Department of Pediatrics, University of California, San Diego, Medical Center, San Diego, California 92103-0831
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ABSTRACT |
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Bumetanide is well
known for its ability to inhibit the nonconductive
Na+-K+-2Cl
cotransporter. We were surprised in preliminary studies to find that
bumetanide in the contraluminal bath also inhibited NaCl absorption in
the human sweat duct, which is apparently poor in cotransporter
activity. Inhibition was accompanied by a marked decrease in the
transepithelial electrical conductance. Because the cystic fibrosis
transmembrane conductance regulator (CFTR) Cl
channel is richly
expressed in the sweat duct, we asked whether bumetanide acts by
blocking this anion channel. We found that bumetanide
1) significantly increased whole
cell input impedance, 2)
hyperpolarized transepithelial and basolateral membrane potentials, 3) depolarized apical membrane
potential, 4) increased the ratio of
apical-to-basolateral membrane resistance, and
5) decreased transepithelial
Cl
conductance
(GCl).
These results indicate that bumetanide inhibits CFTR
GCl
in both cell membranes of this epithelium. We excluded bumetanide
interference with the protein kinase A phosphorylation activation
process by "irreversibly" phosphorylating CFTR [by using
adenosine
5'-O-(3-thiotriphosphate) in the
presence of a phosphatase inhibition cocktail] before bumetanide
application. We then activated CFTR
GCl
by adding 5 mM ATP. Bumetanide in the cytoplasmic bath
(10
3 M) inhibited ~71%
of this ATP-activated CFTR
GCl,
indicating possible direct inhibition of CFTR
GCl.
We conclude that bumetanide inhibits CFTR
GCl
in apical and basolateral membranes independent of phosphorylation. The
results also suggest that
>10
5 M bumetanide cannot
be used to specifically block the
Na+-K+-2Cl
cotransporter.
ion transport inhibitors; anion channel blockers; electrolyte absorption; cystic fibrosis; phosphorylation
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INTRODUCTION |
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CYSTIC FIBROSIS transmembrane conductance regulator
(CFTR) is a protein kinase A (PKA)- and ATP-activated
Cl channel (2, 5, 6, 13,
20). The CFTR
Cl
channel plays a critical role in transepithelial
Cl
absorption and secretion. Absence or functional abnormalities of these
Cl
channels in the epithelial cell membranes of certain exocrine cells
lead to the severe pathology associated with cystic fibrosis (CF).
Furthermore, excessive stimulation of these
Cl
channels by certain bacterial toxins causes severe diarrhea (4). Pharmacological blockers of CFTR
Cl
channels can play a significant role in
1) distinguishing these channels
from other types of
Cl
channels and 2) developing
therapeutic agents to control certain types of diarrhea involving CFTR
Cl
channels.
Previous attempts to find effective pharmacological blockers for CFTR
Cl
channels have met with limited success. Earlier studies suggested that
several pharmacological agents, including
5-nitro-2-(3-phenylpropylamino)benzoic acid,
diphenylamine-2-carboxylate, and glibenclamide, inhibit CFTR
Cl
channel conductance
(GCl)
in heterologous expression systems or in cultured conditions (1, 3, 7,
9, 25, 26). However, all these compounds are relatively poor inhibitors
of CFTR
GCl,
requiring high concentrations for effects (3, 7, 9, 25, 26). These
compounds can also act as metabolic poisons (3, 8), making it difficult
to distinguish between direct effects on the channel and indirect
effects caused by depletion of ATP or cAMP or interference with the
phosphorylation activation of CFTR.
Preliminary studies from our laboratory revealed that the loop diuretic
bumetanide decreased transepithelial electrical conductance (Gt) and
significantly inhibited NaCl absorption in the native sweat duct (14).
