Departments of 1 Pediatrics and 3 Medicine and 2 Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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ABSTRACT |
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ATP and its
metabolites stimulate Cl
secretion in human epithelium in vitro and in vivo. The specific
purinergic receptor subtypes that govern these effects have been
difficult to separate, in part due to multiple parallel pathways for
Cl
secretion in respiratory
and intestinal epithelia. In a simplified model using COS-7 cells, we
demonstrate acquisition of an ATP-, ADP-, AMP-, and adenosine
(ADO)-regulated halide permeability specifically following expression
of wild-type (wt) cystic fibrosis transmembrane conductance regulator
(CFTR). This halide permeability is blocked by the
P1 purinergic receptor antagonist
8-phenyl theophylline, sensitive to the protein kinase A inhibitor
H-89, and associated with a modest, dose-dependent increase in cellular
cAMP concentration. Phorbol esters poorly activate halide permeability
compared with ADO, and ADO-stimulated efflux was not affected by
treatment with the protein kinase C inhibitor bisindolylmaleimide I. The A2 ADO receptor (AR) agonists
5'-N-ethylcarboxamide adenosine
and ADO were strong activators, whereas the
A1 AR agonist
R-phenylisopropyladenosine failed to
activate halide permeability. Metabolic conversion of ADO nucleotides
by surface ecto-5'-nucleotidase to more active (less
phosphorylated) forms contributes to anion transport activation in
these cells. Immunoprecipitation with
anti-A2B AR antibody identified a
31-kDa protein in both COS-7 and human bronchial epithelial cells.
Together, these findings indicate that ADO and its nucleotides are
capable of activating wtCFTR-dependent halide permeability through
A2B AR and that this AR subtype is
present in human bronchial epithelium. We also present data showing
that this pathway can activate clinically significant mutant CFTR
molecules such as R117H.
secretion; genetics; COS-7; cystic fibrosis; G protein-coupled receptor
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INTRODUCTION |
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CYSTIC FIBROSIS TRANSMEMBRANE conductance regulator
(CFTR) is a member of the traffic ATPase family (18), serving diverse roles relevant to ion transport, including endogenous
Cl channel function (5),
downregulation of Na+ transport
(39), and secondary Cl
channel regulation (33). Positive regulation of CFTR
Cl
channel activity is
complex and includes direct ATP binding and hydrolysis at nucleotide
binding domains 1 and 2 (8, 17), phosphorylation of the regulatory
domain by protein kinases A and C (PKA and PKC) (10, 20), and
interactions of the phosphorylated protein with membrane-associated
phosphatases (6). These studies, through addition and subtraction of
regulatory components, have defined the function of CFTR at the
single-channel or whole cell level.
Surface receptors represent physiological and potentially accessible
routes to modulate CFTR activity and
Cl secretion in vivo. For
example, vasoactive intestinal peptide is capable of CFTR activation in
certain epithelia (26, 42).
2-Adrenergic receptor
stimulation is a well-established receptor-coupled method to activate
CFTR-dependent Cl
secretion
in the mammalian airway and is an important mechanism by which to
discriminate between cystic fibrosis (CF) and non-CF phenotypes in vivo
(22). Study of receptor-coupled CFTR activation is somewhat difficult,
however, due in part to the many
Cl
secretory pathways that
may be influenced by surface receptor activation. Furthermore, direct
addition of drugs such as forskolin, membrane-permeant cAMP analogs,
PKA, or other regulatory proteins bypasses events at the cell surface
that may play important roles in CFTR regulation and thus may not fully
predict responses in living cells.
Adenosine (ADO) nucleotides appear to be important regulators of
Cl secretion through
surface receptors. However, stimulation of Cl
secretion in airway and
intestinal epithelia following purinergic receptor activation regulates
Cl
conductive pathways by
both CFTR-dependent and CFTR-independent mechanisms (2-4, 12, 23,
27, 31, 37, 38, 40, 41). Moreover, of the many purinergic receptors and
pathways described, those responsible for signaling through CFTR are
not known with certainty. ADO receptor (AR) activation by ADO and ADO
analogs has been shown to be a potent apical stimulus to elevate
cellular cAMP concentration and activate cAMP-dependent
Cl
secretion in
CFTR-expressing epithelia, including human and canine airway cell
monolayers (21, 23, 31) and the T84 human colonic cell line (2-4,
25, 41). Although ADO-dependent activation of luminal
Cl
secretion in each of
these epithelia appears to be at least in part CFTR dependent, a
mechanistic understanding of AR-coupled Cl
secretion has been
limited by the numerous purinergic receptor subtypes and diverse
Cl
secretory pathways
inherent in these epithelia. Additional studies of purinergic receptor
subtypes capable of signaling
Cl
transport, and in
particular coupling of ARs to CFTR activation, would contribute to a
better understanding of how ADO and its nucleotides regulate epithelial
ion transport.
