Bumetanide blocks CFTR GCl in the native sweat duct

M. M. Reddy and P. M. Quinton

Department of Pediatrics, University of California, San Diego, Medical Center, San Diego, California 92103-0831

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 10-6 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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 of alpha -toxin derived from Staphylococcus aureus) in cytoplasmic Ringer solution containing 140 mM potassium gluconate (KGlu) and 5 mM ATP applied to the basolateral surface of the microperfused sweat duct for 15-30 min. As described earlier (13, 20, 22, 23), alpha -toxin effectively removes the basolateral membrane as a barrier to cAMP and ATP without affecting the functional integrity of the apical membrane (13, 20, 22, 23). This preparation allows exogenous manipulation of intracellular cAMP and ATP so that the properties of the regulation of CFTR GCl in the apical membranes can be studied in relative isolation.

Electrical 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 (Delta 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 alpha -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 MOmega , and tip potential was <4 mV. A piezoelectric hybrid manipulator (model PM-520, Frankenberger) was used to impale the cells.

CELL POTENTIALS. The criteria for accepting results from impaled cells are the same as described earlier (15-17). Basolateral membrane potential (Vb) and Vt were measured with respect to bath ground. Apical membrane potential (Va) was calculated as the difference between Vb and Vt (15-17).

INPUT IMPEDANCE. Input impedance was measured by injecting 0.3-nA constant-current pulses into the impaled cell for 0.5 s by using a single-electrode voltage-clamp amplifier (model 8100-1, Dagon) in switched-clamp or bridge current-clamp mode (15). The magnitude of voltage deflections induced by current pulses reflects the input impedance of the cell membranes.

VOLTAGE DIVIDER RATIO. During a 0.5- to 10-s constant transepithelial current pulse of 50-100 nA, the Vb deflection (Delta Vb) was measured directly with the impaling microelectrode. The Va deflection (Delta Va) was determined by subtracting Delta Vb from Delta Vt. The voltage divider ratio (VDR) was taken as Delta Va/Delta Vb, which is proportional to the ratio of the resistance of the apical membrane (Ra) to the resistance of the basolateral membrane (Rb) or Ra/Rb (Fig. 1) (17). The effect of bumetanide on VDR was assessed during perfusion of the luminal and contraluminal bath with Ringer solution containing 150 mM NaCl without amiloride in the lumen. We separately studied the effect of luminal amiloride on the VDR under similar perfusion conditions but without bumetanide in the contraluminal bath (see Fig. 3B).


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Fig. 1.   A simplified model of voltage divider ratio (VDR) measurements. Apical and basolateral membranes of duct epithelium are represented by 2 series resistances (Ra and Rb, respectively) in parallel with electrical resistance at tight junctions (Rs). Voltage drops across apical (Delta Va) and basolateral (Delta Vb) membranes are proportional to resistances across respective membranes, i.e., VDR = Delta Va/Delta Vb = Delta Ra/Delta Rb. By following changes in VDR as well as absolute amplitudes of Delta Va and Delta Vb, it is possible to predict qualitative changes in electrical conductances at each membrane; e.g., if Delta Va, Delta Vb, and VDR increase after bumetanide, we can conclude that inhibitor increased Ra more than Rb. Increases in Ra reduce relative magnitude of transcellular current (ic) while simultaneously increasing current flow across paracellular shunt (is). Under these conditions, if Rb remains constant or decreases, Delta Vb must decrease. An absolute increase in Delta Vb across Rb in face of decreasing transcellular current (and an increased VDR) would indicate that inhibitor increased Rb along with Ra. Bumetanide in bath increased VDR and transcellular current pulse-induced Delta Vb, indicating that inhibitor affected Cl- conductance (GCl) in both cell membranes.

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) (ATPgamma S), and 0.01 mM cAMP were added to the cytoplasmic bath as needed. Phosphatase inhibitors fluoride (5 mM), vanadate (0.001 mM), and okadaic acid (0.001 mM) were added to the cytoplasm as a phosphatase inhibition cocktail.

