cAMP-independent phosphorylation activation of CFTR by G proteins in native human sweat duct

M. M. Reddy and P. M. Quinton

Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California 92093-0831


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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It is generally believed that cAMP-dependent phosphorylation is the principle mechanism for activating cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. However, we showed that activating G proteins in the sweat duct stimulated CFTR Cl- conductance (GCl) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR GCl is independent of protein kinase A. We activated G proteins and monitored CFTR GCl in basolaterally permeabilized sweat duct. Activating G proteins with guanosine 5'-O-(3-thiotriphosphate) (10-100 µM) stimulated CFTR GCl in the presence of 5 mM ATP alone without cAMP. G protein activation of CFTR GCl required Mg2+ and ATP hydrolysis (5'-adenylylimidodiphosphate could not substitute for ATP). G protein activation of CFTR GCl was 1) sensitive to inhibition by the kinase inhibitor staurosporine (1 µM), indicating that the activation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM) and SQ-22536 (100 µM); and 3) independent of Ca2+, suggesting that Ca2+-dependent protein kinase C and Ca2+/calmodulin-dependent kinase(s) are not involved in the activation process. Activating AC with 10-6 M forskolin plus 10-6 M IBMX (in the presence of 5 mM ATP) did not activate CFTR, indicating that cAMP cannot accumulate sufficiently to activate CFTR in permeabilized cells. We concluded that heterotrimeric G proteins activate CFTR GCl endogenously via a cAMP-independent pathway in this native absorptive epithelium.

heterotrimeric G protein; cystic fibrosis; SQ-22536; dideoxyadenosine; electrolyte transport; absorption; fluid transport regulation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) is known to be a cAMP/ATP-dependent Cl- channel (14, 27, 30). The physiological significance of this channel is emphasized by the fact that abnormalities in this Cl- channel function cause severe life-threatening exocrinopathy including cystic fibrosis (CF) or secretory diarrhea (5, 13, 14). A clear understanding of the physiological mechanisms regulating this vital Cl- channel may aid in the development of therapies for diseases involving abnormal CFTR Cl- channel function.

CFTR is expressed in different exocrine glands [e.g., sweat glands, airways, pancreas, and intestine (13, 14, 30)] performing diverse physiological functions including transepithelial absorption and/or secretion of Cl-. Studies on cultured airway epithelial cells have indicated that activating heteromeric G proteins with guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) inhibits CFTR Cl- currents (28, 29). These studies also suggested that inhibiting these G proteins activates mutant CFTR Cl- currents in CF cells, suggesting that pharmacological manipulation of G proteins may have a significant therapeutic potential in treating CF (28, 29). However, in general, knowledge of physiological regulation of CFTR is minimal. The predominant function of the sweat duct is to absorb NaCl from the primary sweat secreted by the sweat secretory coil. Its single function and homogeneity of cellular structure make the sweat duct an almost an ideal model system in which to study physiological signal transductions that regulate CFTR in the context of native electrolyte absorption.

We have previously shown that a trimeric Gsalpha protein, which is known to activate adenylyl cyclase (AC) and increase cAMP, appears in the sweat duct apical membranes (23). Activating the G proteins with GTPgamma S results in a significant activation of CFTR conductance (GCl) in the basolaterally permeabilized native sweat duct (23). Exogenous application of high concentrations of cAMP activates CFTR GCl in basolaterally alpha -toxin permeabilized sweat ducts (20). These observations are consistent with the widespread notion that cAMP-dependent protein kinase A (PKA) phosphorylation is the principle endogenous mechanism for activating CFTR in the epithelial tissues (14, 27, 30). However, we were puzzled by finding that after the apical G proteins were activated, CFTR GCl activation was dependent only on ATP and did not require exogenous cAMP in the cytoplasmic perfusate medium (23). Furthermore, CFTR appears to be constitutively open in some epithelial cells, including sweat duct and Calu-3 airway epithelial cells (11, 19). However, attempts to deactivate CFTR by pharmacologically inhibiting cAMP production have not been successful (19). These results suggest that the predominant mechanism for activating CFTR in this absorptive epithelium might involve a G protein-activated mechanism that is independent of a cAMP cascade.

G proteins are a family of membrane-bound proteins that exist in both monomeric and heterotrimeric forms (2, 25, 29). The general scheme of signal transduction by heterotrimeric G proteins involves alpha beta gamma heteromers. When a receptor linked to G proteins is activated, the GTP binds to the alpha -subunit of the G protein complex and liberates it from the beta gamma complex. Both alpha -GTP complex and beta gamma complex are known to regulate cellular events (3, 8, 10). G proteins may regulate ion channels by different mechanisms including 1) regulation of AC/cAMP/PKA cascade-dependent phosphorylation; 2) regulating protein kinase C (PKC)-dependent phosphorylation through inositol phosphate metabolites and diacylglycerol, for example; and 3) direct interaction with channel proteins (2, 3, 8, 10).