In this study, we report that ADO and adenosine mono-, di-, and
triphosphates activate wild-type (wt) CFTR specifically through an
A2B AR. The pathway could be
defined unambiguously because COS-7 cells lack endogenous cAMP-,
Ca2+-, ADO-, or ADO
nucleotide-dependent Cl
efflux. We also show that the same receptor is present in human airway
epithelial cells and provide the first example of activation of a
clinically important CFTR mutation (R117H) through this
receptor-coupled pathway.
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METHODS |
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Transient CFTR expression. CFTR was transiently expressed in COS-7 and HeLa cells using a vaccinia-based expression system. This system is a well established method to study the function and processing of wtCFTR and mutant CFTR proteins (9). Cells grown on Vectabond-treated glass coverslips (for fluorescence measurements), 100-mm tissue culture-treated petri dishes, or tissue culture-treated six-well trays (Costar) were infected with vaccinia virus containing the T7 polymerase (vTF7-3; generous gift of Dr. B. Moss, National Institutes of Health; hereafter referred to as vT7) at a multiplicity of infection of 10 for 30 min. After vaccinia infection, wtCFTR or R117H CFTR under control of the T7 promoter in the pTM-1 vector was introduced into cells in complex with 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE; 20 µg DOTAP/DOPE and 5 µg pTM-1 CFTR per 5 × 105 cells) for 4 h. wtCFTR in the pTM-1 vector was the generous gift of Dr. S. Cheng (Genzyme, Cambridge, MA); R117H CFTR in the pTM-1 vector was the generous gift of Dr. Michael Welsh (Howard Hughes Medical Institute, University of Iowa, Iowa City, IA). Cells were then washed in PBS, returned to DMEM plus 10% fetal bovine serum, and studied 18-24 h postinfection.
Functional CFTR assay.
CFTR function in individual cells was assayed using the halide-quenched
dye
6-methoxy-N-(3-sulfopropyl)quinolinium
(SPQ). Briefly, cells were loaded for 10 min with SPQ (10 mM) by
hypotonic shock and then mounted in a specially designed perfusion
chamber for fluorescence measurements. Fluorescence of single cells was measured with a Zeiss inverted microscope, a PTI imaging system, and a
Hamamatsu camera. Excitation was at 340 nm, and emission was measured
at >410 nm. All functional studies were at 37°C. Cells were
bathed in a quenching buffer (NaI) at the beginning of the experiments
and were switched after establishment of a stable baseline to a
halide-free (NO3) dequenching buffer
at 200 s. The SPQ assay used in these studies was configured to detect
I
efflux through CFTR or
other permeability pathways as described previously (10). Cells were
stimulated with agonist, unless otherwise indicated, at 500 s and then
returned to the quenching NaI buffer. Fluorescence was normalized to
the baseline (quenched) value (average fluorescence from 100-200
s), with increases presented as percent increase in fluorescence over
basal. Values used to generate curves are from all dequenching cells
(~90% of all cells studied) in paired experiments performed over
1-2 days. Values are means ± SE. The buffers used in the SPQ
assay were 1) NaI buffer [in
mM: 130 NaI, 5 KNO3, 2.5 Ca(NO3)2,
2.5 Mg(NO3)2,
10 D-glucose, and 10 HEPES (pH
7.30)] and 2)
NaNO3 buffer (identical to NaI
buffer except that 130 mM NaNO3
replaced NaI). Relative permeability coefficients for maximal
I
exit from COS-7 cells
were measured by SPQ as the rate of dequench after agonist stimulation
(mean fluorescence/s for the 100 s of maximal fluorescence change). To
maximize intracellular inhibitor activity for certain experiments (see
Fig. 3), cells were loaded with inhibitors at concentrations above the
inhibition constant values reported in cell-free systems. Specifically,
the staurosporine concentration used (see Fig. 3) was 100- to
1,000-fold above the IC50 values
for purified kinases including PKA, PKC, and protein kinase G
(IC50 values for these enzymes are
0.5-10 nM). H-89 (PKA inhibitor) and bisindolylmaleimide I (BIM-I;
PKC inhibitor) were used (see Fig. 3) at concentrations 100- to
200-fold above those known to inhibit the respective purified enzymes
in cell-free systems (per manufacturer's protocol).
