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.

Dibutyryl cAMP activates bumetanide-sensitive electrolyte transport in Malpighian tubules

1991 ◽  
Vol 261 (3) ◽  
pp. C521-C529 ◽  
Author(s):  
J. L. Hegarty ◽  
B. Zhang ◽  
T. L. Pannabecker ◽  
D. H. Petzel ◽  
M. D. Baustian ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Fluid Secretion ◽  
Ringer Solution ◽  
Malpighian Tubules ◽  
Membrane Voltage ◽  
Dibutyryl Camp ◽  
K Secretion ◽  
Transepithelial Voltage

The effects of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and bumetanide (both 10(-4) M) on transepithelial Na+, K+, Cl-, and fluid secretion and on tubule electrophysiology were studied in isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. Peritubular DBcAMP significantly increased Na+, Cl-, and fluid secretion but decreased K+ secretion. In DBcAMP-stimulated tubules, bumetanide caused Na+, Cl-, and fluid secretion to return to pre-cAMP control rates and K+ secretion to decrease further. Peritubular bumetanide significantly increased Na+ secretion and decreased K+ secretion so that Cl- and fluid secretion did not change. In bumetanide-treated tubules, the secretagogue effects of DBcAMP are blocked. In isolated Malpighian tubules perfused with symmetrical Ringer solution, DBcAMP significantly hyperpolarized the transepithelial voltage (VT) and depolarized the basolateral membrane voltage (Vbl) with no effect on apical membrane voltage (Va). Total transepithelial resistance (RT) and the fractional resistance of the basolateral membrane (fRbl) significantly decreased. Bumetanide also hyperpolarized VT and depolarized Vbl, however without significantly affecting RT and fRbl. Together these results suggest that, in addition to stimulating electroconductive transport, DBcAMP also activates a nonconductive bumetanide-sensitive transport system in Aedes Malpighian tubules.

Electrophysiological evidence for Cl secretion in shark renal proximal tubules

1985 ◽  
Vol 248 (2) ◽  
pp. F282-F295 ◽  
Author(s):  
K. W. Beyenbach ◽  
E. Fromter
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Proximal Tubules ◽  
In Vitro Perfusion ◽  
Electrophysiological Properties ◽  
Electrophysiological Responses ◽  
Tubule Lumen ◽  
Transepithelial Voltage ◽  
Cl Conductance

The electrophysiology of shark proximal tubules (Squalus acanthias) was investigated using conventional microelectrodes and cable analysis. Under in vitro perfusion with symmetrical Ringer solutions, tubule transepithelial resistance was 36.3 +/- 2.3 omega X cm2 (means +/- SE, n = 44). Other electrophysiological variables varied widely under control conditions. In unstimulated tubules (n = 16) the transepithelial voltage (VT,o) was lumen positive (1.2 +/- 0.2 mV), the basolateral membrane potential (Vbl,x) was -61.3 +/- 1.6 mV, and the fractional resistance of the apical membrane (fRa) was 0.67 +/- 0.02. Spontaneously stimulated tubules (n = 28) had lumen-negative VT,o values (-1.5 +/- 0.4 mV), low Vbl,x values (-41.3 +/- 1.7 mV), and low fRa values (0.30 +/- 0.02). The stimulated state can be induced in unstimulated tubules via treatment with cAMP. Multiple microelectrode impalements in a single tubule revealed epithelial cells sharing similar electrophysiological properties. Selective ion substitutions in the tubule lumen and peritubular bath uncovered an increased Cl conductance in the apical membrane of spontaneously and cAMP-stimulated tubules. Anthracene-9-carboxylic acid tended to reverse the stimulated state, and furosemide hyperpolarized Vbl,x. These results constitute the first evidence for secretory Cl transport in a renal proximal tubule. The electrophysiological responses to ion substitutions, stimulators, and inhibitors are strikingly similar to those of known Cl-transporting 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.