It is well known that 106 M
bumetanide inhibits the
Na+-K+-2Cl
cotransporter and often has been used as a specific inhibitor of this
process. However, the
Na+-K+-2Cl
cotransporter is not responsible for transepithelial salt
absorption in the sweat duct (18, 21). Because bumetanide is
structurally related to CFTR
Cl
channel inhibitors such as 5-nitro-2-(3-phenylpropylamino)benzoic acid and diphenylamine-2-carboxylate and blocks other
Cl
channels (9, 28), we surmised that bumetanide inhibits salt transport
in the sweat duct by blocking CFTR
GCl.
Activation of CFTR requires PKA phosphorylation and physiological concentrations of ATP (2, 13, 20). Loop diuretics are known metabolic inhibitors that may indirectly affect CFTR GCl by inhibiting the regulatory process (3, 8). Consequently, we sought to determine whether bumetanide directly affects CFTR or whether it inhibits indirectly by interrupting the CFTR activation process. Here we show that bumetanide blocks CFTR GCl in the apical and basolateral membranes of the native sweat duct and that its inhibition of CFTR GCl is independent of any metabolic effects it may exert.
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METHODS |
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Tissue Acquisition
Sweat glands were obtained as previously described (12, 14, 15, 19, 20) from adult male volunteers without medical history who gave informed consent. The isolated glands were transferred to a cuvette with Ringer solution cooled to 5°C, where the segments of reabsorptive duct (~1 mm long) were separated from the secretory coil of the sweat gland under microscopic control (model SMZ-10, Nikon). With use of a glass transfer pipette, the sweat duct was transferred to a perfusion chamber containing Ringer solution for cannulation and microperfusion at 35 ± 2°C.Selective Permeabilization of the Basolateral Membrane
The basolateral membrane of the sweat duct was selectively permeabilized with a pore-forming agent (1,000 U ofElectrical Measurements
Electrical setup.
After the lumen of the sweat duct was cannulated with a double-lumen
cannula made from theta glass (1.5 mm diameter, Clark Electromedical
Instruments, Reading, UK), a constant-current pulse of 50-100 nA
for 0.5-10 s was injected through one barrel of the cannulating
pipette containing NaCl Ringer solution. The other barrel of the
cannulating pipette served as an electrode for measuring transepithelial potential
(Vt)
with respect to the contraluminal bath and as a cannula for perfusing
the lumen of the duct with selected solutions.
Vt
was monitored continuously using one channel of a WPI-700 dual
electrometer referenced to the contraluminal bath.
Gt
was measured as described earlier from the amplitude of
Vt deflections
(Vt) in
response to 50- to 100-nA transepithelial constant-current pulses by
using the cable equation (12).
Apical GCl.
Cl diffusion potentials
(VCl)
and
GCl
were monitored as indicative of the level of activation of
GCl.
After
-toxin permeabilization of the basolateral membrane, the
epithelium is simplified to a single (apical) membrane with parallel
Na+ and
Cl
conductances (13, 20, 22, 23). Application of amiloride further
simplifies the system to a predominantly
Cl
-selective
membrane. The composition of Ringer solution in bath and lumen was
designed to set up an ion gradient for the only permeable ion,
Cl
[140 mM KGlu
(bath)/150 mM NaCl (lumen)]. Under these conditions the
Vt
and
Gt
can be regarded as
VCl
and
GCl, respectively.
Microelectrode studies.
A Brown-Flaming microelectrode puller (model P-77) was used to pull
microelectrodes from Clark glass capillaries. Microelectrodes were
filled with 4 M potassium acetate. Electrode resistance was 40-80
M, and tip potential was <4 mV. A piezoelectric hybrid manipulator
(model PM-520, Frankenberger) was used to impale the cells.