Human primary airway cell tissue culture. Bronchial tissue from surgical specimens was obtained through the University of Alabama at Birmingham Tissue Procurement Office. Cells were isolated and grown as explant cultures in plastic tissue culture dishes (15), except that serum-free growth medium was modified as described in Ref. 44. Cells were fed serum-free growth medium on alternate days and studied ~2 wk postseeding.
cAMP levels. Cellular cAMP levels were measured using a standardized fluorometric cAMP assay kit (Cayman Chemicals, Ann Arbor, MI); 1 × 106 cells were stimulated with agonist for 10 min and extracted with ice-cold ethanol, and the cAMP levels from the supernatant were determined. The nonxanthine phosphodiesterase inhibitor papaverine was used in these experiments, as xanthines (i.e., IBMX) may have interfering effects on AR activation (19). Experiments were performed in quadruplicate on 3 separate days, with similar results obtained in each case.
Protein detection. Cell lysates from 1 × 106 cells were isolated and immunoprecipitated with isoform-specific rabbit anti-human A2B AR (Alpha Diagnostics, antibody raised against a 16-amino acid sequence corresponding to an extracellular domain of the human A2B AR) linked to protein G-Sepharose beads as previously described (9). Proteins were separated by 12% SDS-PAGE and blotted onto polyvinylidene difluoride membranes. Primary antibody was rabbit anti-human A2B AR, secondary antibody was goat anti-rabbit IgG biotin conjugate (Boehringer Mannheim), and tertiary antibody was neutralite avidin alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). Blots were developed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 4-nitro blue tetrazolium chloride (NBT) in carbonate buffer (pH = 8.8).
Materials.
ATP, ADP, ADO, UTP, IBMX,
5'-N-ethylcarboxamide adenosine
(NECA), adenosine deaminase (ADA), and ,
-methyleneadenosine
5'-diphosphate were obtained from Sigma (St. Louis, MO); AMP,
forskolin, dideoxyforskolin, phorbol 12-myristate 13-acetate (PMA),
staurosporine, H-89 dihydrochloride, 9-(tetrahydro-2'-furyl)adenine (SQ-22536), BIM-I, 8-phenyl
theophylline (8-PT), and
R-phenylisopropyladenosine (R-PIA)
were purchased from Calbiochem (San Diego, CA); SPQ was obtained from
Molecular Probes (Eugene, OR); DOTAP/DOPE was from Avanti Polar Lipids
(Alabaster, AL); A2A AR and
A2B AR antibodies were obtained
from Alpha Diagnostic International (San Antonio, TX); NBT, BCIP, and
goat anti-rabbit IgG biotin conjugate were purchased from Boehringer
Mannheim (Indianapolis, IN); Vectabond was purchased from Vector
Laboratories (Burlingame, CA).
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RESULTS |
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ADO and its nucleotides, including ATP, ADP, and AMP (200 µM), were
strong activators of halide permeability in COS-7 cells expressing
wtCFTR but not in the absence of CFTR expression (Fig. 1, A and
B). Activation by ADO
and AMP was brisk and quantitatively similar to the response following
direct activation of adenylate cyclase plus phosphodiesterase
inhibition (Fig. 1A; forskolin 20 µM plus IBMX 100 µM). In contrast, UTP (200 µM) or ionomycin (2 µM) failed to activate halide permeability in wtCFTR-expressing COS-7
cells, whereas ATP and ADO failed to activate halide permeability in
control COS-7 cells infected with vT7 alone (Fig.
1A). In other control experiments,
inhibition of the
Na+-K+-2Cl
cotransporter by furosemide (200 µM) had minimal effect on halide efflux stimulated either by forskolin plus IBMX or by ADO, suggesting that the cotransporter is not a substantial participant in these two
activation pathways. Maximal activation by either forskolin plus IBMX
or by ADO was decreased by <10% in the presence of furosemide (data
not shown).