Contribution of surface epithelial cells to total conductance of Necturus gastric fundus mucosa

1996 ◽  
Vol 270 (6) ◽  
pp. G902-G908 ◽  
Author(s):  
G. Kottra ◽  
C. Iacovelli ◽  
R. Caroppo ◽  
S. Curci ◽  
P. Bakos ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Voltage Divider ◽  
Gastric Fundus ◽  
Total Conductance ◽  
In Vitro Activation ◽  
Transepithelial Voltage ◽  
Gastric Fundus Mucosa ◽  
Voltage Divider Ratio ◽  
Microelectrode Techniques

Microelectrode techniques were used to quantify the contribution of surface epithelial cells (SEC) to transepithelial conductance (gt) of Necturus gastric fundus mucosa. Transepithelial voltage (Vt) and resistance (Rt) as well as the basolateral cell membrane potential (Vb) and voltage divider ratio of SEC were measured. Freshly mounted preparations did not respond to luminal amiloride (10 microM), but within 2-3 h a significant response developed (delta Vt = 3.8 +/- 1.2 mV, delta Rt = 63 +/- 23 omega cm2, and delta Vb = -6.9 +/- 1.3 mV), indicating activation of an apical Na+ conductance in SEC. Using circuit analysis equations, we calculate that SEC contribute 10.4% to gt under control conditions and 13.0% after Na+ conductance activation. Histamine (0.1 mM), which stimulates the oxyntopeptic cells (OC), increased Vt and decreased Rt but did not significantly alter the membrane resistances of SEC. As a result, the contribution of SEC to gt fell to 7.4 or 9.3%, respectively. The data confirm that SEC are poorly permeable and that the major conductance path across gastric mucosa leads through OC in the glands. The reason for the protracted in vitro activation of the apical Na+ conductance in SEC is not known.

Localization of Cl- conductance in normal and Cl- impermeability in cystic fibrosis sweat duct epithelium

1989 ◽  
Vol 257 (4) ◽  
pp. C727-C735 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Cystic Fibrosis ◽  
Basolateral Membrane ◽  
Voltage Divider ◽  
Sweat Duct ◽  
Basolateral Membranes ◽  
Duct Epithelium ◽  
Voltage Divider Ratio ◽  
Normal Duct ◽  
Cl Conductance ◽  
Cl Permeability

We studied the Cl- permeability properties of apical and basolateral membranes of human reabsorptive sweat duct (RSD) from normal and cystic fibrosis (CF) subjects. In normal ducts, Cl- substitution by impermeant anion gluconate in the lumen increased the voltage divider ratio (VDR) from 4.8 +/- 0.9 to 7.0 +/- 1.1 (n = 8, P less than 0.05), whereas Cl- substitution in the contraluminal bath decreased the VDR from 3.2 +/- 0.7 to 1.9 +/- 0.4 (n = 7, P less than 0.05). These results are consistent with a significant Cl- permeability in both apical and basolateral membranes of normal ducts. Amiloride (10(-4) M) in the lumen of normal ducts resulted in a small increase in VDR from 4.2 +/- 0.6 to 5.0 +/- 0.8 (n = 10, P less than 0.05), whereas the current-induced basolateral membrane voltage deflections (delta Vb) increased from 6.9 +/- 1.3 to 7.7 +/- 1.2 mV, suggesting that inhibition of Na+ permeability decreased basolateral membrane Cl- permeability. In the absence of luminal Cl-, amiloride decreased delta Vb and induced much greater effect on VDR (from 5.2 +/- 1.1 to 10.8 +/- 2.3, n = 9, P less than 0.05) than in the presence of Cl-. Likewise, in the presence of amiloride, Cl- substitution in the lumen had greater effect on VDR (increased from 3.5 +/- 0.5 0.5 to 10.0 +/- 1.5, n = 15, P less than 0.05) than in the absence of amiloride. These results indicate that Na+ conductance in the apical membrane of the normal duct is significantly smaller than Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of histamine on the basolateral K+ conductance of frog stomach oxyntic cells and surface epithelial cells