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Solutions
In experiments using intact nonpermeabilized ducts, the luminal and contraluminal bath perfusion solutions contained (in mM) 150 NaCl, 0.38 KH2PO4, 2.13 K2HPO4, 1.2 MgSO4, 1 Ca(CH3COO)2, and 10 glucose at pH 7.4. Amiloride (0.01 mM) was added to the luminal solution as needed. The cytoplasmic (bath) solution used in experiments with permeabilized ducts contained 140 mM KGlu, 0.38 mM KH2PO4, 2.13 mM K2HPO4, 1.2 mM MgSO4, and 260 µM CaCl2 buffered with 2.0 mM EGTA (Sigma Chemical) to 80 nM free Ca2+ at pH 6.8. ATP (5 mM), 5 mM adenosine 5'-O-(3-thiotriphosphate) (ATPFirst, we tested the electrophysiological effects of 1 mM bumetanide on
the basolateral and luminal surfaces of intact microperfused duct. Then
we studied the effect of bumetanide (1-0.001 mM) as well as its
sister compound furosemide (1 mM) on the CFTR
GCl in the apical membranes of basolaterally -toxin-permeabilized and
microperfused sweat ducts. Bumetanide was mixed in 150 mM NaCl in
Ringer solution (or basic salt solution) to test the effect on the
basolateral and apical membranes of intact ducts. In the experiments
designed to test the effect of bumetanide and furosemide on cAMP- and
ATP-activated CFTR
GCl
from the cytoplasmic side of the apical membrane of permeabilized
ducts, we dissolved these compounds in 140 mM KGlu Ringer solution with
and without cAMP and ATP.
Data Analysis
The relative effect of inhibitors on VCl and GCl was evaluated in terms of percent inhibition in Vt and Gt. Values are means ± SE; n is the number of ducts from a minimum of four human subjects. Statistical significance was determined on the basis of Student's t-test for paired samples. P < 0.05 was taken as being significantly different. Results presented as electrophysiological traces are representative of at least three such experiments on ducts from as many subjects. ![]() |
RESULTS |
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We studied the effect of bumetanide in intact and basolaterally
-toxin-permeabilized sweat ducts. The effects of bumetanide were
fully reversible, even at the highest concentrations used in this study.
Intact Sweat Duct
The onset and washout of bumetanide effects were relatively slow in nonpermeabilized intact sweat duct. Peak effects on the electrical properties of the intact duct were slow to develop (5-10 min), even at 10Gt.
Application of 1 mM bumetanide in 150 mM NaCl Ringer solution to both
epithelial surfaces significantly decreased
Gt from 120 ± 8.7 to 18.9 ± 6.9 mS (n = 3). The
effect of bumetanide on Gt
was Cl
dependent. Bumetanide had no effect on electrical conductance in the
absence of
Cl
in
the medium (data not shown).
Cell membrane input impedance.
The untreated sweat duct cell membrane input impedance was 16.8 ± 1.7 M. Application of 1 mM bumetanide in the bath significantly increased the cell membrane input impedance by 58 ± 18.8% (4 cells from 3 sweat ducts; Fig. 2).
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Electrical potentials.
Under control conditions,
Vt,
Va,
and
Vb
were approximately 13,
28, and
41 mV,
respectively. Bumetanide (1 mM) in the bath significantly
hyperpolarized Vt
and Vb to
30 and
51 mV, respectively, and depolarized
Va to
21
mV (Table 1).
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VDR.
Under control conditions, the apical-to-basolateral membrane VDR was
4.4 ± 1.4 (n = 5). Bumetanide (1 mM) significantly increased the VDR to 5.2 ± 1.4 (n = 5; Fig.
3). Bumetanide also increased the amplitude
of voltage drops across the basolateral membrane (Vb increased
from 6.5 ± 1.4 to 7.6 ± 1.5 mV,
n = 5) and the apical membrane
(
Va increased
from 26.4 ± 6.7 to 36.4 ± 7.6 mV, n = 5; Figs. 1 and 3). For comparison,
the epithelial Na+ channel
inhibitor amiloride (0.01 mM), which is known to selectively increase
Ra,
caused a significant increase in VDR from 2.6 ± 0.5 to 3.4 ± 0.4 (n = 4; Fig. 3).
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Permeabilized Sweat Duct
CFTR GCl in the apical membrane of sweat duct was completely inhibited after
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Irreversible phosphorylation of apical CFTR.