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Stimulatory effects were also observed in T84 cells expressing endogenous wtCFTR, although ADO nucleotide-dependent responses were frequently more rapid in onset and of greater magnitude than pharmacologic stimulation of adenyl cyclase (Fig. 1C). Interpretation of these effects in T84 cells is complicated by the presence of multiple endogenous halide permeability pathways activated by both CFTR-dependent and CFTR-independent nucleotides (2-4, 25, 38, 40, 41). ADO nucleotides may augment halide efflux in T84 cells by stimulation of these additional pathways. In contrast, ADO and its nucleotides failed to activate halide permeability in HeLa cells expressing wtCFTR compared with direct adenyl cyclase stimulation (Fig. 1D). Thus CFTR is necessary but not sufficient to confer ADO- and ADO nucleotide-regulated halide permeability in the COS-7 and HeLa cell lines following transient expression.
Figure 1 suggests that ADO and its nucleotides activate CFTR in COS-7
cells by a purinergic receptor pathway. To differentiate between ADO
(P1)- and ATP
(P2)-selective receptors, we
studied the effects of the methylxanthine AR
(P1) antagonist 8-PT on ADO nucleotide-activated halide permeability. The results in COS-7 cells
expressing wtCFTR (Fig. 2,
A-D)
indicate that ADO nucleotide-activated halide permeability is sensitive
to 8-PT at concentrations known to elicit AR blockade (29). These
findings support AR (P1) rather than ATP receptor (P2)
activation of CFTR in COS-7 cells.
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The effects of kinase inhibition are shown in Fig.
3. In the first set of
experiments (Fig. 3, A and
B), the broad-spectrum kinase
inhibitor staurosporine reduced halide permeability after stimulation
by 20 µM forskolin plus 100 µM IBMX (to raise cellular cAMP) and
ADO (200 µM) in a similar, dose-dependent manner. These results
suggest that the effects of ADO are indirect and include kinase
activation. Because CFTR has been shown to be regulated by adenyl
cyclase, PKA, and PKC, we investigated the effects of a PKA-specific
inhibitor (H-89) and a PKC-specific inhibitor (BIM-I) on ADO- and
forskolin-stimulated efflux. As is shown in Fig.
3C, inhibition of the PKA pathway by
H-89 (4 µM; 2-h incubation) blocked both ADO- and
forskolin-stimulated halide effluxes. In contrast, treatment with the
PKC inhibitor BIM-I had no effect on ADO-stimulated halide efflux (Fig.
3D), whereas similar exposure to
H-89 (8 µM) plus the adenyl cyclase inhibitor SQ-22536 (40 µM)
completely abolished the response to ADO (data not shown). Furthermore,
stimulation of cells with the PKC activator PMA (100 nM; Fig.
3E), at a concentration shown
previously to acutely activate halide efflux in primary canine airway
cells (24) and CFTR-dependent halide efflux in C127i cells (13),
had very little effect on halide efflux compared with ADO. Together,
these results indicate that ADO activation of halide permeability in
CFTR-expressing COS-7 cells is sensitive to adenyl cyclase and
PKA inhibition and suggest that ADO activates wtCFTR in COS-7
cells through a cAMP- and PKA-dependent AR pathway.
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We next investigated ADO nucleotide potency in COS-7 cells expressing
wtCFTR. Determining an analog activity series is a useful way to
functionally identify AR subtypes, for example, in human respiratory
cell monolayers (23). Figure 4 indicates
that NECA and ADO are strong activators of halide permeability, whereas R-PIA is not (5 nM to 5 µM). Together, the selectivity series NECA > ADO > R-PIA supports A2 AR
stimulation governing activation of halide permeability (29).
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A2 AR stimulation functionally
couples to Gs, activating adenyl
cyclase and elevating cellular cAMP (29). We therefore compared cellular cAMP levels in COS-7 cells following incubation with forskolin
(20 µM) or ADO nucleotides (ADO, AMP, ADP, and ATP; each at
concentrations of 200 µM). The results in Fig.
5 demonstrate that ADO and three of its
nucleotides elevate cellular cAMP levels above control values.