The objective of this investigation was to determine whether AC and PKA phosphorylation mediate the G protein regulation of CFTR in NaCl absorption endogenously. We found that phosphorylation is involved in the G protein-induced activation of CFTR in the apical membranes of sweat duct but that, unexpectedly, such activation of CFTR appears to be independent of the AC/cAMP cascade in this native tissue.


    METHODS
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INTRODUCTION
METHODS
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Tissue Acquisition

Sweat glands were obtained from adult male volunteers without medical history who gave informed consent. Individual sweat glands were isolated from the skin in Ringer solution (maintained at ~5°C) by dissection with fine-tipped tweezers under a dissection microscope. The isolated glands were transferred to a cuvette with Ringer solution cooled to 5°C in which the segments of reabsorptive duct (~1 mm in length) were separated from the secretory coil of the sweat gland under microscopic control (model SMZ-10; Nikon). With the use of a glass transfer pipette, sweat ducts were 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 (alpha -toxin; 1,000 U/ml derived from Staphylococcus aureus) in cytoplasmic Ringer solution containing 140 mM K-gluconate and 5 mM ATP applied to the basolateral surface of the microperfused sweat duct for 15-30 min. As described earlier, alpha -toxin effectively remove the basolateral membrane as a barrier to cAMP and ATP without affecting the functional integrity of the apical membrane. This preparation allows free manipulation of intracellular cAMP, ATP, and GTP so that the properties of the regulation of CFTR GCl in the apical membranes can be studied in relative isolation from their endogenous metabolism (17, 20).

Electrical Measurements

Electrical setup. After the lumen of the sweat duct had been cannulated with a double-lumen cannula made from theta - glass, a constant current pulse of 50-100 nA was injected for a duration of 0.5 s 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 by using one channel of a WPI-700 dual electrometer referenced to the contraluminal bath. Transepithelial conductance (Gt) was calculated from the cable equation as described earlier (9, 17) by using the measured amplitude of the Vt deflections in response to the transepithelial constant current pulse.

Apical Cl- conductance. Cl- diffusion potentials (VCl) and GCl were monitored as indicators of the level of activation of GCl. Treatment with alpha -toxin to permeabilize the basolateral membrane simplified the epithelium to a single (apical) membrane with parallel Na+ and Cl- conductances. Application of amiloride further simplified the system to a predominantly Cl--selective membrane. The composition of Ringer solution in bath and lumen was designed to set up a single ion gradient, i.e., exclusively for Cl- [140 mM K-gluconate (bath)/150 mM NaCl (lumen)]. Under these conditions, the Vt and Gt can be regarded as VCl and GCl, respectively (17, 20, 22).

Solutions

The luminal perfusion R solutions contained (in mM) 150 NaCl, 5 K+, 3.5 PO<SUB>4</SUB><SUP>3−</SUP>, 1.2 MgSO4, 1 Ca2+, and 0.01 amiloride, pH 7.4. Cl--free luminal Ringer solution was prepared by complete substitution of Cl- with the impermeant anion gluconate. The cytoplasmic bath solution contained (in mM) 145 K+, 140 gluconate, 3.5 PO<SUB>4</SUB><SUP>3−</SUP>, and 1.2 MgSO4 as well as 260 µM Ca2+ buffered with 2.0 mM EGTA (Sigma) to 80 nM free Ca2+, pH 6.8. Nominally Mg2+-free cytoplasmic bath solution with 5 mM EDTA was used to prepare Mg2+-free solution. Nominally Ca2+-free cytoplasmic bath solution was prepared by adding 2 mM EGTA to Ca2+-free cytoplasmic bath solution. ATP (5 mM), adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S; 5 mM), cAMP (0.1 mM), GTPgamma S (0.1 mM), 5'-adenylylimidodiphosphate (AMP-PNP; 5 mM), AlCl3 (0.1 mM), KF (5 mM), 2',5'-dideoxyadenosine (DDA; 0.05-1 mM), and SQ-22536 (0.1) were added to the cytoplasmic bath as needed.

Data Analysis

VCl and GCl in bar graphs represent peak values that were stable for at least 2 min within ± 2 mV. Data are presented as means ± SE (n = number of ducts from a minimum of 4 human subjects). Statistical significance was determined on the basis of Student's t-test for paired samples. A P value of <0.05 was taken to be significantly different. Data presented as representative examples are taken from similar experiments repeated at least three times.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Effect of GTPgamma S

After basolateral alpha -toxin permeabilization of the sweat duct cytoplasmic nucleotides, responsible for activating CFTR, leak out of the cell and CFTR deactivates spontaneously as indicated by a virtually complete lack of GCl and VCl across the apical membrane. Reactivation of CFTR requires the presence of both 0.1 mM cAMP and 5 mM ATP in the cytoplasmic bath. Removing cAMP deactivates CFTR even in the presence of ATP, showing endogenous dephosphorylation of CFTR. However, application of 10-100 µM GTPgamma S to the cytoplasmic bath CFTR could be activated by ATP alone without requiring the addition of cAMP. After G protein-induced activation, application of 5 mM ATP alone increased GCl and VCl by 44.1 ± 19.5 mS/cm2 and 46.4 ± 4.8 mV, respectively (means ± SE, n = 7 ducts, P < 0.001). The effect of GTPgamma S on CFTR was irreversible for the duration of the experiment (>1 h).1 The effect of GTPgamma S could not be mimicked by 100 µM ATPgamma S, because we could not activate CFTR by subsequent addition of 5 mM ATP alone to the cytoplasmic bath (Fig. 1). The effect of G protein-induced activation on CFTR GCl was comparable to that of 0.1 mM cAMP plus 5 mM ATP (Fig. 1B).