First, 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 alpha -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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Abstract
Introduction
Methods
Results
Discussion
References

We studied the effect of bumetanide in intact and basolaterally alpha -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 10-3 M. Bumetanide at <0.1 mM had little effect on the electrical properties of the intact microperfused sweat duct. Application of bumetanide to the luminal perfusate produced inconsistent and relatively smaller effects on the electrical properties. Using the intact microperfused sweat duct, we also examined the effect of 1 mM bumetanide on the cell input impedance, Vt, Va, and Vb, as well as on the apical-to-basolateral membrane VDR.

Gt. 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 MOmega . 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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Fig. 2.   Effect of bumetanide on cell membrane input impedance. A: an intact nonpermeabilized sweat duct cell impaled with a microelectrode. After stabilization of Vb and transepithelial potential (Vt), a 0.3-nA constant-current pulse for 0.5 s was injected into cell through microelectrode. Amplitude of voltage deflections is proportional to cell input impedance. Bumetanide [10-3 M in basic salt solution (BSS)] in contraluminal bath significantly hyperpolarized Vb and Vt while increasing cell input impedance, as indicated by significantly larger downward voltage deflections during bumetanide application. Bumetanide inhibited ion conductance in epithelial cell membranes. B: summary of data collected from experiments similar to example in A.

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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Table 1.   Effect of bumetanide on electrical potentials of human reabsorptive sweat duct

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 (Delta Vb increased from 6.5 ± 1.4 to 7.6 ± 1.5 mV, n = 5) and the apical membrane (Delta 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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Fig. 3.   Effect of bumetanide on VDR. A: bumetanide (10-3 M in BSS) in contraluminal bath increased Rt and VDR of an intact nonpermeabilized sweat duct. Bumetanide increased VDR while simultaneously increasing Delta Vb and Delta Vt. Bumetanide increased Ra more than Rb. Contraluminal and luminal perfusate Ringer solutions contained 150 mM NaCl without luminal amiloride. B: summary of data on VDR compiled from experiments similar to example in A. Even though bumetanide was applied to basolateral membrane surface, effect of inhibitor was more pronounced on apical membrane. Our results show that bumetanide is permeable to basolateral membrane to block apical cystic fibrosis transmembrane conductance regulator (CFTR) GCl from cytoplasmic side. In comparison, luminal amiloride (without bumetanide in contraluminal bath) also increased VDR.

Permeabilized Sweat Duct

CFTR GCl in the apical membrane of sweat duct was completely inhibited after alpha -toxin permeabilization of the basolateral membrane, because cAMP and ATP wash out of the cytoplasmic compartment (Fig. 4). Exogenous application of cAMP and 5 mM ATP restored CFTR GCl in the apical membrane, as indicated by a large increase in the Cl- diffusion potential and Gt. Bumetanide in the cytoplasmic bath inhibited the cAMP + ATP-activated CFTR GCl in the apical membrane (Fig. 4).


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Fig. 4.   Effect of bumetanide on cAMP + ATP-activated apical CFTR GCl. Basolateral membrane was permeabilized with alpha -toxin. CFTR GCl in apical membrane was activated by exogenous 0.01 mM cAMP + 5 mM ATP. Then effect of 0.001-1 mM bumetanide on CFTR GCl was tested. Bumetanide significantly blocked CFTR GCl, even in presence of cAMP and ATP. Bumetanide does not inhibit CFTR GCl by depleting cell of cAMP or ATP. VCl, Cl- diffusion potential.

