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)

Altered electrical potential profile of human reabsorptive sweat duct cells in cystic fibrosis

1989 ◽  
Vol 257 (4) ◽  
pp. C722-C726 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Cystic Fibrosis ◽  
Membrane Potential ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Potential Profile ◽  
Sweat Duct ◽  
Normal Cells ◽  
Electrophysiological Properties ◽  
Duct Cells ◽  
Normal Duct

The electrophysiological properties of reabsorptive sweat duct (RSD) cells from normal and cystic fibrosis (CF) subjects were studied using intracellular microelectrodes. The apical membrane potential (Va) of CF duct cells was reversed in "polarity" (+28.0 +/- 2.4 mV, n = 46) compared with normal duct cells (-24.9 +/- 0.4 mV, n = 145), and the basolateral membrane potential (Vb) of CF cells was hyperpolarized significantly (-50.1 +/- 1.2 mV, n = 46) in comparison to normal cells (-34.6 +/- 0.4 mV, n = 145). The substitution of the impermeant anion gluconate for Cl- in the lumen of the normal duct depolarized Va from -24.9 +/- 1.1 to 8.9 +/- 3.1 mV (n = 18) and hyperpolarized Vb from -34.3 +/- 1.1 to -55.6 +/- 3.7 mV (n = 18), which mimicked the cell electrical potential profile of CF ducts even in the presence of Cl-. Cl- substitution in the bath depolarized Vb of normal ducts by 22.5 +/- 2.6 mV (n = 24), while hyperpolarizing Va by -3.4 +/- 1.6 mV (n = 24). The response of the electrical profiles of CF cells to Cl- substitution in either the lumen or the bath was significantly reduced compared with normal cells. The effect of the Na+ conductance blocker amiloride (10(-4) M) on Vb was not significantly different in CF (delta Vb = -26.4 +/- 3.8 mV, n = 9) vs. normal (delta Vb = -27.6 +/- 2.5 mV, n = 30) cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of abnormal Cl- impermeability on sweating in cystic fibrosis

1984 ◽  
Vol 247 (1) ◽  
pp. C3-C9 ◽  
Author(s):  
J. Bijman ◽  
P. M. Quinton
Keyword(s):  
Cystic Fibrosis ◽  
Electrical Potential ◽  
Normal Subjects ◽  
Electrochemical Potential ◽  
Sweat Glands ◽  
Low Permeability ◽  
Sweat Duct ◽  
Active Process ◽  
Nacl Transport ◽  
Normal Duct

Parameters of electrolyte transport in single sweat glands in normal subjects and cystic fibrosis (CF) patients were monitored and compared. Results indicate that in both normal and CF sweat ducts, Na+ is reabsorbed by an active process in which Cl- follows passively while K+ is secreted. However, while net NaCl reabsorption is markedly lower, the electrical potential associated with sweat emerging from the sweat duct is significantly more negative in CF than in normal subjects. Comparison of the differences in apparent electrochemical potential experienced by ions in the sweat duct during secretion indicates that Na+ is held out of the lumen of both groups of ducts against a large but similar gradient, but that Cl- is held in the CF duct against a much larger gradient than in the normal duct. These results indicate that the mechanism for Na+ reabsorption is not inhibited in the CF duct, but that the decreased NaCl transport in the defective duct is due to an abnormally low permeability to Cl-. Analysis of the electrical potential as a function of the Cl- gradient in the sweat suggests that the normal and defective route of Cl- uptake may be transcellular.

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.

Interrelationships of ion transport in rat submaxillary duct epithelium

1982 ◽  
Vol 242 (2) ◽  
pp. F132-F139 ◽  
Author(s):  
H. Knauf ◽  
R. Lubcke ◽  
W. Kreutz ◽  
G. Sachs
Keyword(s):  
Ion Transport ◽  
Basolateral Membrane ◽  
Ion Concentration ◽  
Transport Systems ◽  
Salivary Duct ◽  
Basolateral Membranes ◽  
A Value ◽  
Duct Epithelium ◽  
K Secretion

The transport of Na+, K+, Cl-, and HCO3(-) across the epithelium of the rat submaxillary salivary duct is postulated to be due to the coupling of the basolateral Na+-K+-ATPase with various ion transport systems in the luminal and basolateral membranes. Na+ reabsorption depends on the presence of a rheogenic (Na+ conductance) and an electroneutral (Na+:H exchange) pathway, both of which are sensitive to amiloride. K+ secretion is postulated to be mediated by a K+: H+ antiport, coupling between Na+ reabsorption and K+ secretion, thus depending on local H+ ion concentration. The ratio between electroneutral Na+ influx and K+ efflux, therefore, determines the rate of HCO3(-) secretion. In the absence of Na+ influx, although K+ efflux falls, HCO3(-) secretion rises to a value equal to that of K+ secretion. The maintenance of K+ secretion in the absence of luminal Na+ requires an additional Na+-entry step across the basolateral membrane, also postulated to be due to Na+:H+ exchange.