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Fig. 1.   The effect of adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) and guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) on ATP activation of cystic fibrosis transmembrane conductance regulator (CFTR). A: GTPgamma S was applied to the cytoplasmic side in the complete absence of ATP. Excess 100 µM GTPgamma S in the bath was washed out. Under these conditions, application of 5 mM ATP activated CFTR conductance (GCl) independent of cAMP. This effect of GTPgamma S was not mimicked by ATPgamma S, indicating that phosphatase-resistant "irreversible" phosphorylation of CFTR by GTPgamma S was not responsible for the observed results. These results indicate that CFTR GCl is regulated by G proteins in the native sweat duct. B: summary of data collected from experiments similar to that shown in A. Notice that after G protein activation, ATP alone stimulated CFTR GCl comparable to activation with cAMP + ATP before G protein activation (n = 7, P < 0.001) as indicated by similar increases in Cl- diffusion potentials (VCl) and Cl- conductance (CFTR GCl).

Effect of Inhibiting Phosphorylation

Removing Mg2+ from the cytoplasmic bath significantly inhibited ATP activation of CFTR after GTPgamma S was applied to the duct. The magnitude of ATP-induced CFTR GCl and VCl, respectively, was 56.4 ± 14.3 mS/cm2 and 51.7 ± 16.4 mV in the presence of Mg2+ but only 6.8 ± 4.4 mS/cm2 and 3.8 ± 4.1 mV in the nominal absence of Mg2+ (n = 3, P < 0.02; Fig. 2). However, Mg2+ was not required for GTPgamma S activation of G proteins because application of GTPgamma S in the complete absence of Mg2+ resulted in sustained activation of G proteins. This effect was shown by the subsequent, prompt activation of CFTR GCl when ATP and Mg2+ were reintroduced without GTPgamma S (Fig. 2). However, after G proteins were similarly preactivated, the nonhydrolyzable ATP analog AMP-PNP (5 mM) did not activate CFTR (Fig. 3). Likewise, ATP failed to activate CFTR GCl after preactivating G proteins when endogenous kinases were inhibited by the nonselective kinase inhibitor staurosporine (10-6 M) (Fig. 4).


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Fig. 2.   G protein activation of CFTR GCl in the presence of ATP is Mg2+ dependent. A: this experiment tested whether ATP activation of CFTR requires Mg2+. Notice that G protein can be activated by GTPgamma S in the complete absence of Mg2+ as indicated by subsequent ATP activation of CFTR in the presence of Mg2+. B: after the G proteins are activated, ATP activation of CFTR critically requires Mg2+ because we could not activate CFTR GCl in the absence of Mg2+ in the cytoplasmic bath. These results indicate that ATP hydrolysis and a phosphorylation process probably are necessary to activate CFTR GCl. C: summary of data collected from experiments similar to that in B, showing a significant inhibition of G protein /ATP-induced CFTR GCl activity by Mg2+ removal from the cytoplasmic bath (n = 3, P < 0.02).



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Fig. 3.   ATP hydrolysis is required for activating CFTR after G protein activation. This experiment tested whether ATP hydrolysis is required for activating CFTR GCl after G proteins are activated. In this experiment, 100 µM GTPgamma S was applied to the cytoplasmic bath to activate G proteins before application of the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate (AMP-PNP) or physiological ATP. Notice that AMP-PNP had little effect, whereas ATP significantly activated CFTR GCl, indicating that ATP hydrolysis is required for G protein-mediated CFTR GCl activation.



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Fig. 4.   Effect of inhibiting kinases with staurosporine on G protein activation of CFTR. A: in this trace from a permeabilized duct, ATP alone does not activate CFTR even though CFTR is clearly present as shown by its response to cAMP + ATP. Likewise, after spontaneous deactivation, application of GTPgamma S alone did not activate CFTR. B: as shown by this trace, taken subsequently from the same duct as that in A, G proteins were activated by ATP alone (without cAMP) as confirmed by CFTR GCl activation. Application of 10-6 M staurosporine, a nonspecific kinase inhibitor, completely inhibited this ATP activation of CFTR GCl, indicating that a kinase phosphorylation process is involved in the ATP activation of CFTR after G protein activation.