We used a phosphatase inhibitor cocktail consisting of 0.001 mM okadaic
acid + 5 mM fluoride + 0.001 mM vanadate to inhibit endogenous
phosphatases. We also used phosphatase-resistant ATPS as substrate
for PKA phosphorylation of CFTR. Under these conditions, CFTR
was irreversibly phosphorylated after PKA activation by cAMP. We
confirmed that CFTR remained
"irreversibly"1
phosphorylated by adding ATP in the complete absence of cAMP and noting
that application of ATP alone resulted in normal increases in
GCl
(Fig. 5). We then tested the
effect of bumetanide on the irreversibly phosphorylated, ATP-activated
CFTR
GCl.
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Bumetanide concentration response.
Application of 1 mM bumetanide in the cytoplasmic bath inhibited
irreversibly phosphorylated ATP-activated CFTR
GCl.
Bumetanide at <0.01 mM had little effect on the
GCl-activated
CFTR, but significant inhibition occurred at 1 mM bumetanide (Fig. 5).
The concentration-response data revealed that ~300 µM bumetanide
caused a 50% reduction in CFTR
GCl
from control in the absence of bumetanide (Fig.
5B). The bumetanide effect slowly,
but mostly, reversed on washout of the inhibitor (Fig. 5). Preliminary
results suggested that another loop diuretic of the same family,
furosemide (1 mM), apparently caused less inhibition of activated CFTR
GCl
(Fig. 6), as indicated by persistently
larger
Cl
diffusion potential, even in the presence of furosemide.
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DISCUSSION |
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The loop diuretic bumetanide has long been considered a specific
inhibitor of the
Na+-K+-2Cl
cotransporter (3, 24). However, in the human sweat duct, in which the
Na+-K+-2Cl
cotransporter has little, if any, functional role in transepithelial Cl
absorption (18, 21), bumetanide significantly inhibited
Cl
transport and decreased
Gt (14). Because
the human sweat duct exhibits a very high electrical conductance
(>100 mS/cm2) as a result of
abundant expression of CFTR (12), we question whether the inhibitory
effect of bumetanide might be due to inhibition of CFTR
GCl.
Therefore, we addressed the following questions: 1) Where is the bumetanide-sensitive
GCl
located? 2) Does bumetanide block
CFTR
GCl?
3) Is inhibition indirect via
inhibition of phosphorylation of CFTR?
Transcellular vs. Paracellular Effects of Bumetanide
Transcellular and paracellular pathways contribute to ion transport in a number of epithelia (15). A decrease in transepithelial GCl as reported earlier could have been due to a decrease in transcellular CFTR GCl and/or paracellular GCl. There is no evidence of paracellular GCl in the sweat duct (15-17, 19, 20); however, bumetanide caused an ~60% increase in the cell membrane input impedance (Fig. 2), showing that bumetanide must inhibit transcellular GCl located in apical and/or basolateral cell membranes (16, 17).Does Bumetanide Block CFTR GCl?
Almost 90% of the electrical conductance across the sweat duct epithelium is due to CFTR ClBumetanide Inhibits GCl in Both Cell Membranes
Previously we showed that apical and basolateral membranes include a cAMP-activated GCl (19). To determine which cell membrane is bumetanide sensitive in terms of GCl, we measured the cell membrane potentials and the apical-to-basolateral membrane VDR (Table 1, Fig. 3) before and after application of bumetanide.Inhibition of basolateral GCl.
The results showed that bumetanide blocked basolateral membrane
GCl.
Application of bumetanide hyperpolarized
Vb
toward K+ electromotive force of
~72 mV (18). In CF ducts without basolateral GCl,
a similar hyperpolarization of
Vb
was observed (Fig. 3) (16). Bumetanide caused an increase in the
amplitude of the voltage drop
(Vb; Figs. 1
and 3, RESULTS) when transcellular
current was pulsed across
Rb.