Deactivation of CFTR-Cl conductance by endogenous phosphatases in the native sweat duct

1996 ◽  
Vol 270 (2) ◽  
pp. C474-C480 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Cystic Fibrosis ◽  
Okadaic Acid ◽  
Apical Membrane ◽  
Transmembrane Conductance Regulator ◽  
Sweat Duct ◽  
Transmembrane Conductance ◽  
Phosphatase Inhibitors ◽  
Cystic Fibrosis Transmembrane ◽  
Cl Conductance

Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel. However, very little is known about the endogenous mechanism(s) of deactivation of CFTR-Cl conductance (CFTR-GCl) in vivo. We studied the action of endogenous phosphatases in regulation of the adenosine 3',5'-cyclic monophosphate (cAMP)- and ATP-induced CFTR-GCl in the apical membrane of microperfused preparations of basolaterally permeabilized native sweat duct. Activation of CFTR-GCl was monitored by measuring the apical Cl diffusion potentials and GCl, which spontaneously deactivated on removal of cAMP. This spontaneous loss of CFTR-GCl activity could be prevented by a cocktail of phosphatase inhibitors (fluoride, vanadate, and okadaic acid). We studied the effects of each of these phosphatase antagonists on the rate of deactivation of CFTR-GCl after cAMP washout. In contrast to vanadate or fluoride, okadaic acid virtually prevented deactivation of CFTR-GCl after cAMP washout. We conclude that either or both protein phosphatases 1 and 2A are responsible for the dephosphorylation deactivation of CFTR-GCl in vivo.

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.

Chloride secretory response to extracellular ATP in human normal and cystic fibrosis nasal epithelia

1992 ◽  
Vol 263 (2) ◽  
pp. C348-C356 ◽  
Author(s):  
L. L. Clarke ◽  
R. C. Boucher
Keyword(s):  
Cystic Fibrosis ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Chloride Secretion ◽  
Extracellular Atp ◽  
Nasal Epithelium ◽  
Secretory Process ◽  
Cl Secretion ◽  
Epithelial Cultures ◽  
Cl Conductance

Chloride secretion across cystic fibrosis (CF) airway epithelia is effectively regulated by pathways associated with intracellular Ca2+ metabolism, but not by mechanisms dependent on protein kinase A or C. In a search for therapeutically useful agonists, we investigated the effects of extracellular ATP on the Cl- secretory process in human normal and CF nasal epithelial cultures with double-barreled Cl- selective microelectrodes. When applied to the basolateral membrane of normal, but not CF, nasal epithelium, extracellular ATP (10(-4) M) stimulated a small increase in Cl- secretion that was primarily associated with a hyperpolarizing conductance in the basolateral membrane. In contrast, ATP applied to the apical (luminal) membrane of either normal or CF nasal epithelium stimulated a greater increase in Cl- secretion that was associated with activation of an apical membrane Cl- conductance. The increases in Cl- current and apical conductance were greater in CF tissues and attained maximal values similar to normal nasal epithelium. We conclude 1) that basolateral application of ATP indirectly induces Cl- secretion by activating a basolateral (K+) conductance and is an effective secretagogue only in normal nasal epithelium and 2) that luminally applied ATP is an effective Cl- secretagogue that activates the apical membrane Cl- conductance of normal and CF nasal epithelia to an equivalent level.

Regulation of Cl- permeability in normal and cystic fibrosis sweat duct cells

1990 ◽  
Vol 259 (5) ◽  
pp. C842-C846 ◽  
Author(s):  
S. J. Ram ◽  
M. L. Weaver ◽  
K. L. Kirk
Keyword(s):  
Cystic Fibrosis ◽  
Digital Imaging ◽  
Sweat Duct ◽  
Dependent Protein Kinase ◽  
Duct Cells ◽  
Dependent Protein ◽  
Regulatory Defect ◽  
Cells Cultured ◽  
Permeability Regulation ◽  
Cl Permeability