Effect of cAMP-Elevating Agents

We tested the effect of cAMP-elevating agents on both the intact unpermeabilized and the alpha -toxin-permeabilized ducts. In the intact unpermeabilized ducts, CFTR GCl was monitored as the change in Gt (indicated by the magnitude of voltage deflections associated with transepithelial constant current pulses) and Vt (which included either 1) spontaneous potentials in 150 mM NaCl bilaterally or 2) diffusion potentials generated by 150 mM Na-gluconate in the lumen and 150 mM NaCl in the contraluminal bath). Application of forskolin (to activate AC) and IBMX (to inhibit phosphodiesterase) increased CFTR GCl in cAMP-responsive native sweat ducts,2 as indicated by an increase in Gt and corresponding changes in Vt (Fig. 5). In contrast, application of a cocktail of the cAMP-elevating agents forskolin and IBMX to the cytoplasm in the presence of ATP had no detectable effect on CFTR in basolaterally permeabilized sweat duct (Fig. 5). These results may indicate that small solutes such as cAMP cannot accumulate sufficiently to activate CFTR in permeabilized duct. Moreover, after permeabilization, the apical membrane conductance dramatically decreased, consistent with the loss of cytosolic CFTR-activating substances. Exogenous addition of cAMP or GTPgamma S in the presence of ATP rapidly restored CFTR GCl. These results show that permeabilizing the basolateral membrane with alpha -toxin probably depletes the cytoplasm of small molecules such as cAMP, cGMP, ATP, and GTP.


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Fig. 5.   Effects of IBMX and forskolin on CFTR GCl. A: this experiment tested the effect of 10-6 M IBMX on the CFTR GCl of native nonpermeabilized sweat duct. The lumen (L) and contraluminal bath (B) were perfused with 150 mM NaCl. Notice that the transepithelial conductance increased (indicated by decreasing transepithelial voltage deflections in response to 100 nA transepithelial constant current pulses) and transepithelial potential (Vt) decreased because of increased CFTR GCl shunt following IBMX application. B: this experiment tested the effect of 10-6 M forskolin on transepithelial Cl- diffusion potential [generated by 150 mM Na-gluconate (NaGlu) in the lumen and 150 mM NaCl in the contraluminal bath] and CFTR GCl in a nonpermeabilized intact sweat duct. Notice that forskolin increased transepithelial conductance (transepithelial voltage deflections in response to constant current pulses decreased) and luminally directed Cl- diffusion potential (Vt increased), indicating an increase in intracellular cAMP in both A and B. C: a representative example in which the effect of a cocktail of forskolin, IBMX, and ATP on CFTR GCl was studied on the basolaterally alpha -toxin-permeabilized sweat duct. Notice that forskolin + IBMX failed to activate CFTR GCl [indicated by a lack of increase in Cl- diffusion potentials and conductance (as observed in the case of cAMP + ATP application)]. These results suggest that the alpha -toxin prevents adequate intracellular accumulation of newly synthesized cAMP by allowing it to diffuse out of the permeabilized cell or, possibly, by blocking its production.

Effect of Inhibiting AC

Inhibiting AC with 1 mM DDA (a membrane-permeable inhibitor of AC) in the bath did not inhibit CFTR GCl in nonpermeabilized intact duct as indicated by the lack of effect of DDA on transepithelial GCl and VCl (Fig. 6). Application of either DDA (50 µM or 1 mM) or SQ-22536 (another AC inhibitor; 100 µM) in the cytoplasmic bath of basolaterally permeabilized duct also had little effect on G protein-induced activation of CFTR in the presence of ATP (Fig. 7). After G protein-induced activation, ATP increased CFTR GCl and VCl, respectively, by 36.8 ± 6.7 mS/cm2 and 47.1 ± 11.3 mV in the presence of DDA (1 mM) and by 38.9 ± 7.3 mS/cm2 and 53.0 ± 10.5 mV in the absence of DDA (n = 7). These results indicate that AC is not requisite to activate CFTR GCl.


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Fig. 6.   Lack of effect of inhibiting adenylyl cyclase (AC) with 2',5'-dideoxyadenosine (DDA) on CFTR GCl in nonpermeabilized intact duct. A: the sweat duct was perfused with Ringer solutions containing 150 mM NaGlu in the lumen and 150 mM NaCl in the bath. The transepithelial Cl- diffusion potential of approximately -80 mV reflects large spontaneous CFTR GCl in the nonpermeabilized ducts. B: the experiment shows lack of effect of 1 mM DDA on transepithelial Cl- diffusion potentials and conductance. The experimental conditions are the same as described above for A.



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Fig. 7.   Effect of inhibiting AC by DDA and SQ-22536 on G protein activation of CFTR. A: this experiment determined whether the G protein effect on CFTR is mediated by increased cAMP levels due to G protein activation of AC. Pharmacologically inhibiting AC with 2 different inhibitors (DDA and SQ-22536) had little effect on the G protein-induced activation (with GTPgamma S) of CFTR GCl in the presence of ATP. B: lack of effect of 1 mM DDA on G protein-induced activation of CFTR GCl. C: summary of data collected from experiments similar to that shown in B on the lack of effect of 1 mM DDA on G protein-induced activation of CFTR (n = 7).

