luminal 20 mM Ba2+, zero K+. In paired experiments, antidiuretic hormone (ADH) increased Gc from 44.5 +/- 5.6 to 58.9 +/- 8.9 mS X cm-2 (delta = 14.3 +/- 5.5; P less than 0.02); however, in the presence of 10(-4) M luminal furosemide, ADH had no significant effect on Gc (delta = 5.0 +/- 4.3; NS). A set of similarly paired measurements together with paired observations on the effects of bath Cl- deletion, permitted an assessment of the effect of ADH on the magnitude of the fall in Gc on bath Cl- removal (delta GClc, mS X cm-2). delta GClc was clearly larger with ADH, 29.6 +/- 4.3, than without ADH, 19.2 +/- 1.0 (delta = 10.4 +/- 4.9; P less than 0.05). However, with luminal furosemide, ADH had no significant effect on delta GClc (delta = 1.7 +/- 4.5; NS). These results indicate that the ADH-dependent increase in Gc is secondary to increased salt entry across the apical membrane. We computed apical (ga, mS X cm-2) and basolateral (gb, mS X cm-2) membrane conductances from the Gc measurements and apical-to-basolateral membrane resistance ratios (Ra/Rb) obtained from cell impalement: the ADH-dependent Gc increase was due to an increase in gb, which was blocked entirely by luminal furosemide. We propose that ADH increases the number of functioning apical membrane Na+,K+,2Cl- transport units, and that gb increases because cell Cl- activity rises and depolarizes the basolateral membrane. Thus the calculated cellular Cl- activity was 16.3 mM without ADH, and 25 mM with ADH.

Effects of Ba2+ and Cs+ on apical membrane K+ conductance in toad retinal pigment epithelium

1995 ◽  
Vol 268 (5) ◽  
pp. C1164-C1172 ◽  
Author(s):  
B. A. Hughes ◽  
A. Shaikh ◽  
A. Ahmad
Keyword(s):  
Retinal Pigment Epithelium ◽  
Apical Membrane ◽  
Dissociation Constants ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Membrane Resistance ◽  
Conductance Ratio ◽  
Inwardly Rectifying

Intracellular microelectrode techniques were employed to characterize the blocker sensitivity of the K+ conductance (gK) at the apical membrane of the toad retinal pigment epithelium (RPE). Increasing the K+ concentration in the apical bath ([K+]o) from 2 to 5 mM produced a rapid depolarization of the apical membrane potential (VA). The addition of 0.5 mM Ba2+ or 5 mM Cs+ to the apical bath rapidly depolarized VA and increased the transepithelial resistance and ratio of apical-to-basolateral membrane resistance. In the presence of apical Ba2+ or Cs+, the response of VA to delta [K+]o was markedly reduced, indicating that these ions are effective blockers of apical gK. The Ba(2+)- and Cs(+)-induced decreases in the apparent apical-to-basolateral membrane conductance ratio were concentration dependent, with apparent dissociation constants of 17 microM and 0.5 mM, respectively. The apparent blocker sensitivity of apical gK is similar to that previously demonstrated for the inwardly rectifying K+ conductance in isolated toad RPE cells, suggesting that the inwardly rectifying K+ conductance comprises much of apical gK.

cAMP stimulates the Na+-K+ pump in frog retinal pigment epithelium

1988 ◽  
Vol 254 (1) ◽  
pp. C84-C98 ◽  
Author(s):  
B. A. Hughes ◽  
S. S. Miller ◽  
D. P. Joseph ◽  
J. L. Edelman
Keyword(s):  
Apical Membrane ◽  
Pigment Epithelium ◽  
Phase 1 ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Phase 2 ◽  
Induced Voltage ◽  
Significant Drop ◽  
Stimulation Of

Adenosine 3', 5'-cyclic monophosphate (cAMP) induced increases in active Na+ secretion and K+ absorption that were blocked by apical ouabain (10(-4) M), suggesting stimulation of the Na+-K+ pump. cAMP also produced rapid membrane voltage and resistance changes that could be divided chronologically into three phases. In phase 1, the basolateral membrane depolarized at a faster rate than the apical membrane, probably as a result of an increase in basolateral membrane conductance. In phase 2, the apical membrane repolarized toward control faster than the basal membrane, whereas in phase 3 the basolateral membrane repolarized faster than the apical membrane. Apical ouabain completely inhibited the cAMP-induced repolarization of the apical membrane during phase 2. Thus the stimulation of the Na+-K+ pump occurs within minutes of cAMP elevation. Na+ removal from the basal side did not block the cAMP-induced voltage changes, indicating that the initial conductance increase is not due to Na+. In contrast, Na+ removal from the apical bath inhibited all phases of the cAMP response. This suggests that apical membrane Na+-dependent transport mechanisms mediate the stimulation of the Na+-K+ pump. cAMP also caused a significant drop in intracellular K+ activity (approximately 5 mM) that preceded phase 2. This drop could stimulate the Na+-K+ pump, as suggested by previous experiments.

scholarly journals Electrophysiology of flounder intestinal mucosa. I. Conductance properties of the cellular and paracellular pathways.