Bumetanide can cause a significant increase in
Vb with
constant-current pulses only under two conditions: 1) the inhibitor causes an increase
in
Rb
by blocking a
GCl
across that membrane, or 2) it
causes a decrease in
Ra.
In fact, as discussed below, bumetanide increased
Ra.
If other parameters remained constant, this increase in
Ra
should have decreased the magnitude of
Vb (Fig. 1).
The significant increase in
Vb shows that
Rb increased significantly because of bumetanide inhibition of
GCl
in this membrane (17).
Inhibition of apical GCl.
Inhibition of the apical
GCl
depolarizes
Va
toward the Na+ electromotive force
across the Na+-selective membrane
(15-17, 19) and increases
Ra, causing an increase in VDR. Therefore, depolarization of
Va
(Table 1) and an increase in apical VDR (Fig. 3) after bumetanide
application are consistent with inhibition of
GCl
in the apical membrane. However, the weaker effect of bumetanide when
applied to the apical surface (14) raises other significant questions:
Is the basolateral membrane more permeable to bumetanide so that it can
only inhibit the apical
GCl
from the cytoplasmic side? Is bumetanide inhibition of apical
GCl
secondary to
GCl
inhibition at the basolateral membrane because of membrane cross talk
(16-18)? To address these questions, we permeabilized the
basolateral membrane with -toxin to functionally remove it from the
epithelium. Apical CFTR
GCl
was activated by exogenous application of cAMP and ATP, as described
earlier (13, 20). We directly tested the effect of bumetanide in the
cytoplasmic bath on apical CFTR
GCl
(Fig. 4) and found that bumetanide in the cytoplasmic bath inhibits
apical
GCl
in a dose-dependent manner. These results are consistent with an
earlier report that bumetanide inhibits on-cell CFTR
Cl
channels expressed in mouse L cells (27). The present results also show
that the basolateral membrane is more permeable to the inhibitor than
the apical membrane (10, 11) and that bumetanide inhibits CFTR
GCl
mainly from the cytoplasmic side.
Bumetanide Inhibits CFTR GCl Independent of PKA Phosphorylation
Bumetanide might be indirectly inhibiting the GCl by interfering with a regulatory mechanism involving cAMP and Ca2+ (11). Furthermore, loop diuretics act as metabolic inhibitors (3) that could indirectly affect cellular ATP and cAMP levels. Because activation of CFTR GCl is dependent on PKA phosphorylation and ATP (2, 5, 6, 13, 20, 22, 23), we asked whether bumetanide might appear to block CFTR GCl by interrupting the activation process.Irreversible phosphorylation of apical CFTR.
We used the following strategy to rule out the possibility that
bumetanide reduces CFTR
GCl
indirectly through inhibition of PKA phosphorylation or depletes
intracellular ATP levels. We permeabilized the basolateral membrane
with -toxin, as described earlier. CFTR
GCl
was completely abolished immediately after the permeabilization of the
basolateral membrane by loss of endogenous cAMP and ATP through
-toxin pores (Figs. 4 and 5) (13, 20), which promotes complete
dephosphorylation of CFTR. CFTR was then rephosphorylated in the
presence of cAMP and ATP
S, as described earlier, to create the
poorly hydrolyzable phosphate ester (20, 22, 23). We confirmed that
CFTR was irreversibly phosphorylated by addition of 5 mM ATP alone
without cAMP to activate CFTR
GCl. We then tested the effect of bumetanide on this irreversibly
phosphorylated, ATP-activated CFTR
GCl.
Inhibition is independent of metabolic effects. Some of the actions of loop diuretics could be due to metabolic poisoning (3). If bumetanide only inhibited CFTR indirectly by interrupting intracellular cAMP and ATP production or by inhibiting phosphorylation, it should not have had an effect on irreversibly phosphorylated, exogenous ATP-activated CFTR GCl. In fact, we found that bumetanide significantly blocked irreversibly phosphorylated, ATP-activated CFTR GCl in a concentration-dependent manner (Fig. 5). These results showed that, irrespective of potential effects of bumetanide on the process of regulation or ATP production, the inhibitor appears to directly block CFTR.