Effect of Ca2+

We tested whether the G protein effector might require Ca2+ by removing Ca2+ from the cytoplasmic bath. Nominally Ca2+-free EGTA-buffered Ringer solution in the cytoplasm had little effect on either GTPgamma S or AlF<SUB>4</SUB><SUP>−</SUP>-mediated ATP activation of CFTR (Fig. 8).3 After G protein activation, ATP increased CFTR GCl and VCl, respectively, by 29.3 ± 6.3 mS/cm2 and 48.7 ± 6.3 mV in the presence of Ca2+ (80 nM) and by 30.9 ± 2.8 mS/cm2 and 42.0 ± 5.4 mV in the complete absence of Ca2+ (n = 4). Thus it seems unlikely that G protein activation of CFTR requires a Ca2+-dependent pathway.


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Fig. 8.   Lack of effect of Ca2+ removal on G protein-induced activation of CFTR. A: in this experiment we activated G proteins in the nominal absence of Ca2+ in the cytoplasmic bath. G proteins were activated by adding either 100 µM AlCl3 + 5 mM KF or 100 µM GTPgamma S to Ca2+-free (0 Ca2+) cytoplasmic Ringer solution containing 2 mM EGTA. G protein-induced activation by AlF<SUB>4</SUB><SUP>−</SUP> or GTPgamma S in the absence of Ca2+ is indicated by CFTR GCl stimulation by ATP alone without cAMP. B: summary of data collected from experiments similar to that shown in A. The effect of ATP on CFTR GCl after G protein-induced activation in the absence of Ca2+ (in the cytoplasmic bath) was compared with the standard effect of cAMP and ATP in the same ducts. Notice that Ca2+ had little effect on either GTP binding to G protein or ATP activation of CFTR as indicated by a comparable effects of G protein-activated (in the presence of ATP) CFTR GCl with that of cAMP + ATP-activated CFTR GCl (n = 4).

Lack of Synergistic Effect of GTPgamma S With cAMP

The effect of cAMP- and G protein-induced activation on the magnitude of CFTR GCl was comparable. Application of cAMP after G proteins were activated with GTPgamma S did not increase the ATP activation of CFTR GCl (Fig. 9).


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Fig. 9.   Lack of synergistic effects of G protein and cAMP activation on CFTR GCl. In this experiment the G proteins were activated by GTPgamma S. G protein activation was confirmed by CFTR GCl activation by ATP alone. Notice that addition of cAMP to the cytoplasmic bath did not enhance CFTR GCl activity, indicating that the two methods of activating CFTR GCl are not additive and that GTPgamma S activation is probably not simply the result of partially stimulating a cAMP-dependent pathway.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The apical membrane of the reabsorptive sweat duct expresses a number of heterotrimeric G proteins, including Gsalpha , Gialpha , Gqalpha , and Gbeta (unpublished immunocytochemical observations). It is well known that these heterotrimeric G proteins control the activity levels of a number of protein kinases, including those responsible for phosphorylation activation of CFTR such as PKA and PKC (2, 3, 7, 10). However, it is also known that regulation of a number of G protein-mediated ion channels involve direct interaction between the channel protein and the G protein (2, 3, 8, 10). Therefore, we also examined whether the activation of CFTR GCl by the apical G proteins involves phosphorylation or a direct interaction between CFTR and the G protein. Because kinase phosphorylation is involved in the G protein-mediated activation of CFTR, we investigated the possible role of cAMP/PKA cascade in the G protein-mediated activation of CFTR GCl by ATP alone (in the absence of exogenous cAMP) in the permeabilized duct.

G Protein-Induced Activation of CFTR Requires Phosphorylation

Kinase phosphorylation critically requires Mg2+ (20). Removing Mg2+ from the cytoplasmic bath before application of ATP prevented subsequent activation of CFTR by ATP (Fig. 2). However, Mg2+ also plays a critical role in GTPgamma S binding to the G proteins and in ATP hydrolysis (8, 20). The Galpha -GTPgamma S-Mg2+ complex is extremely stable, favoring beta gamma -subunit dissociation and activation of the G protein interaction with the target proteins (8). Therefore, we tested whether the lack of effect of ATP on CFTR in the absence of Mg2+ is due to the failure of GTPgamma S binding to the G protein. We exposed the apical membranes to GTPgamma S for ~1 min in the complete absence of Mg2+ and then washed out GTPgamma S with Mg2+-free solution. Subsequent addition of ATP in the presence of Mg2+ activated CFTR GCl. These results are surprising in light of previous reports that removing Mg2+ destabilizes Galpha -GTPgamma S, increases the rate of dissociation of GTPgamma S from Galpha , and increases the association of alpha -subunits with beta gamma -subunits, there by deactivating the G protein (8). These results suggest that 1) the apical G proteins may be unique in not requiring Mg2+ for GTPgamma S binding to the Galpha or 2) other divalent cations such as Ca2+ may replace Mg2+ in facilitating GTPgamma S binding to Galpha . Therefore, the failure of CFTR GCl to activate with ATP in the absence of Mg2+ is most likely due to a need for higher Mg2+ levels for phosphorylation or hydrolysis of CFTR than for GTPgamma S binding to the G protein.