1985 ◽  
Vol 85 (6) ◽  
pp. 843-864 ◽  
Author(s):  
D R Halm ◽  
E J Krasny ◽  
R A Frizzell
Keyword(s):  
Membrane Potential ◽  
Apical Membrane ◽  
Driving Forces ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Membrane Conductance ◽  
Equivalent Circuit Analysis ◽  
K Secretion ◽  
Paracellular Pathways ◽  
K Concentration

We evaluated the conductances for ion flow across the cellular and paracellular pathways of flounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels. An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (psi a) and, at [K]m less than 40 mM, the K dependence of psi a was abolished by 1-2 mM mucosal Ba. The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (psi b) with reduced serosal Cl concentrations, [Cl]s. psi b was unaffected by changes in [K]s or [Na]s. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (Gp) normally accounts for 96% of transepithelial conductance (Gt). The high Gp attenuates the contribution of the cellular pathway to psi t while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes. Thus, psi a and psi b (approximately 60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membrane. Equivalent circuit analysis suggests that apical conductance (Ga approximately equal to 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb approximately equal to 1.5 mS/cm2) would account for only 50% of net Cl absorption. This, together with our failure to detect a basolateral K conductance, suggests that Cl absorption across this barrier involves KCl co-transport.

Central role of the apical membrane H+-ATPase in electrogenesis and epithelial transport in Malpighian tubules

2000 ◽  
Vol 203 (9) ◽  
pp. 1459-1468 ◽  
Author(s):  
K.W. Beyenbach ◽  
T.L. Pannabecker ◽  
W. Nagel
Keyword(s):  
Epithelial Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Coupling ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Proton Channel ◽  
Transepithelial Transport

The effects of bafilomycin A(1), a blocker of V-type H(+)-ATPases, were investigated in Malpighian tubules of Aedes aegypti. Bafilomycin A(1) reduced rates of transepithelial fluid secretion and the virtual short-circuit current (vI(sc)) with an IC(50) of approximately 5 micromol l(−)(1). As vI(sc) decreased, the electrical resistance increased across the whole epithelium and across the apical membrane, indicating effects on electroconductive pathways. Bafilomycin A(1) had no effect when applied from the tubule lumen, pointing to the relative impermeability of the apical membrane to bafilomycin A(1). Thus, bafilomycin A(1) must take a cytoplasmic route to its blocking site in the proton channel of the H(+)-ATPase located in the apical membrane of principal cells. The inhibitory effects of bafilomycin A(1) were qualitatively similar to those of dinitrophenol in that voltages across the epithelium (V(t)), the basolateral membrane (V(bl)) and the apical membrane (V(a)) depolarized towards zero in parallel. Moreover, V(bl)always tracked V(a), indicating electrical coupling between the two membranes through the shunt. Electrical coupling allows the H(+)-ATPase to energize not only the apical membrane, but also the basolateral membrane. Furthermore, electrical coupling offers a balance between electroconductive entry of cations across the basolateral membrane and extrusion across the apical membrane to support steady-state conditions during transepithelial transport.

Apical membrane chloride channels in a colonic cell line activated by secretory agonists

1988 ◽  
Vol 254 (4) ◽  
pp. C505-C511 ◽  
Author(s):  
D. R. Halm ◽  
G. R. Rechkemmer ◽  
R. A. Schoumacher ◽  
R. A. Frizzell
Keyword(s):  
Cell Line ◽  
Chloride Channels ◽  
Single Channel ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Anion Channel ◽  
Cell Types ◽  
Cl Channel ◽  
Evoked Activity ◽  
Apical Membranes

