Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California 92093-0831
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ABSTRACT |
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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
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INTRODUCTION |
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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) (GTP
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 Gs 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 GTP
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
-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 heteromers. When a receptor linked to G proteins is
activated, the GTP binds to the
-subunit of the G protein complex
and liberates it from the
complex. Both
-GTP complex and
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.
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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 (Electrical Measurements
Electrical setup.
After the lumen of the sweat duct had been cannulated with a
double-lumen cannula made from 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
-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 POData 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. ![]() |
RESULTS |
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Effect of GTPS
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Effect of Inhibiting Phosphorylation
Removing Mg2+ from the cytoplasmic bath significantly inhibited ATP activation of CFTR after GTP
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Effect of cAMP-Elevating Agents
We tested the effect of cAMP-elevating agents on both the intact unpermeabilized and the
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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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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 GTP
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Lack of Synergistic Effect of GTPS With cAMP
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DISCUSSION |
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The apical membrane of the reabsorptive sweat duct expresses a
number of heterotrimeric G proteins, including Gs,
Gi
, Gq
, and G
(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 GTPWe tested this possibility further by studying the effect of the
nonhydrolyzable ATP analog AMP-PNP on CFTR GCl
after activating G proteins with GTPS (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.
GTPS Does Not Irreversibly Phosphorylate CFTR
PKA Phosphorylation Is Not Required to Activate CFTR
If G protein-mediated activation of CFTR requires phosphorylation but is not thiophosphorylated by GTPNo 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
-toxin removes the basolateral membrane as a functional barrier so
that intracellular cAMP cannot accumulate. After
-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 (
-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
-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
-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 GTP
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 GTPWhat 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 thatWhy Are CFTR Cl Channels Constitutively Open in
the Duct?
What Triggers the G Protein-Mediated Activation of CFTR GCl?
Unpublished results involving the use of immunocytochemical labeling techniques revealed the presence of GsImplications 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 ClConclusion
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 |
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We are grateful to Kirk Taylor and Michael Adams for expert technical assistance and to the numerous volunteer subjects who supported these investigations.
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FOOTNOTES |
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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 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.
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