We tested this possibility further by studying the effect of the nonhydrolyzable ATP analog AMP-PNP on CFTR GCl after activating G proteins with GTPgamma S (21). As shown in Fig. 3, only ATP, not AMP-PNP, activated CFTR GCl, confirming that ATP hydrolysis is required at one or more steps in the G protein cascade that activates CFTR. Because ATP hydrolysis is involved at two different kinetic steps, one requiring and the other independent of phosphorylation (20, 21), we tested whether ATP hydrolysis reflects the phosphorylation process. Although staurosporine is nonspecific, Fig. 4 shows that this inhibitor apparently prevented ATP activation of CFTR GCl, presumably by blocking endogenous kinase activity. These results indicate that a kinase-dependent phosphorylation step is required in G protein-induced activation of CFTR.

GTPgamma S Does Not Irreversibly Phosphorylate CFTR

CFTR GCl clearly can be activated by PKA, which is ATP and Mg2+ dependent (20, 21). Previous studies also showed that CFTR can be irreversibly phosphorylated by using ATPgamma S as substrate for PKA phosphorylation (20, 21). Under these conditions, CFTR GCl remained activated as long as ATP was present because the thiophosphorylated CFTR cannot be dephosphorylated (20, 21). Because application of GTPgamma S also activated CFTR GCl as long as ATP was present (Fig. 1), it is possible that GTPgamma S might thiophosphorylate CFTR by either a constitutively active kinase or a G protein-activated kinase using GTPgamma S as substrate. The possibility that an endogenously active kinase is responsible for CFTR phosphorylation is ruled out by the facts that 1) an equimolar concentration of ATPgamma S in the absence of cAMP failed to thiophosphorylate CFTR (CFTR was not subsequently activated by ATP alone; Fig. 1) and 2) previous studies showed that once CFTR was irreversibly thiophosphorylated, CFTR remained activated independent of Mg2+ (21). However, normal ATP phosphorylation activation of CFTR GCl critically depended on the presence of Mg2+ in the cytoplasmic bath. Removing Mg2+ after prior activation of CFTR GCl in the presence of Mg2+ and ATP deactivated CFTR GCl, possibly because endogenous phosphatase dephosphorylation overtook the Mg2+-dependent phosphorylation process (Figs. 2 and 3) or because CFTR gating is Mg2+ dependent at some level. Also, in the presence of staurosporine, ATP failed to activate CFTR GCl previously exposed to GTPgamma S. If CFTR had been irreversibly phosphorylated by GTPgamma S, inhibiting the kinase by staurosporine would not have prevented ATP activation of CFTR GCl, as shown in Fig. 4. Together, these results argue that G protein-induced activation in the presence of GTPgamma S and ATP does not cause irreversible thiophosphorylation of CFTR. In contrast, they suggest that activating the G proteins activates an as yet unknown kinase phosphorylation of CFTR.

PKA Phosphorylation Is Not Required to Activate CFTR

If G protein-mediated activation of CFTR requires phosphorylation but is not thiophosphorylated by GTPgamma S, as concluded above, then, we asked, does PKA mediate the phosphorylation activation? The apical membrane of this absorptive epithelium shows immunocytochemical labeling consistent with the presence of Gsalpha (unpublished observation). Furthermore, Gsalpha commonly stimulates AC to increase intracellular cAMP levels in a number of tissues (2, 3, 10). It is therefore tempting to assume that an apical Gsalpha activates an AC/cAMP/PKA-dependent phosphorylation activation of CFTR GCl. However, the following studies indicate that G protein-induced activation of CFTR does not involve the cAMP regulatory cascade.

No cAMP accumulation in permeabilized duct cells. The intact nonpermeabilized sweat duct has significant K+ and Cl- conductances in the basolateral membrane and Na+ and Cl- conductances in the apical membrane (17-20). Complete substitution of NaCl in the contraluminal bath with equimolar K-gluconate significantly depolarizes the basolateral membrane and transepithelial potentials (20, 21). Permeabilizing the basolateral membrane with alpha -toxin removes the basolateral membrane as a functional barrier so that intracellular cAMP cannot accumulate. After alpha -toxin, first, K+ and Cl- diffusion potentials across the basolateral membrane were abolished (Vt of about +11 mV reflects the junction potential), and the K+ conductance inhibitor (Ba2+) or Na+-K+-pump inhibitor (ouabain) had no effect on basolateral membrane potential after permeabilization (20); second, isoproterenol (beta -adrenergic agonist) variably induced activation of CFTR GCl (19) [possibly by increasing intracellular cAMP levels via a G protein-coupled mechanism (7, 31)] but did not have an effect on the Cl- conductance of permeabilized ducts (results not shown). More specifically, the AC activator forskolin and the phosphodiesterase inhibitor IBMX together activated CFTR GCl in some nonpermeabilized ducts but never in alpha -toxin-permeabilized ducts (Fig. 5). These results suggest that any newly synthesized cAMP does not accumulate sufficiently inside the cell to activate PKA phosphorylation of CFTR (Fig. 5). This conclusion is further corroborated by the fact that during alpha -toxin permeabilization, the apical CFTR GCl becomes almost completely deactivated but can be reactivated quickly by the addition of exogenous cAMP and ATP to the cytoplasmic bath perfusate (Fig. 1) (20).