We characterized the anion channel responsible for the increase in apical membrane Cl secretion using a model salt-secreting epithelium, the T84 colonic cell line. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated secretagogues, prostaglandin E2, forskolin, and 8-bromo-cAMP, evoked activity of an outwardly rectifying Cl channel in previously quiet cell-attached membrane patches. The channel remained active in excised, inside-out membranes, where its single-channel conductance was 40-45 pS at 0 mV with 160 mM NaCl in pipette and bath. Selectivities were PCl/PNa = 50 and for halides I(1.8)/Br(1.4)/Cl(1.0)/F(0.4). This halide sequence illustrates that the ability of various anions to undergo transepithelial secretion is determined by the selectivity of the basolateral membrane Cl entry step rather than by the apical Cl channel. Open-channel probability increased with depolarization, an effect that would adjust the rate of Cl exit across secretory cell apical membranes with agonist-induced changes in apical membrane potential. Comparison with the properties of Cl channels detected in other cell types suggests that this cAMP-stimulated Cl channel is uniquely present in the apical membranes of salt-secreting epithelial cells.

Muscarinic stimulation of gallbladder epithelium. II. Fluid transport, cell volume, and ion permeabilities

1993 ◽  
Vol 265 (6) ◽  
pp. C1613-C1619 ◽  
Author(s):  
G. A. Altenberg ◽  
M. Subramanyam ◽  
L. Reuss
Keyword(s):  
Cell Volume ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Time Dependent ◽  
Water Volume ◽  
K Channels ◽  
Membrane Conductances ◽  
Muscarinic Stimulation ◽  
Stimulation Of

Activation of muscarinic receptors in the fluid-absorptive epithelium of the Necturus gallbladder elevates cytosolic Ca2+ concentration, transiently hyperpolarizes the cell membrane voltages, and decreases the apparent fractional resistance of the apical membrane [G. A. Altenberg, M. Subramanyam, J. S. Bergmann, K. M. Johnson, and L. Reuss. Am. J. Physiol. 265 (Cell Physiol. 34): C1604-C1612, 1993]. In these studies, we show that at the peak of the hyperpolarization both apical and basolateral membrane resistances (Ra and Rb, respectively) decreased, but in 2-3 min Ra returned to control values while Rb rose to a level approximately 60% higher than control. The acetylcholine (ACh)-induced decrease in Ra is caused by activation of apical membrane maxi K+ channels secondary to elevation of cytosolic Ca2+ concentration. The increase in Rb is due to decreases in K+ and Cl- conductances. ACh had no effects on cell KCl content or water volume, although K+ conductance transiently increased. These results can be explained by the changes in basolateral membrane conductances. ACh did not alter fluid absorption. In conclusion, ACh has complex time-dependent effects on K+ and Cl- electrodiffusive permeabilities without measurable changes in cell volume or in the rate of transepithelial fluid transport.

Effects of oxytocin on cation content and electrophysiology of frog skin epithelium

1988 ◽  
Vol 255 (3) ◽  
pp. C357-C367 ◽  
Author(s):  
H. F. Schoen ◽  
A. Kaufman ◽  
D. Erlij
Keyword(s):  
Frog Skin ◽  
Rana Catesbeiana ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Short Circuit Current ◽  
Flame Photometry ◽  
Intracellular Na Activity ◽  
Current Voltage

We measured effects of oxytocin on current-voltage (I-V) relations of frog (Rana catesbeiana) skins impaled with an intracellular microelectrode. In both Cl- and Cl(-)-free (SO4(2-) solutions, oxytocin caused an approximate doubling short-circuit current (Isc) and a depolarization of the cell membrane. Increase in apical membrane slope conductance, chord conductance, and permeability after oxytocin correlated with the increase in amiloride-sensitive Isc. Oxytocin also increased basolateral membrane conductance (gb). In Cl-, the shift in the voltage intercept of the apical membrane I-V relation (Ea) implied increased intracellular Na+ activity (a(Na)c) after oxytocin. In isolated frog skin epithelia, a similar increase in intracellular [Na+] after oxytocin was demonstrated by flame photometry. In SO4(2-), changes caused by oxytocin in both Ea and in flame photometrically determined cell [Na+] were minimal. The voltage intercept of the basolateral membrane I-V relations (Eb) was shifted by oxytocin in both Cl- and SO4(2-) solutions. Assuming that the basolateral membrane is selectively permeable to K+, changes in K+ obtained from Eb were in disagreement with those obtained by flame photometry. These results suggest that 1) the increase in a(Na)c caused by oxytocin is not essential to produce either the increase in gb or Isc and 2) ions other than K+ make an important contribution to basolateral membrane conductance.

Sign in / Sign up

Export Citation Format

Share Document