Relative Potency of Bumetanide
Bumetanide is a potent inhibitor of the Na+-K+-2ClConclusions
Bumetanide blocks CFTR GCl in apical and basolateral membranes of the native sweat duct. Bumetanide inhibition of CFTR GCl does not involve PKA phosphorylation or altered levels of cytosolic ATP but appears to be due to direct interaction with the channel protein. Bumetanide must be used with caution as a selective anion transport inhibitor, since it inhibits GCl and Cl ![]() |
ACKNOWLEDGEMENTS |
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The authors are grateful to Kirk Taylor for expert technical assistance and the numerous subjects who volunteered for skin biopsy.
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FOOTNOTES |
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This study was supported by grants from Cystic Fibrosis Research, Inc., and the National Cystic Fibrosis Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1
Proteins phosphorylated with ATPS are not
strictly irreversibly phosphorylated, but phosphatase dephosphorylation
of this group is so slow that, especially in the presence of
phosphatase inhibitors, it can be treated as irreversible.
Address for reprint requests: P. M. Quinton, Dept. of Pediatrics-0831, UCSD Medical Center, 220 Dickinson St., University of California, San Diego, San Diego, CA 92103-0831.
Received 6 April 1998; accepted in final form 28 September 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alton, E. W.,
and
A. J. Williams.
Modification of gating of an airway epithelial chloride channel by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB).
J. Membr. Biol.
28:
141-151,
1992.
2.
Anderson, M. P.,
H. A. Berger,
D. P. Rich,
R. J. Gregory,
A. E. Smith,
and
M. J. Welsh.
Nucleoside triphosphates are required to open the CFTR chloride channel.
Cell
67:
775-784,
1991[Medline].
3.
Cabantchik, Z. I.,
and
R. Greger.
Chemical probes for ion transporters of mammalian cell membranes.
Am. J. Physiol.
262 (Cell Physiol. 31):
C803-C827,
1992
4.
Fondacaro, J. D.
Intestinal ion transport and diarrheal disease.
Am. J. Physiol.
250 (Gastrointest. Liver Physiol. 13):
G1-G8,
1986[Medline].
5.
Gunderson, K. L.,
and
R. R. Kopito.
Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane regulator channel gating.
J. Biol. Chem.
269:
19349-19353,
1994
6.
Hwang, T.-C.,
G. Nagel,
A. C. Nairn,
and
D. C. Gadsby.
Regulation of CFTR Cl channels by phosphorylation and ATP hydrolysis.
Proc. Natl. Acad. Sci. USA
91:
4698-4702,
1994[Abstract].
7.
Keeling, D. J.,
A. G. Taylor,
and
P. L. Smith.
Effects of NPPB (5-nitro-2(3-phenylpropylamino)benzoic acid) on chloride transport in intestinal tissues and T84 cell line.
Biochim. Biophys. Acta
1115:
42-48,
1991[Medline].
8.
Lukacs, G. L.,
A. Nanda,
O. D. Rotstein,
and
S. Grinstein.
The chloride channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) uncouples mitochondria and increases the proton permeability of the plasma membrane in phagocytic cells.
FEBS Lett.
288:
17-20,
1991[Medline].
9.
McCarty, N. A.,
S. McDonough,
B. N. Cohen,
J. R. Riordan,
N. Davidson,
and
H. A. Lester.
Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl channel by two closely related arylaminobenzoates.
J. Gen. Physiol.
102:
1-23,
1993[Abstract].
10.
McGahan, M. C.,
T. Yoria,
and
P. J. Bentley.
The mode of action of bumetanide: inhibition of chloride transport across the amphibian cornea.
J. Pharmacol. Exp. Ther.
203:
97-102,
1977[Abstract].
11.
Patricia, R.,
O. A. Candia,
and
P. S. Reinach.
Mode of inhibition of active chloride transport in the frog cornea by furosemide.
Am. J. Physiol.