No effect of inhibiting AC on G protein-induced activation of CFTR GCl. CFTR GCl is maximally activated in a majority of the isolated microperfused sweat ducts (19). One possible explanation for such persistent activation of CFTR GCl could be that intracellular cAMP levels are elevated because of continuous G protein stimulation of AC. If this were the case, CFTR GCl should be deactivated by inhibiting AC. There are about 10 different isoforms of AC in mammalian tissues (31). DDA and SQ-22536 inhibit all known forms of AC and block cAMP production. Thus we tested the effect of AC inhibitors on the Cl- conductance of intact nonpermeabilized ducts. CFTR GCl remained high and unaffected by DDA (even at 1 mM), suggesting that intracellular cAMP is not responsible for the constitutive, persistent activation of CFTR in the native sweat duct (Fig. 6). We also tested the effect of DDA and SQ-22536 in the cytoplasmic bath on GTPgamma S/ATP activation of CFTR GCl in the permeabilized duct to be certain that the inhibitors diffused into the cell and that the microdomains of AC/PKA did not escape inhibition. Figure 7 shows that these inhibitors did not prevent G protein-induced activation of CFTR GCl. These results strongly indicate that G protein-mediated signal transduction associated with CFTR GCl activation does not involve an AC/cAMP cascade in this salt-absorbing epithelium.

Phosphorylation is Ca2+ Independent

G proteins also effect signal transduction through phospholipase C and PKC (12). Because Ca2+ plays a significant role in PKC- and calmodulin-dependent kinases, we tested the effect of removing Ca2+ on both cAMP- and GTPgamma S-mediated activation of CFTR GCl. Figure 8 shows that removing Ca2+ did not have an effect on the magnitude of G protein-mediated ATP activation of CFTR GCl, indicating the finding that Ca2+-dependent pathways do not play a direct role in the G protein-induced activation of CFTR.

What Are the Alternative Phosphorylation Pathways?

Some G protein-coupled signal transduction mechanisms involve cGMP (8, 12). We and others have shown that cGMP activates CFTR GCl in this tissue (6, 17, 30). However, it is not presently clear how G protein activation of CFTR GCl would involve phosphorylation by a cGMP-dependent kinase (G-kinase). G proteins generally activate cGMP phosphodiesterase, which would decrease, not increase, intracellular cGMP (8, 12). Furthermore, even if G protein-induced activation increased intracellular cGMP production, it is unlikely that this intracellular cyclic nucleotide would accumulate in this permeabilized tissue any better than cAMP to effect a G-kinase phosphorylation activation of CFTR. In fact, after permeabilization, cGMP and ATP were required exogenously to activate CFTR GCl, and CFTR GCl was promptly deactivated when cGMP was washed out from the cytoplasmic bath, indicating that alpha -toxin pores are highly permeable to these nucleotides (17). cAMP activates CFTR GCl in a number of epithelial tissues (13, 14, 26, 32). If GTPgamma S activation of CFTR GCl involves another component, independent of cAMP (23), it might show additive effects on GCl stimulation. However, simultaneous application of cAMP and GTPgamma S did not increase CFTR GCl (Fig. 9). These results cannot distinguish whether cAMP and G proteins activate CFTR via common phosphorylation or act independently to maximally activate by either mechanism. Alternatively, the G proteins might activate PKA (hence, CFTR phosphorylation) by a mechanism that is independent of AC/cAMP cascade in this tissue. Other kinases including but not limited to a Ca2+-independent PKC isoform may also play a role. At this time, we cannot further identify the G protein-mediating kinase involved in activating CFTR in the sweat duct. Further investigation is needed to determine which of the numerous protein kinase(s) is specifically involved in G protein-induced phosphorylation activation of CFTR in the sweat duct.

Why Are CFTR Cl- Channels Constitutively Open in the Duct?

