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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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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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
106 M forskolin plus 106 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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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 GTPS 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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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 (-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, -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 
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 PO
, 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, 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) (ATPS; 5 mM), cAMP (0.1 mM),
GTPS (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.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of GTPS
-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 GTPS 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 GTPS on CFTR was
irreversible for the duration of the experiment (>1
h).1 The effect of GTPS
could not be mimicked by 100 µM ATPS, 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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Effect of Inhibiting Phosphorylation
Removing Mg2+ from the cytoplasmic bath significantly inhibited ATP activation of CFTR after GTPS 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 GTPS activation of G proteins because application of GTPS 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 GTPS (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 (106 M) (Fig.
4).
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Effect of cAMP-Elevating Agents
We tested the effect of cAMP-elevating agents on both the intact unpermeabilized and the-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 GTPS in the presence of
ATP rapidly restored CFTR GCl. These results
show that permeabilizing the basolateral membrane with -toxin
probably depletes the cytoplasm of small molecules such as cAMP, cGMP,
ATP, and GTP.
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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 GTPS
or AlF
-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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Lack of Synergistic Effect of GTPS With cAMP
S did
not increase the ATP activation of CFTR GCl
(Fig. 9).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 GTPS binding to the G proteins and in ATP hydrolysis (8,
20). The G-GTPS-Mg2+ complex is extremely
stable, favoring -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 GTPS binding to
the G protein. We exposed the apical membranes to GTPS for ~1 min
in the complete absence of Mg2+ and then washed out GTPS
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 G-GTPS, increases the rate
of dissociation of GTPS from G, and increases the association of
-subunits with -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 GTPS
binding to the G or 2) other divalent cations such as
Ca2+ may replace Mg2+ in
facilitating GTPS binding to G. 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 GTPS 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 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
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 GTPS also activated
CFTR GCl as long as ATP was present (Fig. 1), it
is possible that GTPS might thiophosphorylate CFTR by either a
constitutively active kinase or a G protein-activated kinase using
GTPS 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 ATPS 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 GTPS. If CFTR had been irreversibly
phosphorylated by GTPS, 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 GTPS 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 GTPS, 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 Gs (unpublished
observation). Furthermore, Gs 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
Gs 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
-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 GTPS/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 GTPS-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-toxin pores are highly permeable to these
nucleotides (17). cAMP activates CFTR
GCl in a number of epithelial tissues (13, 14, 26, 32). If GTPS 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 GTPS 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?
-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 GTPS 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 Gs,
Gi, and Gq 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 GTPS 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
GTPS 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 |
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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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TOP
ABSTRACT
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METHODS
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DISCUSSION
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