Interestingly, ADO and related nucleotides elevated cellular cAMP
levels far less than direct adenyl cyclase stimulation, despite strong
activation of halide permeability by each of these agonists (Fig. 1,
A and
B). These findings suggest that
activation of CFTR by this receptor-coupled mechanism may be greater
than what would be expected based strictly on elevation of cellular
cAMP. To further investigate the relationship between receptor-coupled
cAMP elevation and CFTR activation, we performed dose-response studies
of the effect of forskolin and ADO on cAMP levels and anion transport
(Fig. 6,
A-C).
Figure 6A compares forskolin- and
ADO-stimulated cAMP dose response curves and demonstrates that
forskolin can elevate cAMP levels nearly 10-fold higher than ADO, with
a half-maximal level achieved at ~5 µM. In contrast, cAMP levels
after ADO, although significantly less than those achieved following
forskolin stimulation, demonstrate dose dependency, with half-maximal
levels achieved at ~0.1 µM (Fig.
6B). In functional SPQ experiments
(Fig. 6C), ADO is substantially more
potent (activation of I
efflux begins at ~0.1 µM) than direct adenyl cyclase stimulation with forskolin (activation of
I
efflux begins at ~10
µM) following the acute addition of agonists. The cumulative halide
permeability measurements by SPQ such as those shown in Fig. 6 do not
account for desensitization of cell surface receptors or other aspects
of cellular tachyphylaxis. However, in all of our experiments (Figs. 1,
3, 6C, and 9), ADO elicited anion
transport effects roughly equivalent to or above those of forskolin,
even though cAMP levels in these cells are much less elevated following
ADO stimulation compared with forskolin (Figs. 5 and 6,
A and
B). These results therefore raise
the possibility that total cellular cAMP concentration may not directly
predict CFTR activation and that mechanisms in addition to cAMP may
contribute to ADO-dependent activation of wtCFTR (see
DISCUSSION).
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The preceding experiments suggest that
A2 AR activation mediates the ADO
nucleotide-stimulated halide permeability in COS-7 cells.
A2 ARs have been subclassified
into A2A and
A2B subtypes (29), both of which
can signal through adenyl cyclase. COS-7 cells are not known to
endogenously express A2 ARs, but
at least one report suggests that ADO can elevate cellular cAMP in this cell line (35). To clarify the A2
AR subtype responsible for our findings, we performed
immunoprecipitation studies in COS-7 and normal human bronchial
epithelial cells using isoform-specific anti-A2A and
anti-A2B antibodies. In both cell
types (Fig.
7A), an
~31-kDa protein was recognized by the
anti-A2B antibody, indicating the
presence of the A2B AR in COS-7
cells and normal human bronchial epithelia. In contrast, the
A2A AR polyclonal antibody failed to identify the A2A AR in either
cell type (data not shown).
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These studies indicate that ADO and ADO phosphates activate wtCFTR by
utilizing an A2B AR in COS-7
cells. However, whether the stimulatory effects of ADO nucleotides are
produced by these nucleotides or by their metabolites is unknown.
Previous studies of A2
AR-activated Cl secretion
in colonic and airway epithelia suggest that surface ecto-5'-nucleotidase activity influences ADO nucleotide-activated Cl
secretion by
metabolizing ADO phosphates to ADO (37, 38, 40, 41). We therefore
investigated the effects on ADO nucleotide-stimulated halide efflux of
1) ecto-5'-nucleotidase
inhibition and 2) ADO depletion by
conversion to inosine. In the first experiment, COS-7 cells were
stimulated with ATP in the presence of the ecto-5'-nucleotidase inhibitor
,
-methyleneadenosine 5'-diphosphate. The ATP
formulation used in these experiments does not contain measurable
levels of ADO as judged by HPLC (Sigma technical specifications).
Figure 8A
indicates that inhibition of ecto-5'-nucleotidase activity attenuated the ATP-activated halide permeability, suggesting that ADO
and/or adenosine mono- or diphosphates generated by surface nucleotidase contribute to CFTR activation. In the second experiment, COS-7 cells were stimulated with freshly prepared AMP (200 µM) pretreated with ADA (1 IU/ml). ADA catalyzes the conversion of ADO to
the inactive nucleotide inosine. Figure
8B indicates that treatment of an AMP
solution with ADA attenuates the AMP-activated halide permeability.