Reabsorptive cells of the human sweat gland normally exhibit a high basal Cl- permeability but are markedly impermeable to Cl- in cystic fibrosis (CF). We examined the possibility that the reduced basal Cl- permeability of CF sweat duct cells in primary culture is due to a defective regulation of plasma membrane Cl- permeability by prostaglandin E2 (PGE2), which is endogenously produced by cultured sweat duct cells. The macroscopic Cl- permeabilities of normal and CF sweat duct cells were assessed using a halide-specific fluorescent dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, in combination with fluorescence digital-imaging microscopy. The Cl- and Br- permeabilities of normal sweat duct cells were markedly reduced by inhibiting endogenous PGE2 production with indomethacin. This inhibition of Cl- permeability by indomethacin was largely reversed by the addition of PGE2 (10 nM to 1 microM), but not forskolin. Conversely, PGE2 failed to stimulate the low Cl- permeabilities of sweat duct cells cultured from CF subjects. Our results support the following conclusions: 1) a defective regulation of Cl- permeability in CF is a feature of reabsorptive as well as secretory epithelial cells, and 2) the nature of this regulatory defect extends beyond altered Cl- permeability regulation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase.

Furosemide blocks basolateral membrane Cl- permeability in gallbladder epithelium

1990 ◽  
Vol 258 (6) ◽  
pp. C1150-C1158 ◽  
Author(s):  
J. S. Stoddard ◽  
G. A. Altenberg ◽  
M. L. Ferguson ◽  
L. Reuss
Keyword(s):  
Anion Exchange ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Ringer Solution ◽  
Combined Effect ◽  
Gallbladder Epithelium ◽  
K Permeability ◽  
Cl Conductance ◽  
Stimulation Of ◽  
Cl Permeability

In Necturus gallbladders bathed in a NaCl Ringer solution buffered with 10 mM HCO3(-)-1% CO2, furosemide (added to the serosal solution) caused a concentration-dependent hyperpolarization of both cell membranes that was slow and reversible. At 10(-3) M furosemide, the basolateral membrane voltage (Vcs) increased significantly from -71 +/- 3 to -85 +/- 3 mV, the depolarization of Vcs elicited by a 10-fold rise in serosal [K+] increased from 34 +/- 4 to 50 +/- 1 mV, the depolarization elicited by lowering serosal [Cl-] from 98 to 8.1 mM was reduced from 15 +/- 1 to 1 +/- 1 mV, and the depolarization in response to lowering serosal [HCO3-] from 10 to 1 mM was reduced from 13 +/- 1 to 5 +/- 0.4 mV. Furosemide could in principle decrease the basolateral membrane Cl- conductance (Gcl), increase the basolateral membrane K+ conductance, or have a combined effect. To distinguish among these possibilities, we estimated the resistance of the basolateral membrane (Rb) by means of two-point intraepithelial cable analysis experiments. Furosemide increased Rb by 22%, which indicates that furosemide reduces basolateral membrane Gcl. The effect cannot be attributed to inhibition of apical membrane anion exchange by serosal addition of furosemide, because base secretion from cells to lumen is unchanged. We conclude that furosemide blocks reversibly basolateral membrane electrodiffusive Cl- permeability. A concomitant stimulation of basolateral membrane electrodiffusive K+ permeability is also possible.

Hydrolytic and nonhydrolytic interactions in the ATP regulation of CFTR Cl- conductance

1996 ◽  
Vol 271 (1) ◽  
pp. C35-C42 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Cystic Fibrosis ◽  
Significant Role ◽  
Atp Hydrolysis ◽  
Transmembrane Conductance Regulator ◽  
Sweat Duct ◽  
Transmembrane Conductance ◽  
Atp Regulation ◽  
Cystic Fibrosis Transmembrane ◽  
Cl Conductance

Previously, we showed in the native sweat duct that, in the presence of 0.1-0.5 mM ATP, nonhydrolyzable ATP analogue adenosine 5'-adenylylimidodiphosphate (AMP-PNP) can activate cystic fibrosis transmembrane conductance regulator Cl- conductance (CFTR GCl) (15). The objective of this study is to determine if 1) nonhydrolytic ATP binding alone can activate CFTR GCl after stable phosphorylation [in the presence of adenosine 5'-O-(3-thiotriphosphate) and phosphatase inhibition cocktail] of CFTR or 2) an ATP hydrolysis (in addition to phosphorylation) is required to support subsequent nonhydrolytic ATP regulation of CFTR GCl. We show that stably phosphorylated CFTR could only be activated by AMP-PNP in the presence of a small background ATP concentration. However, AMP-PNP can sustain previously activated CFTR GCl in the absence of ATP, even though Mg2+ is required for phosphorylation activation of CFTR GCl. However, once stably phosphorylated, ATP activation of CFTR GCl is independent of Mg2+. Our results show that both hydrolytic and nonhydrolytic interactions regulate CFTR GCl in vivo. Nonhydrolytic ATP interaction plays a significant role in both activation and deactivation of CFTR GCl.

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