1990 ◽  
Vol 258 (4) ◽  
pp. G631-G636
Author(s):  
L. Debellis ◽  
S. Curci ◽  
E. Fromter
Keyword(s):  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Voltage Divider ◽  
Cell Types ◽  
Membrane Resistance ◽  
Rana Esculenta ◽  
Frog Stomach ◽  
K Concentration ◽  
Voltage Divider Ratio ◽  
Ion Conductances

The transepithelial potential difference (Vt) and resistance (Rt) and the basolateral cell membrane potential (Vs) of oxyntic cells (OC) and surface epithelial cells (SEC) were measured in isolated stomachs of Rana esculenta. At rest, Vs of OC and SEC was virtually identical [-66.3 +/- 4.5 (SD) (n = 10) and -67.3 +/- 5.9 mV (n = 9)] and both cells responded to increasing serosal K+ concentration from 4 to 13 mmol/l with virtually the same depolarization (delta Vs,K) of +16.2 +/- 2.0 and +16.0 +/- 2.9 mV, respectively, while Vt declined by approximately half as much. Histamine (0.1 mmol/l) reduced Vt and Rt and increased the voltage divider ratio in both cell types, indicating a fall in basolateral membrane resistance. In the OC, this increase was neither associated with a significant alteration of Vs nor with a change in delta Vs,K. In the SEC, however, histamine markedly increased Vs to -75.5 +/- 7.3 mV (n = 9) as well as delta Vs,K to +18.5 +/- 2.6 mV, which was paralleled by an increase in delta Vt,K from 9.8 +/- 3.9 to +12.8 +/- 4.2 mV. The data indicate that 1) both OC and SEC respond to histamine, 2) both OC and SEC contain a basolateral K+ conductance that increases under histamine (in OC probably, in parallel with other ion conductances), and 3) in Rana esculenta the SEC contribute substantially to Vt.

Conductive properties of papillary surface epithelium

1994 ◽  
Vol 266 (2) ◽  
pp. F259-F265 ◽  
Author(s):  
W. B. Reeves
Keyword(s):  
Cell Membrane ◽  
Apical Membrane ◽  
Ussing Chamber ◽  
Basolateral Membrane ◽  
Transepithelial Resistance ◽  
Surface Epithelium ◽  
Renal Papilla ◽  
Transepithelial Voltage ◽  
K Concentration ◽  
Conductive Properties

The surface epithelium of rabbit renal papilla was dissected free from its supporting tissue and mounted in an Ussing chamber. The conductive properties of the epithelium and of the apical and basolateral cell membranes were examined with KCl-filled microelectrodes. The transepithelial voltage was 0.07 +/- 0.15 mV, and the transepithelial resistance was 107 +/- 15 omega.cm2 (n = 29). The fractional resistance of the apical membrane (fRa) was 0.93 +/- 0.01 (n = 103 cells, 29 tissues). The apical membrane was not conductive to Na+, K+, or Cl-. An increase in the K+ concentration of the basolateral solution from 5 to 50 mM depolarized the basolateral membrane voltage (Vb) from -59 +/- 1.6 to -31.2 +/- 2.2 mV (n = 28 cells) and increased fRa from 0.935 +/- 0.01 to 0.962 +/- 0.01 (P < 0.001, n = 21 cells). Likewise, 5 mM barium in the basolateral solution depolarized Vb from -57.7 +/- 2.0 to -29.8 +/- 2.2 mV (n = 21 cells). A tenfold decrease in the Cl- concentration of the basolateral solution caused an 8.3 +/- 1.9 mV depolarization in Vb. Thus the basolateral cell membrane is conductive to K+ and Cl-. Exposure of the apical membrane to amphotericin B demonstrated that the transepithelial resistance is determined primarily by the paracellular pathway.

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.

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.

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.

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