CFTR GCl is constitutively activated in sweat duct cells and under some culture conditions in Calu-3 cells derived from the airways, as well (11, 19). There are at least three distinct possibilities that could explain this phenomenon. First, it is possible that the endogenous levels of cAMP are consistently elevated so that PKA keeps CFTR phosphorylated. However, this does not seem to be a viable explanation because we could not deactivate CFTR after inhibiting cAMP production, conditions that should lead to dephosphorylation. Overnight incubation of ducts in a cocktail containing inhibitors of cAMP production, including propranolol (to block beta -adrenergic receptor) and indomethacin (to block cyclooxygenase and prostaglandin synthesis), did not inhibit CFTR GCl (15, 19). In addition, inhibiting AC in intact nonpermeabilized sweat ducts with DDA or SQ-22536 did not inhibit the constitutively open CFTR GCl (Fig. 6), suggesting that elevated cAMP levels are less likely to be the cause of constitutively active CFTR GCl. Second, very low endogenous phosphodiesterase or phosphatase activities in the intact duct cells may allow CFTR to remain activated. However, we know that CFTR GCl is rapidly dephosphorylated by active endogenous phosphatases in permeabilized cells (22) so that low phosphodiesterase/phosphatase activities seem not to explain constitutive CFTR GCl activation. Third, chronically activated receptors may constitutively stimulate apical G proteins to activate CFTR as long as appropriate levels of ATP exist in the cell. The observation that once GTPgamma S was bound to the G proteins, CFTR remained activated in the presence of ATP alone is consistent with, but not proof of, this notion.

What Triggers the G Protein-Mediated Activation of CFTR GCl?

Unpublished results involving the use of immunocytochemical labeling techniques revealed the presence of Gsalpha , Gialpha , and Gqalpha in the apical membrane. These observations suggested that G proteins in the apical membrane control CFTR in the sweat duct. As discussed above, if activation of the apical G proteins is, in fact, responsible for constitutively opening CFTR Cl- channels in the apical membrane, we must ask, what sustains stimulation of the G proteins in the intact duct? Luminal perfusate (NaCl-containing Ringer solution) is devoid of neurohumoral agents that might otherwise stimulate G proteins. Early reports indicated that changes in the ionic environment might regulate G protein activation. Changes in the cytosolic Cl- concentration were reported to alter G protein regulation of epithelial Na+ channel function in salivary duct epithelial cell (4). Changes in Cl- concentrations appeared to inhibit GTPase activity [hence, to activate G protein (8)]. We therefore tested the effect of increasing cytosolic Cl- concentration from 0 to 140 mM on G protein activation of CFTR. We found that cytosolic Cl- had little effect on CFTR GCl activation by GTPgamma S in the presence of ATP (results not shown). It is known that luminal [NaCl] changes from isotonic to <15 mM as a function of secretory rate (1, 24). We asked whether changes in [Na+] have an effect on G protein activation of CFTR. We found that removing Na+ (by substituting with K+) did not prevent GTPgamma S activation of CFTR GCl (results not shown). Further studies are required to determine the mechanisms that activate apical G proteins and CFTR.

Implications for Cystic Fibrosis

CFTR GCl is significantly reduced or almost completely absent in most CF-affected epithelium (13, 14, 30). Until now, it has been widely believed that cAMP-dependent phosphorylation of CFTR is the predominant physiological mechanism for activating CFTR GCl in a number of epithelial cells in airways, pancreas, intestine, and sweat glands (13, 14, 30). However, our results here suggest that the G protein-induced signal transduction leading to the activation of CFTR may not involve AC/cAMP cascade. Earlier studies on cultured airway epithelial cells indicated that activating the G proteins inhibits CFTR Cl- channels (29). However, it is unclear whether such inhibition is a generalized phenomenon applicable to all the transporting cells (i.e., secretory as well as absorptive cells) within the airways or whether the cells performing absorptive function in the airways exhibit G protein-induced activation of CFTR similar to that in sweat duct cells. Potential therapeutic strategies aimed at modulating the G protein regulation of CFTR within the airways must take into account possible differential effects of G proteins on CFTR as a dependent function of vectorial transport (absorption vs. secretion) in different cell types.

Conclusion

G proteins activate CFTR GCl in the native sweat duct. Kinase phosphorylation is involved in the G protein-mediated CFTR GCl activation, but the AC/cAMP cascade may not play a direct role in this regulatory process.


    ACKNOWLEDGEMENTS

We are grateful to Kirk Taylor and Michael Adams for expert technical assistance and to the numerous volunteer subjects who supported these investigations.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-51899 and by grants from the National Cystic Fibrosis Foundation and Gillette Co.

Address for reprint requests and other correspondence: P. M. Quinton, Dept. of Pediatrics-0831, School of Medicine, Univ. of California, San Diego, La Jolla, CA 92093-0831.

1 We use "irreversible" to mean irreversible in practice, i.e., so slowly reversible that it appears irreversible within the time frame of our observations.

2 Not all intact ducts respond to cAMP-mediated agonist because CFTR is usually spontaneously activated in the duct, presumably to its maximal activated state.

3 AlF<SUB>4</SUB><SUP>−</SUP> is commonly used to activate heterotrimeric G proteins as opposed to monomeric forms.

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. Section 1734 solely to indicate this fact.

Received 9 June 2000; accepted in final form 10 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 280(3):C604-C613
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