Together, these two experiments suggest that metabolic conversion of
ADO nucleotides to more active (less phosphorylated) forms contributes
to the A2B AR activation of halide
permeability in our experiments. This correlates well with studies of
other model epithelia, in which ADO phosphate stimulation of
A2 ARs in T84 cells has been shown
to depend in part on conversion to ADO by surface
ecto-5'-nucleotidases (25, 37, 41).
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The results presented here demonstrate that
A2B AR stimulation elevates cAMP
levels and strongly activates halide permeability in COS-7 cells
expressing wtCFTR. We next asked whether a clinically relevant CFTR
mutant protein could be activated following
A2B AR stimulation. We chose to
study the R117H CFTR, which is known to localize to the cell surface
and maintain normal PKA-dependent activation but which has reduced
single-channel Cl
conductance (34, 43). Figure 9 compares 10 µM ADO- and 10 µM forskolin-stimulated halide permeability in COS-7
cells expressing R117H CFTR, indicating similar strong responses. These
results show that a surface-localized conduction mutant can be
activated in vitro through an A2
AR mechanism that should also be available in vivo. Furthermore,
activation of R117H CFTR by ADO is qualitatively similar to that
obtained by pharmacologic stimulation of adenyl cyclase.
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DISCUSSION |
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It has been shown that ADO and its nucleotides regulate
Cl channel activity in
human airway and colonic epithelia, and this model provides a mechanism
by which the local cellular environment may control luminal
Cl
secretion. The present
studies, using a simplified system, demonstrate coupling of the
A2B AR to CFTR activation and
indicate that ADO and its nucleotides can be potent agonists for
CFTR-dependent halide efflux.
ADO nucleotides elicit Cl
secretion in CF cells and tissues by many pathways, although the
underlying mechanisms are not fully understood. ATP and UTP have been
shown to activate a Ca2+-dependent
Cl
conductive pathway
independent of CFTR through stimulation of P2u receptors (12, 27, 40). ATP is
also capable of activating wtCFTR utilizing a cAMP-independent
purinergic receptor pathway in C127i epithelioid cells
(7). Mucosal AR activation stimulates Cl
secretion in normal, but
not CF, primary airway cell monolayers, although the CFTR genotypes in
these studies were not specified (23). The
A1 AR antagonist
8-cyclopentyl-1,3-dipropylxanthine promotes cAMP-activated whole cell
Cl
currents in normal and
CF respiratory cells (28) and may be capable of directly activating
surface-localized
F508 CFTR (14). Transcripts of both
A1 and
A2 receptors are expressed in
airway epithelium, but the subtypes mediating receptor-coupled
Cl
secretion are not known
(28, 29, 36). In summary, the positive regulation of CFTR and other
Cl
conductive pathways by
ADO and related nucleotides appears to be important to the regulation
of epithelial Cl
transport.
Clinical trials based on previous observations with ATP, UTP, and
8-cyclopentyl-1,3-dipropylxanthine illustrate the importance of a
thorough understanding of the processes by which this class of
compounds activate Cl
secretion.
Although AR-coupled cAMP accumulation has been demonstrated in the
COS-7 cell line previously (35), no studies have shown that COS-7 cells
endogenously express either A1 or
A2 ARs. The results in Fig. 6
suggest that 1) ADO is a potent
agonist for wtCFTR activation, 2)
CFTR activity may be stimulated with relatively lower cellular cAMP
levels in the setting of A2B AR
activation, and 3) receptor-coupled
elevation of cellular cAMP may activate CFTR more efficiently than
non-receptor-mediated (forskolin-induced) elevation of cellular cAMP.
These results also raise the possibility that non-cAMP-dependent
signaling pathways, such as PKC, might contribute to some of the
AR-coupled CFTR activation noted in our experiments. Additional
pathways regulating Cl
secretion have been described in CF and non-CF cells in the past, including mediators that potentially have synergistic or permissive effects with cAMP-activated
Cl
secretion (2-4, 7,
12, 14, 27, 28, 40). The evidence presented in our experiments,
however, suggests that in COS-7 cells a primary mechanism is through
the A2B AR and cAMP (Figs. 3-5).
Four human AR subtypes have been identified, cloned, and characterized
in terms of pharmacology, distribution, and cellular function (29). The
two A2 AR subtypes,
A2A AR and
A2B AR, differ in size, agonist
potency series, and tissue distribution, but both classically coupled
to Gs and adenyl cyclase (29).