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 ATPgamma S 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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Fig. 5.   Concentration-response relationship for bumetanide inhibition of CFTR GCl after irreversible phosphorylation of CFTR in a permeabilized duct. A: CFTR was phosphorylated in presence of adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) + cAMP + phosphatase inhibition cocktail (PIC). Irreversible phosphorylation of apical CFTR was indicated by activation of GCl by ATP alone without cAMP. Then effect of different concentrations of bumetanide on irreversibly phosphorylated ATP-activated CFTR GCl was tested. Bumetanide affected CFTR GCl only at high concentrations (>0.01 mM). Bumetanide at 1 mM inhibited >70% of apical CFTR GCl, even after irreversible phosphorylation. Bumetanide inhibits CFTR GCl irrespective of any potential effects on process of phosphorylation. B: concentration-response curve for bumetanide inhibition of CFTR GCl. Reduction of total CFTR GCl is expressed as Gi/Gmax, where CFTR GCl in presence of a given concentration of bumetanide (Gi) is compared with CFTR GCl in absence of bumetanide (Gmax). Ki, concentration of bumetanide causing a 50% reduction from control values.

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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Fig. 6.   Effect of furosemide in cytoplasmic bath on irreversibly phosphorylated CFTR in a permeabilized duct. First, CFTR was irreversibly phosphorylated by incubating apical membrane preparations of duct in cytoplasmic perfusate containing 5 mM ATPgamma S + 0.01 mM cAMP + PIC. Irreversible phosphorylation of CFTR (see Fig. 5) was confirmed by activating GCl activity by ATP alone without cAMP. Then effect of 1 mM furosemide on irreversibly phosphorylated, ATP-activated apical CFTR GCl was tested. Furosemide also inhibited irreversibly phosphorylated apical CFTR GCl. However, these preliminary results suggested that furosemide apparently caused less inhibition of CFTR GCl than bumetanide at same concentration (see Fig. 5).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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 Cl- channels in the apical and basolateral membranes (12, 19). Our results indicate that bumetanide blocks ~85% of the Gt in the intact microperfused sweat duct. The absence of functional CFTR Cl- channels in the plasma membranes of CF sweat ducts causes effects similar to those induced by bumetanide: increased Vt, decreased Gt by ~90%, and decreased Cl- absorption (12). These observations imply that bumetanide primarily inhibits transepithelial GCl in the sweat duct, and since the GCl of the sweat duct is almost, if not exclusively, due to CFTR (12, 16, 17, 19), we conclude that bumetanide inhibits this channel.

Bumetanide 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 (Delta Vb; Figs. 1 and 3, RESULTS) when transcellular current was pulsed across Rb. Bumetanide can cause a significant increase in Delta 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 Delta Vb (Fig. 1). The significant increase in Delta 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 alpha -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.

We previously reported that CFTR Cl- channels reside in apical and basolateral membranes of this absorptive epithelium (16-18). Therefore, it is not surprising that the GCl in both cell membranes also share common pharmacological properties, which supports our earlier contention.

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 alpha -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 alpha -toxin pores (Figs. 4 and 5) (13, 20), which promotes complete dephosphorylation of CFTR. CFTR was then rephosphorylated in the presence of cAMP and ATPgamma 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+-2Cl- cotransporter compared with furosemide (3, 10). Preliminary data indicated that bumetanide appears to be more effective than furosemide in inhibiting phosphorylated, ATP-activated CFTR GCl in the apical membranes of permeabilized sweat ducts (Figs. 5 and 6). Present results are consistent with an earlier observation that bumetanide is a more potent inhibitor of wild-type on-cell CFTR Cl- channels in ex vivo systems than furosemide (27). These observations suggest that both of these anion transport proteins share pharmacological characteristics. However, when blocking the Na+-K+-2Cl- cotransporter (3, 24), bumetanide is effective at much lower concentrations (10-6 M) than our results here. Therefore, although bumetanide may be used to selectively block the cotransporter, the reverse is not true. Because Cl- is the common ion transported by both proteins, inhibition of both proteins by the same inhibitor further supports the notion that bumetanide interacts directly with the transport molecule. Nonetheless, careful attention to its application and interpretation is warranted if it is to be used as a selective inhibitor.

Conclusions

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- cotransporter mechanisms.

    ACKNOWLEDGEMENTS

The authors are grateful to Kirk Taylor for expert technical assistance and the numerous subjects who volunteered for skin biopsy.

    FOOTNOTES

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 ATPgamma S 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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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