245 (Renal Fluid Electrolyte Physiol. 14):
F660-F669,
1983[Medline].
12.
Quinton, P. M.
Missing Cl conductance in cystic fibrosis.
Am. J. Physiol.
251 (Cell Physiol. 20):
C649-C652,
1986
13.
Quinton, P. M.,
and
M. M. Reddy.
Control of CFTR-Cl conductance by energy levels and non-hydrolytic ATP binding.
Nature
360:
79-81,
1992[Medline].
14.
Reddy, M. M.,
and
P. M. Quinton.
Effects of bumetanide on chloride transport in human eccrine sweat ducts.
Isr. J. Med. Sci.
23:
1210-1213,
1987[Medline].
15.
Reddy, M. M.,
and
P. M. Quinton.
Intracellular potentials of microperfused human sweat duct cells.
Pflügers Arch.
410:
471-475,
1987[Medline].
16.
Reddy, M. M.,
and
P. M. Quinton.
Altered electrical potential profile of human reabsorptive sweat duct cells in cystic fibrosis.
Am. J. Physiol.
257 (Cell Physiol. 26):
C722-C726,
1989
17.
Reddy, M. M.,
and
P. M. Quinton.
Localization of Cl conductance in normal and Cl
impermeability in cystic fibrosis sweat duct epithelium.
Am. J. Physiol.
257 (Cell Physiol. 26):
C727-C735,
1989
18.
Reddy, M. M.,
and
P. M. Quinton.
Intracellular potassium activity and the role of potassium in transepithelial salt transport in human reabsorptive sweat duct.
J. Membr. Biol.
119:
199-210,
1991[Medline].
19.
Reddy, M. M.,
and
P. M. Quinton.
cAMP activation of CF affected Cl conductance in both cell membranes of an absorptive epithelium.
J. Membr. Biol.
130:
49-62,
1992[Medline].
20.
Reddy, M. M.,
and
P. M. Quinton.
Rapid regulation of electrolyte absorption in sweat duct.
J. Membr. Biol.
140:
57-67,
1994[Medline].
21.
Reddy, M. M.,
and
P. M. Quinton.
Intracellular Cl activity: evidence of dual mechanisms of Cl
absorption in human sweat duct.
Am. J. Physiol.
267 (Cell Physiol. 36):
C1136-C1144,
1994
22.
Reddy, M. M.,
and
P. M. Quinton.
Deactivation of CFTR GCl by endogenous phosphatases in the native sweat duct.
Am. J. Physiol.
270 (Cell Physiol. 39):
C474-C480,
1996
23.
Reddy, M. M.,
and
P. M. Quinton.
Hydrolytic and nonhydrolytic interactions in the ATP regulation of CFTR GCl in the native sweat duct.
Am. J. Physiol.
271 (Cell Physiol. 40):
C35-C42,
1996
24.
Schlatter, E.,
R. Greger,
and
C. Wiedke.
Effect of "high ceiling" diuretics on the active salt transport in the thick ascending limb of Henle's loop of rabbit kidney. Correlation of chemical structure and inhibitory potency.
Pflügers Arch.
396:
210-217,
1983[Medline].
25.
Schultz, B. D.,
D. G. A. D. G. Deroos,
C. J. Venglarik,
A. K. Singh,
R. A. Frizzell,
and
R. J. Bridges.
Glibenclamide blockade of CFTR chloride channels.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L192-L200,
1996
26.
Sheppard, D. N.,
and
M. J. Welsh.
Effect of ATP sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.
J. Gen. Physiol.
100:
573-591,
1992[Abstract].
27.
Venglarik, C. J.
Furosemide and bumetanide block CFTR-Cl channels (Abstract).
Pediatr. Pulmonol. Suppl.
14:
R74,
1997.
28.
Wu, G.,
and
O. P. Hamill.
NPPB block of Ca2+-activated Cl currents in Xenopus oocytes.
Pflügers Arch.
420:
227-229,
1992[Medline].