A2 AR activation has been found to
regulate diverse tissue processes, including respiratory and colonic
ion transport (2-4, 23, 25, 31).
A2A AR transcripts have been
identified in human lung, brain, heart, and kidney (29), whereas
transcripts from the smaller A2B
AR (330 amino acids; protein ~31-33 kDa) have been identified in
many tissues, including human brain, ileum, colon, and the human
colonic T84 cell line (29, 30, 32, 36, 38). Neutrophil-derived AMP has
been shown to mediate A2B AR
activation, cAMP accumulation, and
Cl secretion following
conversion to ADO by surface-localized CD73 (ecto-5'-nucleotidase) in T84 cells (25, 37, 38). The
A2 AR subtype(s) responsible for
ADO-stimulated Cl
secretion
in human airway cells, however, has not been defined. Our results
demonstrate that A2B ARs mediate
halide permeability effects in COS-7 cells following wtCFTR expression,
and indicate the presence of A2B
AR protein in primary human bronchial epithelial cells.
A2B receptor stimulation of COS-7
cells expressing either wtCFTR or R117H CFTR indicates that this G
protein-coupled receptor can effectively activate CFTR-dependent halide
transport (Fig. 9). The R117H CFTR represents a class IV CFTR mutation,
characterized by intact protein production, maturation, surface
localization, and regulation but defective single-channel
Cl conduction (34, 43).
Therefore, this mutation represents an ideal candidate to investigate
the effects of A2B AR stimulation. The phenotype of patients possessing the R117H mutation is unusual. These patients are predominantly pancreas sufficient but do suffer the
typical respiratory sequelae of cystic fibrosis (16). It has therefore
been suggested that the R117H mutation may rest at the boundary of
required CFTR function in two organ systems, providing adequate
function to protect the exocrine pancreas but failing to provide the
necessary function in the lungs to protect the airways from the
pulmonary manifestations of cystic fibrosis. Although
Cl
transport in cells
expressing the common
F508 CFTR trafficking mutant may not be
expected to be stimulated by A2B
AR activation, our studies raise the possibility that the function of
R117H CFTR and possibly other class IV surface-localized CFTR mutations
might be augmented through pharmacologic activation of
A2B AR. Further studies will be
required to test this possibility.
In summary, these experiments are the first to describe
1) AR-coupled CFTR activation by ADO
and adenosine mono-, di-, and triphosphates in a cell line devoid of
endogenous competing Cl
transport pathways, 2)
A2B AR regulation of
CFTR-dependent halide transport, 3)
A2B AR protein in COS-7 and native
human bronchial epithelia, and 4)
A2B receptor activation of R117H
CFTR. The findings help clarify the positive regulatory effects that
ADO and its nucleotides can confer to wtCFTR and R117H CFTR through the
A2B AR. COS-7 cells possess
properties that may be similar to native secretory epithelia, including
expression of G protein-coupled receptors. Previous studies indicate
that several mutant CFTRs can be pharmacologically activated in
cell-free systems with PKA or PKC plus ATP, by direct stimulation of
adenyl cyclase, or by phosphatase inhibition. None of these methods,
however, utilize endogenous receptor-coupled pathways that are present
in vivo. The studies presented here provide a means to evaluate whether mutant CFTRs localized to the plasma membrane can be activated by
A2B receptor coupling. For
example, additional experiments in COS-7 cells have recently allowed us
to identify two other surface-localized CFTR mutants (A455E and G1349D)
that can functionally couple to
A2B AR activation (11). We
speculate that patients possessing these and other surface-localized
mutant CFTRs might uniquely benefit from therapies that exploit
AR-coupled pathways to activate CFTR.
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ACKNOWLEDGEMENTS |
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We are indebted to Jan Tidwell and Kynda Roberts for help in preparing the manuscript.
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FOOTNOTES |
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J. P. Clancy is a Leroy P. Matthews Award recipient, funded through the Cystic Fibrosis Foundation (CLANCY96LO). These experiments were also supported by Cystic Fibrosis Foundation Research Development Program, Component II, R464, and by National Institutes of Health Grants P30-HD-28831 and RO1-DK-49057.
Addresss for reprint requests: J. P. Clancy, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, 1918 University Blvd. (782 MCLM), Birmingham, AL 35294-0005.
Received 15 September 1997; accepted in final form 3 November 1998.
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